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Arbejdsrapport fra Miljøstyrelsen, 51/2003

Appendices 1-18 to: Report on the Health Effects of Selected Pesticide Coformulants

Contents

Appendix 1: Manganese (II) sulphate, Manganese (II) sulphide1   General description

Because limited data are available specifically for manganese sulphate and manganesse sulphide, and most manganese compounds seem to cause the same adverse effects, this paper includes several studies with other inorganic manganese compounds.s

Manganese displays oxidation states: +2, +3, +4, +6 and +7. Compounds in which manganese is in its +2 oxidation state are the most stable ones, but bivalent manganese can be readily oxidized to the +3 and +4 states. (Beliles 1994).

 In this paper, the following manganese compounds are addressed:

Manganese (II) sulphate (MnSO₄)
Manganese (II) sulphide (MnS),
Manganese (II) chloride (MnCl₂)
Manganese (II, III) tetroxide (Mn₃O₄)
Manganese (III) trioxide (Mn₂O₃)
Manganese (III) phosphate (MnPO₄)
Manganese (IV) dioxide (MnO₂)

1.1  Identity

Molecular formula:          a) MnSO₄
                                      b) MnS

Structural formula:           a)

                                      b)
                                     

Molecular weight:            a) 151.00
                                       b) 87.00

CAS-no.:                        a) 7785-87-7
                                       b) 18820-29-6

Synonyms:                      a) Manganese sulphate
                                          Sulphuric acid, manganese (2⁺) salt (1:1)

                                       b) Manganese monosulphide

1.2   Physical / chemical properties

Description:                    a)  Manganese sulphate forms several hydrates. The
                                      article of commerce is usually a mixture of the
                                      tetra- and pentahydrates. The monohydrate exists
                                      in the form of pale red crystals.

                                     b)  Manganese sulphide is a pink, green or brown-
                                     green powder. Three crystalline modifications
                                     exist: a-form, green cubic crystals; b-form, red
                                     cubic crystals; g-form, red hexagonal crystals.

                                     In moist conditions manganese sulphide readily
                                     oxidizes in air to the sulphate.               

Melting point:                  a) 700°C

Boiling point:                   a) 850°C (decomposes)

Density:                          a) 3.25 g/ml    

Vapour pressure:             -

Concentration of

saturated vapours:           -

Solubility:                        a) Water:  520 g/l (at 5°C), 700 g/l (at 70°C) 

                                      b) Insoluble in water. Soluble in diluted acids.

References:                    ATSDR (2000), ChemFinder (2001), HSDB (2000),

                                      Merck Index (1996)

2   Toxicokinetics

Manganese is a natural component of the environment and an essential element in the nutrition of humans and animals. No formal recommended dietary allowance exists for manganese. However, the Scientific Committee for Food of the EU has estimated 1 to 10 mg Mn/day as an acceptable range of intake. This level is equivalent to the usual daily intake. Manganese is involved in basic enzymatic processes including oxidative phosphorylation, decarboxylation, hydrolysis, Krebs cycle reactions, and the metabolism of carbohydrates. The concentration of manganese in the body is under normal circumstances held relatively constant by alterations in the rate and amount of manganese absorbed from the gastrointestinal tract, and by its distribution and rate of excretion (homeostasis). In children under one year of age, the homeostatic mechanisms may not have fully developed. (ATSDR 2000, Beliles 1994, SCF 2000).

2.1   Absorption

Manganese absorption occurs primarily through the diet. However, absorption via the lungs can be significant for occupationally exposed persons or for those exposed to excess levels of airborne manganese, such as downwind of a manganese point source (ATSDR 2000).

2.1.1   Inhalation

Inhalation of inorganic manganese may be in the form of aerosols or attached to particles, which may reach the lungs depending on the aerosol or particle size, shape, density, hygroscopicity, and electric charge. The deposition pattern in the respiratory tract of inhaled inorganic manganese is related to particle size. In humans, large particles (aerodynamic diameter of 5-30 mm) are mainly deposited in the upper airways while smaller particles (5 mm or less) may reach the lower airways. Particles that are deposited in the lower airways are probably mainly absorbed, while particles deposited in the upper airways may be moved by mucociliary transport to the throat, where they are swallowed and enter the stomach. A study has reported that more than 60% of particulate manganese trioxide inhaled by human volunteers was transferred to the gastrointestinal tract. No studies measuring the absolute amount of absorbed manganese following inhalation have been found. Manganese chloride is more readily absorbed than manganese dioxide following intratracheal instillation indicating that the absorption following inhalation is higher for more soluble forms of inorganic manganese. (ATSDR 2000, Beliles 1994).

2.1.2   Oral intake

Of ingested inorganic manganese only about 3-5% is absorbed from the gastro-intestinal tract in humans. There are many factors influencing the absorption such as water solubility of the compounds in ques­tion, age, and the level of manganese and other trace elements such as iron in the gut. (ATSDR 2000, Beliles 1994).

Studies with rodents show that the absorption and/or retention of manganese are higher in neonates, but returns to the level of older animals at approximately post-gestational day 17-18. Available studies do not provide adequate data to determine when this transition takes place in human infants. (ATSDR 2000, Beliles 1994).

In persons with iron defi­ciency, manganese absorption is increased probably because iron and manganese are absorbed by the same transport system in the gut. In some animal studies, calcium and phosphorus have depressed the manganese uptake. In rats fed a diet deficient in manganese, the absorption was at least two-fold higher than in rats ingesting an adequate amount of manganese. (ATSDR 2000, Beliles 1994).

2.1.3   Dermal contact

Manganese uptake across intact skin would be expected to be extremely limited (ATSDR 2000).

2.2   Distribution

In healthy humans, tissue concentrations of manganese range mainly between 0.1 and 1 mg Mn/kg wet weight with the highest levels in the liver, pancreas, and kidney and the lowest levels in bone and fat. (ATSDR 2000, Beliles 1994).

2.2.1   Inhalation

Seventy male CD rats were exposed nose-only for 90 minutes to an aerosol of radiolabelled manganese chloride particles at 0.54 mg Mn/m³. Half of the animals had both nostrils patent while the other half had the right nostril plugged to prevent nasal deposition of manganese chloride on the occluded side. At 0, 1, 2, 4, and 8 days post-exposure, the left and right sides of the nose and brain, including the olfactory pathway and striatum were sampled and analysed for the level of manganese. High levels of manganese were observed in the olfactory bulb and tract/tubercle on the side or sides with an open nostril within 1-2 days following inhalation consistent with direct olfactory transport of inhaled manganese from the nasal cavity to the brain bypassing the blood-brain barrier. The difference between the manganese level on the occluded and unoccluded side of the olfactory pathway was more than 90%. Only minor differences in the manganese level were seen between the left and right sides of the striatum in unilaterally occluded rats. Besides the brain, other organs (lungs, liver, kidneys, pancreas) also had increased tissue manganese concentrations following inhalation exposure. (Brenneman et al. 2000).

Groups of 6 adult male Sprague-Dawley rats were exposed to airborne manganese phosphate particles at 0, 0.03, 0.3 or 3 mg Mn/m³ for 6 hours/day for 2 weeks. Increased manganese concentrations were reported in olfactory bulb, lung, and striatum at the middle dose and also in cerebellum, femur, skeletal muscle, plasma, and erythrocytes at the high dose at the cessation of exposure. The liver manganese concentration was unaffected by inhalation exposure to manganese phosphate. (Vitarella et al. 2000).

Sprague-Dawley rats got an intranasal instillation with 0.004 mg Mn/kg b.w. of manganese chloride. The olfactory bulb contained the vast majority of measured manganese at 1, 3, and 7 days post-dosing (90, 69, and 47%, respectively) with a value decreasing to 16% at 12 weeks. At the same time-points, the basal forebrain contained 6, 21, and 28% of measured manganese, respectively. Manganese levels in the cerebral cortex, hypothalamus, striatum, and hippocampus were also maximal at 7 days post-dosing. Liver and kidney each contained about 1% of the measured manganese during the first 7 days. (Tjälve et al. 1996 – quoted from ATSDR 2000).

Following intranasal injection of manganese chloride into one nostril, manganese was dose-dependently accumulated in the olfactory bulb and tubercle (Gianutsos et al. 1997 – quoted from ATSDR 2000).

Rats were instilled intratracheally with manganese chloride or manganese dioxide at 1.22 mg Mn/kg b.w. once a week for 4 weeks. Compared to control rats, an increase of the manganese level was measured in striatum, blood, cortex, and cerebellum. (Roels et al. 1997 – quoted from ATSDR 2000).

Preferential accumulation of manganese in specific locations of the brain (including the caudate nucleus, globus pallidus, and the substantia nigra) was noted in 1 monkey exposed to an aerosol of manganese chloride (9-17 mg Mn/m³) several hours/day for 3-5 months (Newland et al. 1989 – quoted from ATSDR 2000).

2.2.2   Oral intake

Dietary manganese, absorbed mainly as Mn(II), enters portal circulation from the gastrointestinal tract and is bound to a-macroglobulin or albumin in the plasma. After transportation to the liver, the major portion of Mn(II) is secreted in bile, but some may be oxidized to Mn(III), which enters the circulation conjugated with plasma transferrin. (Aschner & Aschner 1991 – quoted from ATSDR 2000).

Studies in animals indicate that prolonged oral exposure to manganese compounds results in increased manganese levels in the tissues, but that the magnitude of the increase diminishes over time (ATSDR 2000).

Manganese in the form of manganese chloride or manganese dioxide is distributed more readily to the brain following intratracheal exposure compared to oral exposure by gavage (Roels et al 1997 – quoted from ATSDR 2000).

Mice and rats chronically fed manganese sulphate generally had elevated tissue levels of manganese. The manganese levels in the liver and kidney were higher than the levels in the brain. (NTP 1993 – quoted from ATSDR 2000).

Excess manganese (following oral exposure in humans with liver diseases) was accumulated in certain regions of the brain (basal ganglia, especially the globus pallidus and the substantia nigra) (ATSDR 2000). 

2.2.3   Dermal contact

No data were found.

2.2.4   Other routes

Studies with intraperitoneal and intravenous injections of inorganic manganese to monkeys, calves and rats have been performed. The distribution of manganese may vary depending on the route of exposure. For instance it has been shown that calves injected intravenously had a higher concentration of manganese in the liverand pancreas than calves fed manganese. Rats injected intraperitoneally had more manganese in the pancreas and less in the liver than rats fed manganese. Administration of manganese dioxide intraperitoneally to rats resulted in greater concentration of manganese in the brain than did manganese chloride. In a monkey study with intraperitoneal administration, manganese was found in all tissues studied with the highest level in pancreas, kidney and liver and the lowest level in the blood. (ATSDR 2000). 

2.3   Elimination

In general, biliary excretion is the main pathway by which absorbed manganese reaches the intestines where most of the element is excreted in the faeces. However, some of the manganese in the intestine is reabsorbed through enterohepatic circulation. Small amounts of manganese can also be found in urine, sweat, and milk. (ATSDR 2000).

2.3.1   Inhalation

In humans inhaling manganese chloride or manganese trioxide, about 60% of the material originally deposited in the lungs was excreted in the faeces within 4 days. In several studies, occupationally exposed men had significantly higher urine manganese levels compared to unexposed men. (ATSDR 2000).

Rats instilled intratracheally with manganese chloride or manganese tetroxide excreted about 50% of the dose in the faeces within 3-7 days (Drown et al. 1986 – quoted from ATSDR 2000).

Monkeys exposed to an aerosol of manganese chloride excreted most of the manganese with a half-life of less than half a day. However, a portion of the compound was retained in the lung and brain and was cleared with a half-life of 12-250 days. No further details were given. (Newland et al. 1987 – quoted from ATSDR 2000).

2.3.2   Oral intake

Humans who ingested tracer levels of radioactive manganese excreted the manganese with a half-life of 13-37 days (ATSDR 2000).

2.3.3   Dermal contact

No data were found.

2.3.4   Other routes

Rats exposed to manganese chloride by intravenous injection excreted 50% of the dose in the faeces within 1 day and 85% by day 23. Only minimal levels were excreted in the urine (<0.1% of the dose within 5 days). (Klaassen 1974, Dastur et al. 1971 – both quoted from ATSDR 2000).

2.4   Mode of action

The central nervous system is the primary target of manganese toxicity. In humans manganese causes neurochemical and neuropathological changes predominantly in the globus pallidus in the basal ganglia resulting in symptoms similar to but still different from parkinsonism. Although it is known that manganese is a cellulartoxicant that can impair transport systems, enzyme activities, and receptor functions, the principal manner in which manganese neurotoxicity occurs has not been clearly established. Several possibilities have been proposed. These include enhancement of the oxidation of dopamine and other catecholamines, which increases production of free radicals and reactive oxygen species, thereby causing oxidative stress and damage to the cell, following the depletion of cellular antioxidant defence systems. Other hypotheses involve damage predominantly to the mitochondria. (ATSDR 2000).

 3   Human toxicity

3.1   Single dose toxicity

No data were found.

3.2     Repeated dose toxicity

3.2.1     Inhalation

3.2.1.1   Respiratory effects

Inhalation exposure to manganese dusts such as manganese dioxide or manganese tetroxide has lead to an inflammatory response in the lungs mainly of workers exposed to fairly high concentrations (Abdel Hamid et al. 1990, Akbar-Khanzadeh 1993, Kagamimori et al. 1973, Lloyd Davies 1946, Nogowa et al. 1973, Roels et al. 1987a, WHO 1987 – all quoted from ATSDR 2000). 

3.2.1.2   Cardiovascular effects

Three occupational studies indicate that manganese may affect the cardiovascular system following inhalation by lowering the blood pressure. Specific data on exposure levels are lacking. (Saric & Hrustic 1975, Jiang et al. 1996a, Hobbesland et al. 1997b – all quoted from ATSDR 2000).

3.2.1.3   Endocrine effects

Only two studies have measured endocrine effects in humans exposed to inorganic manganese. Foundry workers exposed for approximately 10 years to 0.04-1.1 mg Mn/m³ (particulate matter) and 0.05-0.9 mg Mn/m³ (fumes) had elevated serum prolactin and cortisol levels. (Alessio et al. 1989 – quoted from ATSDR 2000).

Workers from a ferroalloy plant also had elevated serum prolactin levels (Smargiassi & Mutti 1999 – quoted from ATSDR 2000).

3.2.1.4   Neurological effects

From studies of humans exposed to manganese dusts (mainly manganese dioxide) in mines and factories, clear evidence exist that exposure to manganese for longer periods can lead to a series of serious and ultimately disabling neurological effects. This disease, termed manganism, typically begins with feelings of weakness and lethargy typically after several years of exposure but some individuals may begin to show signs after as few as 1-3 months of exposure. As the disease progresses, a number of other neurological signs such as slow and clumsy gait, speech disturbances, a mask like face, and tremors may become manifest. These signs are frequently accompanied by apathy and dullness along with impotence and loss of libido. (Abdel-Hamid et al. 1967, Emera et al. 1971, Mena et al. 1967, Nelson et al. 1993, Rodier 1955, Saric et al. 1977a, Schuler et al. 1957, Shuqin et al. 1992, Smyth et al. 1973, Tanaka & Lieben 1969, Whitlock et al. 1966 – all quoted from ATSDR).Neurological symptoms may improve in some cases (Shuqin et al. 1992, Smyth et al. 1973 – both quoted from ATSDR 2000). However, in most cases the symptoms are irreversible (Cotzias et al. 1968, Huang et al. 1998 – both quoted from ATSDR 2000).

Frequently psychological disturbances such as hallucinations and psychosis emerge (Cook et al. 1974 – quoted from ATSDR 2000). As the disease progresses, patients develop severe hypertonia and muscle rigidity and may be permanently disabled (Rodier 1955 – quoted from ATSDR 2000).

In neurobehavioral tests, people with manganism, generally have significantly poorer eye-hand coordination, hand steadiness, reaction time and postural stability, and a lower level of cognitive flexibility (Chia et al. 1993, 1995, Crump & Rosseau 1999, Iregren 1990, Lucchini et al. 1995, 1999, Mergler et al. 1994, Roels et al. 1987, 1992, 1999, Wennberg et al. 1991 – all quoted from ATSDR 2000).

One occupational study has reported a lack of significant neurological effects (Gibbs et al. 1999 – quoted from ATSDR 2000).

Reliable dose-response data on the inhalation exposure levels leading to neurological injury in humans are not extensive. However, recent epidemiological data indicate that concentrations of manganese in respirable dust in the range of 0.027 to 0.215 mg Mn/m³ and in total dust in the range of 0.14 to 1.59 mg Mn/m³ in the workplace can result in measurable neurological effects. (Roels et al. 1992 – quoted from ATSDR 2000).

People in a Canadian community near a former manganese alloy production plant were exposed to environmental manganese pollution. Both men and women were adversely affected by exposure to manganese as evidenced in performance in neurobehavioral tests and increased neuropsychiatric symptoms. The poorest performance occurred in those with the highest blood manganese level and neurological effects associated with higher levels of blood manganese were more likely to be observed in persons older than 50 years of age. (Baldwin et al. 1999, Beuter et al. 1999, Bowler et al. 1999, Mergler et al. 1999 – all quoted from ATSDR 2000).

3.2.2   Oral intake

3.2.2.1   Neurological effects

Only limited evidence exist that oral exposure to excess manganese in humans leads to neurological effects similar to those reported for inhalation exposure. Although people exposed to excess manganese via food or drinking water for a longer period exhibited neurological symptoms resembling manganism, data were not sufficient in any of the studies to conclude that the symptoms could be attributed solely to manganese exposure. (Banta & Markesbery 1977, Cawte et al. 1987, Goldsmith et al. 1990, He et al. 1994, Holzgraefe et al. 1986, Iwami et al. 1994, Kawamura et al. 1941, Kilburn 1987, Kondakis et al. 1989, Zhang et al. 1995 – all quoted from ATSDR 2000).

Neurological effects in children are described in the chapter on reproductive and developmental effects.

3.2.3   Dermal contact

No data were found.

3.3   Toxicity to reproduction

3.3.1   Inhalation

Decreased libido and impotence are common symptoms in male workers with clinical signs of manganism following exposure to high levels of manganese dusts in the workplace for 1-35 years (Emera et al. 1971, Jing et al. 1996b, Mena et al. 1967, Rodier 1955, Schuler et al. 1957 – all quoted from ATSDR 2000).

In one of the newer studies, the mean total manganese dust concentration was 0.145 mg Mn/m³ as manganese dioxide (Jing et al. 1996b).

The number of children born to occupationally exposed males (inhalation of 0.97 mg Mn/m³ of manganese dust for 1-19 years) was lower than average (Lauwerys et al. 1985 –quoted from ATSDR 2000).

In another occupational study with 70 men, inhalation of manganese dust as manganese dioxide at a median concentration of 0.71 mg Mn/m³ in total dust for an average of 6.2 years had no effect on the fertility (Gennart et al. 1992 – quoted from ATSDR 2000).

One study has reported increased semen liquefaction time, decreased sperm count and decreased sperm viability in workers exposed to manganese dioxide dust at a total concentration of 0.14 –5.5 mg Mn/m³ for one or more years. Workers exposed to manganese fumes at 6.5 – 82.3 mg Mn/m³ for one or more years had decreased sperm viability. However, an increased level of other metals may have influenced the reproductive effect seen. (Wu et al. 1996 – quoted from ATSDR 2000).

The reported reproductive effects may in part occur as a secondary result of neurotoxicity (ATSDR 2000). 

3.3.2   Oral intake

Incidences of stillbirths and malformations have been studied in an Australian aboriginal population living on an island where environmental levels of manganese are high. However, data from a suitable control group are lacking, and the study population is so small that it is not possible to judge if the incidence of developmental abnormalities (and neurological disorders) is higher than average. The route of exposure was assumed to be primarily oral but inhalation exposure was not ruled out. (Kilburn 1987 – quoted from ATSDR 2000).

Two studies in school children showed that increased exposure (at least 0.24 mg Mn/l water for 3 years or more) to manganese in drinking water and food was associated with poorer performance in school and on neurobehavioral tests as compared to children exposed to lower levels (at most 0.04 mg Mn/l water). The children with increased manganese exposure had significantly decreased serum levels of serotonin, noradrenaline, dopamine and acetylcholinesterase. The manganese level of the hair was inversely related to the performance in the neurobehavioral tests. However, the children may have been exposed to other metals that may have influenced the developmental effects seen (He et al. 1994, Zhang et al. 1995 – both quoted from ATSDR 2000).A higher manganese level has also been found in the hair of learning disabled children. The route of excess exposure may be through ingestion of increased amounts, metabolic disturbances, improper balance of other nutrients (such as iron), or decreased ability to clear manganese. (Collipp et al. 1983, Pihl & Parkes 1977 – both quoted from ATSDR 2000).

3.3.3   Dermal contact

No data were found.

3.4   Mutagenic and genotoxic effects

An increase in chromosomal aberrations was found in a group of workers, which had been exposed to a mixture of manganese, nickel, and chromium by inhalation for 10-24 years. The median manganese concentrations during the survey were 0.18 mg Mn/m³ for respirable dust and 0.71 mg Mn/m³ for total dust. No information was available on the genotoxicity of manganese alone. (Elias et al. 1989 – quoted from ATSDR 2000).

3.5   Carcinogenic effects

No data were found.

4   Animal toxicity

4.1   Single dose toxicity

Acute inhalation exposure to high concentrations of manganese dusts (manganese dioxide) can cause an inflammatory response in the lung (Maigetter et al. 1976, Shiotsuka 1984 – both quoted from ATSDR 2000).

Oral doses of manganese salts given by gavage can cause death in animals (LD₅₀-values for rats in the range of 275 to 1082 mg Mn/kg b.w.) (Holbrook et al. 1975, Kostial et al. 1978, 1989, Singh & Junnarkar 1991, Smyth et al. 1969 – all quoted from ATSDR 2000).

4.2   Repeated dose toxicity

4.2.1   Inhalation

4.2.1.1   Respiratory effects

Inhalation exposure to manganese dusts such as manganese dioxide or manganese tetroxide has in several studies lead to an inflammatory response in the lungs of animals exposed for periods ranging from 1 day to 10 months at manganese concentrations ranging from 0.7 to 69 mg Mn/m³ (Bergstrom 1977, Camner et al. 1988, Shiotsuka 1984, Suzuki et al. 1978, Ulrich et al. 1979a, 1979b – all quoted from ATSDR 2000).

4.2.1.2   Neurological effects

In several animal studies, intermediate or chronic inhalation exposure of monkeys and rats to manganese dusts has not produced neurological signs similar to those seen in humans (Bird et al. 1984, EPA 1983c, Ulrich et al. 1979a, 1979b – all quoted from ATSDR 2000).
However, in a chronic study with rhesus monkeys, decreased levels of dopamine were found in several regions of the brain (caudate and globus pallidus) (Bird et al. 1984 – quoted from ATSDR 2000).

Behavioural tests have detected signs of neurological effects in mice, although these are only seen at relatively high exposure levels of about 60-70 mg Mn/m³ (Lown et al. 1984, Morganti et al. 1985 – both quoted from ATSDR 2000).

4.2.2   Oral intake

4.2.2.1   Gastrointestinal effects

Adverse gastrointestinal effects including hyperplasia and erosion of the forestomach have been reported in B6C3F₁ mice fed manganese sulphate at a dose level of 585 mg Mn/kg b.w. per day for males and 731 mg Mn/kg b.w. per day for females for 2 years but not in F344 rats fed similar doses (NTP 1993 - quoted from ATSDR 2000).

4.2.2.2   Haematological effects

Some alterations in haematological parameters (including decreases in red blood cell, leukocyte, and neutrophil counts) have been reported in rats and mice following oral exposure. The effects varied according to species, duration of exposure and form of manganese administered. (ATSDR 2000).

No significant haematological effects were observed in F344 rats or B6C3F₁ mice exposed for 2 years to average oral doses of 331 or 905 mg Mn/kg b.w. per day (as manganese sulphate), respectively (Hejtmancik et al. 1987a, 1987b - quoted from ATSDR 2000).

4.2.2.3   Hepatic effects

Minor histological and weight changes of the liver have been reported in mice and rats fed high doses of manganese chloride, manganese tetroxide, and manganese sulphate for different time periods (ATSDR 2000).

A decreased liver weight occurred in male F344 rats fed 33 mg Mn/kg b.w. per day as manganese sulphate for 13 weeks. However, no hepatic changes were reported in a 2-year study in which F344 rats were fed up to 232 mg Mn/kg b.w. per day as manganese sulphate, and B6C3F₁ mice were fed up to 731 mg Mn/kg b.w. per day as manganese sulphate. (NTP 1993 – quoted from ATSDR 2000). 

4.2.2.4   Renal effects

An increased severity of chronic progressive nephropathy was noted in male F344 rats fed 200 mg Mn/kg b.w. per day as manganese sulphate for 2 years (NTP 1993 – quoted from ATSDR 2000).
However, in no other animal studies were any significant renal histopathological changes observed including another 2-year study where B6C3F₁ mice and F344 rats were fed 905 or 331 mg Mn/kg b.w. per day as manganese sulphate, respectively (Hejtmancik et al. 1987a, 1987b - quoted from ATSDR 2000).

4.2.2.5   Endocrine effects

In a 2-year B6C3F₁ mouse study, thyroid follicular hyperplasia and dilation were observed in males fed 584 mg Mn/kg b.w. per day as manganese sulphate, and thyroid follicular hyperplasia was observed in females fed 64 mg Mn/kg b.w. per day. However, no endocrine effects were observed in F344 rats fed up to 232 mg Mn/kg b.w. per day as manganese sulphate for 2 years or in F344 rats and B6C3F₁ mice fed up to 1950 Mn/kg b.w. per day as manganese sulphate for 13 weeks. (NTP 1993 – quoted from ATSDR 2000).

4.2.2.6   Immunological effects

In a 13-week F344 rat study with feeding of manganese sulphate, an increased neutrophil count at 33 mg Mn/kg b.w. per day in males, a decreased lymphocyte count at 130 mg Mn/kg b.w. per day in males, and a decreased total leukocyte count at 155 mg Mn/kg b.w. per day in females were observed. However, no immunological effects were observed in F344 rats fed up to 232 mg Mn/kg b.w. per day or in B6C3F₁ mice fed up to 731 Mn/kg b.w. per day as manganese sulphate for 2 years. (NTP 1993 – quoted from ATSDR 2000).

4.2.2.7   Neurological effects

Oral studies in animals exposed to manganese have sometimes revealed biochemical or neurobehavioral evidence of neurological effects (Bonilla 1978b, 1980, Bonilla & Prasad 1984, Chandra 1983, Chandra & Shukla 1981, Deskin et al. 1981, Eriksson et al. 1987a, Gianutsos & Murray 1982, Gray & Laskey 1980, Komuara & Sakamoto 1991, 1992, Kristensson et al. 1986, Lai et al. 1984, Nachtman et al. 1986, Subhash & Padmashree 1991 – all quoted from ATSDR 2000).

Although decreased motor activity (Gray & Laskey 1980, Komura & Sakamoto 1991 – both quoted from ATSDR 2000) has been observed in mice at high oral doses (280 mg Mn/kg b.w. per day for 100 days), motor deficits similar to clinical effects in humans are seldom observed in rodents and tend to be transient effects (Kristensson et al. 1986 – quoted from ATSDR 2000).

However, neurological effects (weakness, muscular rigidity, and marked degeneration with de-pigmentation of neurons in the region of substantia nigra) similar to those seen in workers exposed to manganese have developed in monkeys given 25 mg Mn/kg b.w. per day (as manganese chloride) for 18 months (Gupta et al. 1980 – quoted from ATSDR 2000 and SCF 2000).

Neurological effects in neonatal animals are described in the section on reproductive and developmental effects.

4.2.3   Dermal contact

No data were found.

4.2.4   Other routes

Monkeys exposed to manganese injected either intravenously or subcutaneously exhibited neurological symptoms very similar to those observed in workers exposed to manganese. In addition, they accumulated manganese in the basal ganglia, as do humans exposed to excess manganese. (Eriksson et al. 1992, Newland & Weiss 1992, Olanow et al. 1996 – all quoted from ATSDR 2000).

4.3   Toxicity to reproduction

4.3.1   Inhalation

Female mice exposed to manganese dioxide by inhalation for 18 weeks (before conception and through gestation) had an increased number of pups per litter (Lown et al. 1984 – quoted from ATSDR 2000).  

In utero exposure to manganese (as manganese dioxide) did not alter gross locomotor activity, different behavioural parameters and learning performance in mice pups. The dams had been exposed to manganese at an average concentration of 61 mg Mn/m³ 7 hours/day, 5 days/week for 16 weeks prior to conception and to either filtered air or manganese during the gestation period. The only effect observed was a reduced weight in pups of mothers exposed to manganese before conception and filtered air after conception. (Lown et al. 1984 – quoted from ATSDR 2000).Rabbits exposed to a single dose of 158 mg Mn/kg b.w. of manganese dioxide by intratracheal instillation experienced severe degenerative changes in the seminiferous tubules leading to infertility over a period of 1-8 months (Chandra et al. 1973, Seth et al. 1973 – both quoted from ATSDR 2000).

4.3.2   Oral intake

No gross, histopathological or organ weight changes were observed in reproductive organs of F344 rats fed manganese sulphate in doses up to 232 mg Mn/kg b.w. per day or B6C3F₁ mice fed up to 731 mg Mn/kg b.w. per day for 2 years, or in F344 rats fed up to 618 mg Mn/kg b.w. per day or B6C3F₁ mice fed up to 1950 mg Mn/kg b.w. per day for 13 weeks (NTP 1993 – quoted from ATSDR 2000).

Groups of female Sprague-Dawley rats were fed manganese sulphate in their diet at doses of 0.75, 4.5, 10, 29, 94 or 187 mg Mn/kg b.w. per day for 8 weeks prior to mating and until gestational day 21. No effect was found on maternal weight gain, implantation number, resorptions, or percentage of dead foetuses. The pups from dams administered 94 mg Mn/kg b.w. per day had significantly decreased weights as compared to other groups. No gross malformations were observed in the foetuses. Manganese doses of 94 and 187 mg Mn/kg b.w. per day resulted in a significant increase in liver manganese concentrations in pregnant females compared to non-pregnant females. Also pregnant rats had consistent liver iron concentrations, whereas non-pregnant rats suffered a dose-dependent decrease in liver iron concentrations. The highest dose in dams caused a significant increase in foetal manganese and a decrease in foetal iron content. (Jarvinen & Ahlstrom 1975 – quoted from ATSDR 2000).

Sperm head abnormalities and the percentage of abnormal sperm was significantly elevated in mice exposed by gavage to manganese sulphate at 23-198 mg Mn/kg b.w. per day for 21 days (Jordar & Sharma 1990 – quoted from ATSDR 2000).

Studies in mice and rats with oral intake of manganese tetroxide indicate that manganese ingestion at doses from about 1050 mg Mn/kg b.w. per day can lead to delayed maturation of the reproductive function in male offspring probably due to decreased testosterone secretion by Leydig cells (Gray & Laskey 1980, Laskey et al. 1982, Laskey et al. 1985  – all quoted from ATSDR 2000).
However, sperm count and fertility did not appear to be affected at manganese doses as high as 1050 mg Mn/kg b.w. per day (Laskey et al. 1982 – quoted from ATSDR 2000).
A diet low in iron worsened the reproductive effects (Laskey et al. 1982 – quoted from ATSDR 2000).

Studies in pregnant rats indicate that manganese ingested in the form of manganese chloride (Grant et al. 1997, Kontur & Fechter 1985, Pappas et al. 1997 – all quoted from ATSDR 2000) or manganese tetroxide (Laskey et al. 1982 – quoted from ATSDR 2000) via the diet or drinking water does not result in female reproductive effects at doses up to 620 mg Mn/kg b.w. per day (manganese chloride) or 1050 mg Mn/kg b.w. per day (manganese tetroxide) throughout gestation.

However, a dose of 33 mg Mn/kg b.w. per day of manganese chloride administered by gavage throughout gestation caused an increase in postimplantation loss in rats but not in rabbits (Szakmary et al. 1995 – quoted from ATSDR 2000).A significant decrease in the number of pregnancies were observed in rats mated at day 90-100 postpartum following exposure to manganese tetroxide in utero and from day 14-15 postpartum to an oral dose of 3500 mg Mn/kg b.w. per day. No such effect was observed at the lower dose level of 1050 mg Mn/kg b.w. per day. (Laskey et al. 1982 – quoted from ATSDR 2000).

Several developmental studies have been performed with manganese chloride in rats exposed for different periods of time in utero and/or postnatally up to 60 days of age either via the drinking water (Kontur & Fechter 1988, Pappas et al. 1997, Ali et al. 1983a, 1985, Dorman et al. 2000 – all quoted from ATSDR 2000) or by gavage (Kristensson et al. 1986, Chandra & Shukla 1978, Deskin et al. 1980, 1981, Szakmary et al. 1995, Grant et al. 1997 – all quoted from ATSDR 2000).

Different neurochemical alterations such as a decreased dopamine level and changes in the level of enzymes involved in the synthesis or metabolism of the neurotransmitter have been observed after postnatal dosing mainly in the gavage studies at doses from about 1 mg Mn/kg b.w. per day (Kristensson et al. 1986, Chandra & Shukla 1978, Deskin et al. 1980, 1981 – all quoted from ATSDR 2000).

The dopamine level was not affected in two studies (Kontur & Fechter 1988, Pappas et al. 1997– both quoted from ATSDR 2000) where rats were dosed in utero (and in one of the studies also postnatally) via drinking water to the dams with 620 and 1240 mg Mn/kg b.w. per day, respectively.

However, the dopamine level was increased in rats dosed in utero and postnatally via drinking water to the dams with 420 mg Mn/kg b.w. per day (Ali et al. 1985 – quoted from ATSDR 2000) and in neonatal rats dosed with 22 mg Mn/kg b.w. per day with a micropipette for the first 21 days of their life (Dorman et al. 2000 – quoted from ATSDR 2000 ).

Retardation of development of the skeleton and internal organs as well as a significant increase in external malformations, such as clubfoot, was observed in rats exposed in utero via gavage to the dams of 33 mg Mn/kg b.w. per day. However, the external malformations were not observed in pups allowed to grow to 100 days of age indicating that these effects were self-corrected. No further details were given. (Szakmary et al. 1995 – quoted from ATSDR 2000).
No malformations were observed at a dose of 22 mg Mn/kg b.w. per day (Szakmary et al. 1995, Grant et al. 1997 – both quoted from ATSDR 2000).

In most of the studies, no clinical or behavioural signs of neurotoxicity were evident. Male pups exposed in utero and postnatally via drinking water to the dams with 620 mg Mn/kg b.w. per day, did not perform different than control rats in a number of behavioural tests that measured spontaneous motor activity, memory, and cognitive ability (Pappas et al. 1997 – quoted from ATSDR 2000).
However, the offspring of rats who drank 240 mg Mn/kg b.w. per day had delayed air righting reflexes, and pups of dams fed a protein-deficient diet as well as administered manganese chloride had significant delays in age of eye opening and development of the auditory startle reflex (Ali et al. 1983a – quoted from ATSDR 2000).
In neonatal rats dosed with 22 mg Mn/kg b.w. per day with a micropipette for the first 21 days of their life, a significant increase in amplitude of the auditory startle reflex was induced (Dorman et al. 2000 – quoted from ATSDR 2000).

4.3.3   Dermal contact

No data were found.

4.3.4   Parenteral administration

Intraperitoneal injection of pregnant mice with 12.5 mg Mn/kg b.w. per day, as manganese sulphate, on days 8-10 of gestation resulted in exencephaly and embryo-lethality. No further details were given. (Webster & Valois 1987 – quoted from ATSDR 2000). 

Similar degenerative changes in testes have been reported in rats and mice following intraperitoneal injection of manganese sulphate (Chandra et al. 1975, Singh et al. 1974 – both quoted from ATSDR 2000) and in rabbits following intravenous injection of manganese chloride (Imam & Chandra 1975 – quoted from ATSDR 2000). 

Postimplantation loss has occurred in several studies in rats and mice injected subcutaneously or intravenously during gestation with manganese chloride at doses from about 1 mg Mn/kg b.w. per day (Colomina et al. 1996, Sanchez et al. 1993, Treinen et al. 1995 – all quoted from ATSDR 2000). 

Increased skeletal abnormalities and delayed ossification has been found in the offspring of rats and mice injected subcutaneously or intravenously during gestation with manganese chloride at doses from about 1 mg Mn/kg b.w. per day. It was not stated whether maternal toxicity occurred in the studies. (Colomina et al. 1996, Grant et al. 1997, Sanchez et al. 1993, Treinen et al. 1995– all quoted from ATSDR 2000). 

4.4   Mutagenic and genotoxic effects

4.4.1   In vitro studies

While manganese sulphate was negative in one Ames test with 5 strains (TA97, TA98, TA100, TA1535, TA1537) of Salmonella typhimurium with or without metabolic activation (Mortelsman et al. 1986 – quoted from ATSDR 2000), it was positive in another Ames test with strain TA97 without metabolic activation (Pagano & Zeiger 1992 – quoted from ATSDR 2000).

Manganese chloride was negative in Ames test in strains TA98, TA100, and TA1535 but it was positive in strain TA1537 (without metabolic activation) and conflicting results were obtained for strain TA102 (Wong 1988, De Meo et al. 1991 – both quoted from ATSDR 2000).

Manganese sulphate was positive in a fungal gene conversion/reverse mutation assay (Singh 1984 – quoted from ATSDR 2000).

Manganese sulphate induced sister chromatid exchanges (with and without metabolic activation) and chromosomal aberrations (without metabolic activation) in Chinese hamster ovary cells (Galloway et al. 1987 – quoted from ATSDR 2000).

In in vitro studies without metabolic activation, manganese chloride produced gene mutations in cultured mouse lymphoma cells (Oberly et al. 1982 – quoted from ATSDR 2000), caused DNA damage in human lymphocytes in the single cellgel assay (De Meo et al. 1991 – quoted from ATSDR 2000), and caused cell transformations in Syrian hamster embryo cells (Casto et al. 1979 –quoted from ATSDR 2000).

4.4.2   In vivo studies

In vivo assays in mice showed that oral doses of manganese sulphate and potassium permanganate caused an increased incidence of micronuclei and chromosomal aberrations in bone marrow (Jordar & Sharma 1990 – quoted from ATSDR).

Oral doses of manganese chloride did not cause chromosomal aberration in the bone marrow or spermatogonia of rats (Dikshith & Chandra 1978 – quoted from ATSDR).

Manganese sulphate was negative for sex-linked recessive lethal mutations in male germ cells and manganese chloride for somatic mutations in fruit flies (Valencia et al. 1985, Rasmuson 1985 – both quoted from ATSDR 2000).

4.5   Carcinogenic effects

Chronic (2 years) oral exposure of F344 rats to manganese sulphate led to an increased incidence of pancreatic tumours (adenomas and carcinomas) in males dosed with up to 331 mg Mn/kg b.w. per day. Although the tumour incidence in the dosed groups was low and not dose-responsive (4 out of 50 in all 3 dose groups), the authors concluded that the tumours were compound related because the incidence of these tumours in the controls was zero. (Hejtmancik et al. 1987a – quoted from ATSDR 2000).

In B6C3F₁ mice, chronic oral exposure to manganese sulphate resulted in small increases in pituitary adenomas in females at 905 mg Mn/kg b.w. per day but not in males at 722 mg Mn/kg b.w. per day (Hejtmancik et al. 1987b – quoted from ATSDR 2000).

Chronic oral exposure of B6C3F₁ mice and F344 rats to manganese sulphate in doses up to 731 and 232 mg Mn/kg b.w. per day, respectively, resulted in a significantly increased incidence of follicular cell hyperplasia and a marginally increased incidence of thyroid gland follicular cell adenomas in high dose mice. No increased tumour incidence was found in any of the rats or in the mice in the middle dose group (228 mg Mn/kg b.w. per day). (NTP 1993 – quoted from ATSDR 2000 and SCF 2000).

5   Regulations

5.1   Ambient air

Denmark (C-value):         0.001 mg Mn/m³ (MST 2002).

WHO (2000):                 0.00015 mg Mn/m³.

US-EPA (1993):             RfC 0.00005 mg Mn/m³ (IRIS 2000).

5.2   Drinking water

Denmark:                        0.05 mg Mn/l (MM 2001).

WHO (1996):                 0.5 mg Mn/l.

US-EPA (1993):             0.05 mg Mn/l (HSDB 2000).

5.3   Soil

Denmark:                        -

The Netherlands:             -

5.4   Occupational Exposure Limits

Denmark:                        0.2 mg Mn/m³ (At 2002).

ACGIH:                          0.2 mg Mn/m³ (HSDB 2000).

The occupational exposure limits in different countries around the world varies between 0.2 and 5 mg Mn/m³ (IRIS 2000).

5.5   Classification

Manganese sulphate is classified for effects following repeated exposure (Xn;R48/20/22 - harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed) and for environmental effects (N;R51/53 – toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment). (MM 2002).

5.6   IARC

-

5.7   US-EPA

Manganese is not classifiable with regard to human carcinogenicity (Group D) (EPA 1993b – quoted from ATSDR 2000).

5.8   ATSDR

Minimal risk level (MRL): 0.00004 mg Mn/m³ (chronic inhalation).

 6   Summary 6.1   Description

Manganese sulphate and manganese sulphide occur as powders or crystals of several hydrates. Manganese sulphate has a high water solubility (greater than 500 g/l) at room temperature, while manganese sulphide is insoluble in water. In moist conditions, manganese sulphide readily oxidizes in air to the sulphate.

Manganese is a natural component of the environment and an essential element in the nutrition of humans and animals.

6.2   Toxicokinetics

Manganese absorption occurs primarily through the diet. Of ingested inorganic manganese about 3-5% is absorbed in humans. The absorption is influenced by such factors as the water solubility of the compounds in ques­tion, age, and the amount of manganese as well as of other trace elements such as iron, calcium and phosphorus present in the gut. Absorption via the lungs can be significant for occupationally exposed persons or for those exposed to excess levels of airborne manganese. The absorption following inhalation is higher than that after oral administration for the more soluble forms of inorganic manganese.

In healthy humans, manganese is distributed to the tissues with the highest levels found in the liver, pancreas, and kidney and the lowest levels in bone and fat.Excess manganese (following oral exposure in humans with liver diseases) was accumulated in certain regions of the brain (basal ganglia, especially the globus pallidus and the substantia nigra).
Mice and rats chronically fed manganese sulphate generally had elevated tissue levels of manganese. The manganese levels in the liver and kidney were higher than the levels in the brain.

Following inhalation in rats, most manganese was distributed to the olfactory bulb and the brain but also to other organs such as the lungs, the liver, the kidneys, and the pancreas. One study indicates that manganese may be distributed directly to the brain from the nasal cavity via the olfactory pathway by-passing the blood-brain barrier. Preferential accumulation of manganese in specific locations of the brain (including the caudate nucleus, globus pallidus, and the substantia nigra) was noted in one monkey exposed to an aerosol of manganese chloride several hours/day for 3-5 months.

Manganese is excreted with a half-life of 13-37 days in humans. In general, biliary excretion is the main pathway by which absorbed manganese reaches the intestines where most of the element is excreted in the faeces.
An inhalation study in monkeys showed that a portion of the manganese was retained in the lung and brain and was cleared with a half-life of up to 250 days. Small amounts of manganese can also be found in urine, sweat, and milk.The central nervous system is the primary target of manganese toxicity. Several hypotheses have been proposed for the toxicological mechanism behind this toxicity.

6.3   Human toxicity

6.3.1   Single dose toxicity

No data were found.

6.3.2   Repeated dose toxicity

From studies of humans exposed to manganese dusts (mainly manganese dioxide) in mines and factories, clear evidence exist that inhalation exposure to high levels of manganese for longer periods can lead to neurological effects. The disease, termed manganism, is characterised by weakness, muscle rigidity, tremor, apathy, speech disturbances, a mask-like face, and slow clumsy movement of the limbs typically after several years of exposure but some individuals may begin to show signs after as few as 1-3 months of exposure. In most cases the symptoms are irreversible. People with manganism generally have significantly poorer eye-hand coordination, hand steadiness, reaction time and postural stability, and a lower level of performance in cognitive flexibility on neurobehavioral tests.

Recent epidemiological data indicate that concentrations of manganese in respirable dust in the range of 0.027 to 0.215 mg Mn/m³ and in total dust in the range of 0.14 to 1.59 mg Mn/m³ in the workplace can result in measurable neurological effects.

Occupational studies indicate that manganese inhaled in high concentrations may elevate the serum prolactin and cortisol levels, lower the blood pressure, and cause inflammation in the lungs.

Only limited evidence exist that oral exposure to excess manganese in humans leads to neurological effects similar to those reported for inhalation exposure.

6.3.3   Toxicity to reproduction

Decreased libido and impotence are common symptoms in male workers with clinical signs of manganism following exposure to high levels of manganese dusts. In addition increased semen liquefaction time, decreased sperm count, decreased sperm viability, and decreased fertility have been reported in some studies.

Two studies of school children showed that increased oral exposure to manganese in drinking water and food was associated with poorer performance in school and on neurobehavioral tests as compared to children exposed to lower levels. However, the children may have been exposed to other metals that may have influenced the developmental effects seen.

Incidences of stillbirths and malformations have been studied in an Australian aboriginal population living on an island where environmental levels of manganese are high. However, data from a suitable control group are lacking.

6.3.4   Mutagenic and genotoxic effects

An increase in chromosomal aberrations was found in a group of workers, which had been exposed to a mixture of manganese, nickel, and chromium by inhalation for 10-24 years.

6.3.5   Carcinogenic effects

No data were found.

6.4   Animal toxicity

6.4.1   Single dose toxicity

Acute inhalation exposure to high concentrations of manganese dusts can cause an inflammatory response in the lung. Oral doses of manganese salts given by gavage can cause death in animals.

6.4.2   Repeated dose toxicity

Oral and inhalation studies in animals exposed to manganese have sometimes revealed biochemical or neurobehavioral evidence of neurological effects. Monkeys exposed to manganese via inhalation, oral intake or injection exhibit neurological symptoms very similar to those observed in workers exposed to manganese. They accumulate manganese in the basal ganglia, as do humans exposed to excess manganese. However, motor deficits similar to clinical effects in humans are seldom observed in rodents and tend to be transient effects.

Animal studies indicate that manganese inhaled in high concentrations may cause inflammation in the lungs. Mice and/or rats exposed orally to high doses of manganese sulphate for 2 years may experience gastrointestinal (e.g. hyperplasia and erosion of the forestomach), renal (increased severity of chronic progressive nephropathy) and/or endocrine (thyroid follicular hyperplasia and dilation) effects.

6.4.3   Toxicity to reproduction

A high systemic concentration of manganese may cause reproductive and developmental effects as evidenced by degenerative changes in testes of male animals injected, instilled or gavaged with manganese and by embryo-lethality, postimplantation loss, and structural abnormalities and delays in pups in studies where manganese was injected during gestation at doses from about 1 mg Mn/kg b.w. per day or gavaged at doses from about 33 mg Mn/kg b.w. per day.

Studies in mice and rats with oral intake of manganese indicate that ingestion at doses from about 1050 mg Mn/kg b.w. per day can lead to delayed maturation of the reproductive function in male offspring probably due to decreased testosterone secretion by Leydig cells. However, sperm count and fertility did not appear to be affected at manganese doses as high as 1050 mg Mn/kg b.w. per day. A diet low in iron worsened the reproductive effects.

No female reproductive effects were found in rats fed manganese sulphate in their diet at doses up to 187 mg Mn/kg b.w. per day (highest dose tested) for 8 weeks prior to mating and until gestational day 21 or in pregnant rats ingesting other inorganic manganese substances at even higher doses.No gross malformations were observed in the foetuses of the rats fed manganese sulphate in their diet at doses up to 187 mg Mn/kg b.w. per day (highest dose tested) for 8 weeks prior to mating and until gestational day 21. The highest dose did cause a significant increase in foetal manganese and a decrease in foetal iron content.

In most studies, no biochemical or behavioural signs of neurotoxicity were evident in pups. Male pups exposed in utero and postnatally via drinking water to the dams with 620 mg Mn/kg b.w. per day, did not perform different than control rats in a number of behavioural tests that measured spontaneous motor activity, memory, and cognitive ability, and did not have any neurochemical alterations. In neonatal rats dosed with 22 mg Mn/kg b.w. per day with a micropipette for the first 21 days of their life, the dopamine level was increased and a significant increase in amplitude of the auditory startle reflex was induced. A decreased dopamine level and changes in the level of enzymes involved in the synthesis or metabolism of the neurotransmitter have been observed after postnatal dosing but mainly in the gavage studies at doses from about 1 mg Mn/kg b.w. per day.

In utero exposure to manganese did not alter gross locomotor activity, different behavioural parameters and learning performance in mice pups whose mothers had been exposed to manganese at an average concentration of 61 mg Mn/m³ 7 hours/day, 5 days/week for 16 weeks prior to conception and to either filtered air or manganese during the gestation period. The only effect observed was a reduced weight in pups of mothers exposed to manganese before conception and filtered air after conception.

6.4.4   Mutagenic and genotoxic effects

In vitro, manganese sulphate was negative in one Ames test with 5 strains of Salmonella typhimurium with or without metabolic activation. It was positive in another Ames test with strain TA97 without metabolic activation, in a fungal gene conversion/reverse mutation assay, in the sister chromatid exchange test in CHO cells with and without metabolic activation, and in a test for chromosomal aberrations in CHO cells without metabolic activation.

In vivo assays in mice showed that oral doses of manganese sulphate caused an increased incidence of micronuclei and chromosomal aberrations in bone marrow. Manganese sulphate was negative for sex-linked recessive lethal mutations in male germ cells in fruit flies.

6.4.5   Carcinogenic effects

Exposure to high oral doses of manganese sulphate for 2 years may cause small increases in thyroid gland follicular cell adenomas (in mice at 731 mg Mn/kg b.w. per day), pituitary adenomas (in female mice at 905 mg Mn/kg b.w. per day), and pancreatic adenomas and carcinomas (in male rats at all doses up to 331 mg Mn/kg b.w. per day). However, rats and mice exposed to about 230 mg Mn/kg b.w. per day for 2 years showed no increases in tumour incidence in one of the studies.

Manganese is an essential element in the nutrition of humans and animals. The concentration of manganese in our body following oral exposure is normally wellregulated. However, the homeostatic system may become overloaded which may result in adverse effects. Most of the observed adverse effects following exposure to manganese have been observed in workers exposed by inhalation.

Neurotoxicity following inhalation is the critical effect of manganese. Clear evidence exist from studies of human workers that inhalation exposure to manganese for longer periods can lead to serious neurological effects (resembling parkinsonism) that in most cases are irreversible. These effects may occur at concentrations of manganese in respirable dust in the range of 0.027 to 0.215 mg Mn/m³ and in total dust in the range of 0.14 to 1.59 mg Mn/m³. Studies in rats indicate that manganese may be distributed directly to the brain from the nasal cavity via the olfactory pathway, which may in part explain why it is mainly neurological effects that are seen after inhalation exposure. In addition, one study in monkeys that had been exposed to an aerosol of manganese chloride showed that most manganese was excreted fairly quickly but a portion of the manganese was retained in the lungs and brain and was cleared with a longer half-life.
Other effects observed in workers exposed to manganese by inhalation included an elevated serum prolactin and cortisol level, a lowered blood pressure, and inflamed lungs (also seen in rodents). The different systemic effects seen in rodents exposed orally to manganese occurred at relatively high concentrations.
Monkeys exposed to manganese exhibit neurological symptoms resembling those observed in workers exposed to manganese but in rodents this is seldom the case. For this reason, studies in rodents may not necessarily be relevant models for the toxicity of manganese in humans even though neurochemical changes are sometimes revealed in rodents exposed to manganese.

Only limited evidence exist that oral exposure to excess manganese in humans lead to neurological effects similar to those reported for inhalation exposure.

Manganese has the potential to cause reproductive and developmental effects as evidenced by reproductive effects seen in workers exposed to manganese by inhalation and by reproductive and developmental effects seen in rodent studies. However, most of the reproductive and developmental effects seen in rodent studies occurred after exposure via gavage or injection where a high systemic concentration of manganese would be found. In most studies, no biochemical or behavioural signs of neurotoxicity were evident in pups even though the absorption and/or retention of manganese are known to be higher in neonates. However, the dopamine level and the amplitude of the auditory startle reflex was increased in neonatal rats dosed with 22 mg Mn/kg b.w. per day with a micropipette for the first 21 days of their life. A reduced weight of pups of mothers exposed to 61 mg Mn/m³ for 16 weeks before conception was the only effect observed in these pups.

It is a cause of concern that manganese was positive in several in vitro and in vivo genotoxic assays. Small increases in thyroid gland follicular cell adenomas, pituitary adenomas, and pancreatic adenomas and carcinomas did occur in rodents exposed orally to relatively high doses of manganese for 2 years.

7   References

At (2002). Grænseværdier for stoffer og materialer. At-vejledning C.0.1, oktober 2002. http://www.at.dk/Overblik/atviden/vejled/c01/C01.htm#Indhold

ATSDR (2000). Toxicological Profile for Manganese. U.S. Department of Health & Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry.

Beliles RP (1994). Manganese. In: The metals, in Clayton GD & Clayton FE. Patty‘s Industrial Hygiene and Toxicology. 4th edition, Vol IIC. New York, John Wiley & Sons, 2106-2124.

Brenneman KA, Wong BA, Buccellato MA, Costa ER, Gross EA and Dorman DC (2000). Direct olfactory transport of inhaled manganese (⁵⁴MnCl₂) to the rat brain: Toxicokinetic investigations in unilateral nasal occlusion model. Toxicol Appl Pharmacol 169, 238-248

ChemFinder (2001). Manganese sulphate and manganese sulphide. Http://www.chemfinder.com.

HSDB (2000). Manganese sulphate. In: Hazardous Substances Data Base. Last revised: 10/2000.

IRIS (2000). Manganese. In: Integrated Risk Information System. Database quest, last revised: 10/2000. US-EPA.

Merck Index (1996). Manganese sulphate and manganese sulphide. In: 12th. ed., Rahway, New Jersey, Merck & Co., Inc., 5772.

MM (2002). The Statutory Order from the Ministry of the Environment no. 439 of June 3, 2002, on the List of Chemical Substances.

MM (2001). Bekendtgørelse om vandkvalitet og tilsyn med vandforsyningsanlæg. Miljø- og Energiministeriets bekendtgørelse nr. 871 af 21. september 2001.

MST (2002). B-værdivejledningen. Vejledning Nr. 2 2002, Miljøstyrelsen, Miljøministeriet.

SCF (2000). Opinion of the Scientific Committee on Food on the tolerable upper intake level of manganese. Http://europa.eu.int/comm/food/fs/sc/scf/out80f_en.pdf.

Vitarella D, Wong BA, Moss OR and Dorman DC (2000). Pharmacokinetics of inhaled manganese phosphate in male Sprague-Dawley rats following subacute (14-day) exposure. Toxicol Appl Pharmacol 163, 279-285.

WHO (1996). Manganese. In: Guidelines for drinking-water quality. Second edition, Vol. 2. World Health Organization, Geneva, 276-284.

WHO (2000).  Manganese. In: Air Quality Guidelines for Europe, Second edition. WHO Regional Publications, European Series No. 91.

WHO (1981). Manganese. Environmental Health Criteria 17. World Health Organisation, International Programme on Chemical Safety, Geneva.

Appendix 2: Diammonium sulphate
8   General description

8.1   Identity

Molecular formula:          H₈N₂O₄S

Structural formula:

Molecular weight:            132.14

CAS-no.:                        7783-20-2

Synonyms:                      Ammonium sulphate

                                      Mascagnite

                                      Sulphuric acid, diammonium salt

8.2   Physical / chemical properties

Description:                    Colourless orthorhombic crystals or white granules with no odour.

Melting point:                  Decomposes above 235°C

Density:                          1.77 g/ml (at 20°C)

Vapour pressure:             -

Concentration of

saturated vapours:           -

Solubility:                        Water:  754-770 g/l (at 20°C)

                                      Insoluble in acetone, alcohol and ammonia.

References:                    ChemFinder (2001), HSDB (2000), IUCLID (2000), Merck Index (1996).

Other chemical substances mentioned in this paper:

Sodium sulphate (Na₂SO₄)
Sodium bisulphate (NaHSO₄)
Ammonium bisulphate (NH₄HSO₄)
Sulphuric acid (H₂SO₄)
Sulphur dioxide (SO₂)
Ammonium nitrate (NH₄NO₃)
Ozone (O₃)

9   Toxicokinetics

9.1   Absorption, distribution, and elimination

9.1.1   Inhalation

Inhaled diammonium sulphate particles (0.2 mg/m³) with a mass median aerodynamic diameter (MMAD) of 0.3 to 0.6 mm reached the lungs of Syrian hamsters. However, a substantial proportion of the compound was found in the nose. (US Department of Commerce - quoted from IUCLID 2000).

The clearance of diammonium sulphate (inhaled at a concentration of 0.2 mg/m³) from the hamster lungs (via the blood and urinary tract) has been determined to be about 20 minutes. The results of clearance studies with guinea pigs and rabbits suggested that there was no species difference. No further details were given. (US Department of Commerce - quoted from IUCLID 2000).

9.1.2   Oral intake

Systemic effects and death following oral exposure to diammonium sulphate indicate that it may be absorbed from the gastrointestinal tract (IUCLID 2000, Sato et al. 1999).

Soluble sulphate salts are rather slowly absorbed from the alimentary tract. Because of their osmotic activity, they draw water into the lumen of the bowel and produce purging. (Gosselin et al. 1984 – quoted from HSDB 2000).

9.1.3   Dermal contact

No data have been found.

9.1.4   Other routes

No data have been found.

9.2   Mode of action

Acute intoxication with ammonium may affect the cerebral nervous system by inhibition of the energy metabolism such as a decrease in ATP in the brain or by inhibition of glutamic acid metabolism resulting in a disturbance of neurotransmitters (Jablonska 1975, Hindfelt et al. 1977, McCandless & Schenker 1981, Schenker & Brady 1988 - all quoted from Sato et al. 1999).

10   Human toxicity

10.1   Single dose toxicity

10.1.1   Inhalation

Groups of 9-15 healthy non-smoking male volunteers between the ages of 18 and 40 were exposed to diammonium sulphate aerosols (or to other gaseous or aerosol pollutants alone or to mixtures of these) at a concentration of 0 or 0.133 mg/m³ (MMAD = 0.55 mm) for 4 hours. During the exposure each person had two 15 minutes exercise sessions on a treadmill. Environmental conditions were mildly stressful with a temperature of 30°C and a relative humidity of 60%. A battery of 19 measurements of pulmonary function was performed just prior to exposure; 2 hours into the exposure, following the first exercise session; 4 hours into the exposure, following the second exercise session; and 24 hours after exposure. Diammonium sulphate under the conditions of the experiment had no effect on the pulmonary function. Ozone in a concentration of 0.4 ppm affected the pulmonary function. The effect of exposure to a combination of ozone and diammonium sulphate seemed slightly greater (although not statistically significant) than exposure to ozone alone. (Stacy et al. 1983).

Twenty healthy non-smoking male and female volunteers aged 21-34 years were exposed by inhalation to clean air, diammonium sulphate aerosols at a concentration of 0.5 mg/m³ (MMAD = 1.0 mm), 1 ppm sulphur dioxide and combined diammonium sulphate/sulphur dioxide. Exposures lasted for 4 hours which included two light to moderate exercise stints for 15 minutes each. The study ran for 3 days/week for 3 consecutive weeks where each volunteer served as his or her own control. Clean air was breathed on day 1 and 3 of each week. Diammonium sulphate (week 1), sulphur dioxide (week 2) or the combination (week 3) was breathed on day 2. No significant changes were measured in pulmonary function or bronchial reactivity following exposure to diammonium sulphate, sulphur dioxide or the combination. The most common symptoms were upper respiratory and eye irritation. Three volunteers reported upper respiratory irritation during the exposure to diammonium sulphate alone and nine during the combined exposure. Two volunteers reported eye irritation during diammonium sulphate exposure alone and three during the combined exposure. (Kulle et al. 1984).

Eight healthy and nine asthmatic non-smoking volunteers aged 60-76 years were exposed by inhalation to clean air, diammonium sulphate at a concentration of 0.070 mg/m³ and sulphuric acid. Exposures to each of the test atmospheres (clean air, diammonium sulphate, or sulphuric acid) were separated by at least 1 week and lasted for 40 minutes composed of 30 minutes at rest and 10 minutes of light exercise on a treadmill. Diammonium sulphate exposure had no effect on pulmonary function parameters (forced expiratory volume in one second, forced vital capacity, and total respiratory resistance) in either group. (Koenig et al. 1993).

Sixteen healthy and seventeen asthmatic non-smoking volunteers with a mean age of 27 were exposed by inhalation to sodium chloride aerosols (control),diammonium sulphate aerosols at a concentration of about 0.1 or 1.0 mg/m³ (MMAD = 0.5-1 mm) and other sulphates (sodium bisulphate, ammonium bisulphate, sulphuric acid) by double blind randomisation. Exposures to each of the test atmospheres (control, diammonium sulphate, sodium bisulphate, ammonium bisulphate, or sulphuric acid) were separated by at least 3 hours and lasted for 16 minutes. To determine if sulphate inhalation caused increased reactivity to a known bronchoconstrictor, all volunteers inhaled carbachol following each 16 minutes exposure. Diammonium sulphate (and the other sulphates) produced a small but significant decrease in flow rates on maximum and partial expiratory flow-volume curves and potentiated the bronchoconstrictor action of carbachol at 1 mg/m³ both in asthmatic and healthy volunteers. The most significant changes were seen with the most acidic sulphates (sulphuric acid and ammonium bisulphate), which also produced a significant decrease at 1 mg/m³ in the mean specific airway conductance and in the forced vital capacity in one second in asthmatic volunteers. No significant changes on the pulmonary function occurred following exposure to 0.1 mg/m³ of sulphates. (Utell et al. 1982).

10.1.2   Oral intake

An 85-year old woman was found dead lying on the ground outside her house. The autopsy could not determine the definite cause of her death since routine poison analysis revealed no toxicological substances in her blood and the pathological examination showed nothing unusual. However, examination at the police laboratory of the solution in the beer can found next to her showed that it was very likely diammonium sulphate. Measurements showed a significant increase of ammonium and sulphate ions in serum and gastric contents. Therefore it was concluded that the cause of death was acute intoxication due to ingestion of diammonium sulphate. (Sato et al. 1999). 

10.1.3   Dermal contact

No data have been found.

10.1.4   Other routes

No relevant data have been found.

10.2   Repeated dose toxicity

Ten healthy and six asthmatic non-smoking volunteer men with a mean age of 42.3 were exposed by inhalation to purified air, diammonium sulphate aerosols at a concentration of 0.1 mg/m³ (MMAD = 0.3 mm) and other sulphates (ammonium bisulphate, sulphuric acid) in a blind experimental fashion with 1-2 days of exposure to air followed by 2-3 consecutive days of sulphate aerosol exposure. Exposures lasted for 2 hours with light exercise on bicycle ergometers for the first 15 minutes of each half hour. No consistent changes in pulmonary function or in symptoms occurred in the healthy or asthmatic men when exposed to diammonium sulphate aerosols. (Avol et al. 1979).

10.3   Toxicity to reproduction

No data have been found.

10.4   Mutagenic and genotoxic effects

No data have been found.

10.5   Carcinogenic effects

No data have been found.

11   Animal toxicity

11.1   Single dose toxicity

11.1.1   Inhalation

No mortality or signs of gross toxicity was observed in 6 young adult male rats that were exposed to diammonium sulphate aerosols at a concentration of 1000-1200 mg/m³ (particle size averaged 2-3 mm) 8 hours/day for 3 consecutive days. Eight of 20 guinea pigs exposed to 800-900 mg/m³, 1/6 exposed to 600-700 mg/m³, and 0/6 exposed to 500-600 mg/m³ for 8 hours died during exposure. The survivors recovered with no noticeable after effects. (Pepelko et al. 1980).  

No effect on pulmonary macrophage numbers and shape was found in hamsters exposed to 0.86 mg/m³ of diammonium sulphate with a MMAD of 0.3 mm for 12 hours (US Department of Commerce - quoted from IUCLID 2000).

Groups of 6-8 adult sheep were exposed by inhalation to diammonium sulphate aerosols in a concentration of 0.1 or 4 mg/m³ (MMAD = 0.5 or 1.5 mm) or to other sulphates (ammonium bisulphate, sulphuric acid) for 4 hours. The sheep first inhaled physiologic buffer, then the known bronchoconstrictor carbachol, and finally half an hour later sulphate aerosols. Some sheep were exposed to more than one salt. These exposures were separated by at least 1 week. The mean pulmonary flow resistance was not altered following exposure to diammonium sulphate. The airway responsiveness to inhaled carbachol was enhanced at some time within the 24-hour period immediately following exposure to diammonium sulphate with a MMAD of 1.5 mm. (Abraham et al. 1983).

A group of five male, mixed breed rabbits were exposed by oral inhalation to clean air, diammonium sulphate aerosols in a concentration of 2.0 mg/m³ (MMAD = 0.4 mm) and to other sulphates (ammonium bisulphate, sodium sulphate). Exposures for individual rabbits were separated by at least 1 week and lasted for 1 hour. The bronchiolar mucociliary clearance was not affected by the treatment with diammonium sulphate. (Schlesinger 1984).

The bronchiolar or tracheal mucociliary clearance was not affected either in donkeys, rats or sheep inhaling diammonium sulphate with a MMAD of 0.1-0.4 for 1-4 hours in doses of 0.3-3.0, 3.6 or 1.1 mg/m³, respectively (Schlesinger et al. 1978, Phalen et al. 1980, Sackner et al. 1981 – all quoted from Schlesinger 1984).

11.1.2   Oral intake

The oral LD₅₀-values reported for diammonium sulphate range from 2840 to 4250 mg/kg b.w. for rats (3 values reported) and from 610 to 640 mg/kg b.w. for mice (2 values reported) (IUCLID 2000).

Groups of 2-3 Japanese white rabbits were anaesthetised and dosed with 0 or 1500 mg/kg b.w. of diammonium sulphate in saline through a gastric probe. The three rabbits dosed with diammonium sulphate showed similar symptoms such as mydriasis, irregular respiratory rhythms, local and general convulsions, until they fell into respiratory failure with cardiac arrest about an hour after the dosing. Electroencephalogram showed slow, suppressive waves initially and 15-20 minutes later a high-amplitude slowing wave pattern in correspondence to local convulsions. Electrocardiogram did not show any change of wave pattern except for bradycardia immediately after the general convulsions and until cardiac arrest. The blood pressure fell down rapidly after the general convulsions. The concentration of ammonium and sulphate ions in serum increased remarkable and blood gas analysis showed severe metabolic acidosis. Biochemical analysis of blood and histological examination of brain, heart, lung, spleen, kidney, liver and stomach revealed no significant changes except for a moderate increase in K⁺ just before cardiac arrest. (Sato et al. 1999). 

11.1.3   Dermal contact

Diammonium sulphate was not irritating to intact or abraded rabbit skin that was tested for 20 or 8 hours, respectively. No further details were given. (BASF 1969 – quoted from IUCLID 2000).

11.1.4   Other routes

11.1.4.1     Ocular irritation

Diammonium sulphate was not irritating to rabbit eyes. No further details were given. (BASF 1969 – quoted from IUCLID 2000).

11.2   Repeated dose toxicity

11.2.1   Inhalation

Young male Sprague-Dawley rats were pre-treated intratracheally with either physiologic saline (to model normal lungs) or porcine pancreatic elastase (to model chronic pulmonary impairment). Groups of 30 rats from each pre-treatment group were exposed to filtered air (control), to 1 ppm sulphur dioxide, to wet diammonium sulphate aerosols in concentrations of 0.5 mg/m³ (MMAD = 0.44 mm), or to combined sulphur dioxide and diammonium sulphate for 5 hours/day, 5 days/week for 4 or 8 months. Half of the rats exposed for 8 months were held for an additional 3-month recovery period.
At 4 months, bronchial epithelial hyperplasia and alveolar mean and median cord (a long flexible structure) length significantly increased in the saline/diammonium sulphate and saline/diammonium sulphate/sulphur dioxide groups but decreased in the elastase/diammonium sulphate and elastase/ diammonium sulphate/sulphur dioxide groups compared to air controls. The number of non-ciliated epithelial cells in the bronchioles was significantly increased in the saline/diammonium sulphate group and focal haemosiderosis was significantly increased in the elastase/diammonium sulphate/sulphur dioxide group.
At 8 months, alveolar interstitial fibrosis tended to be greater in aerosol-exposed rats with either normal or impaired lungs compared to air controls, but was significant only in the saline/diammonium sulphate exposed group. Other significant effects observed at 8 months following exposure to diammonium sulphate and compared to air controls included increased lung volume, emphysema and focal haemosiderosis in elastase treated rats and an increased count of non-ciliated epithelial cells in the bronchioles in saline treated rats.

Effects of elastase treatment (e.g. greater lung volumes, emphysema, and alveolar interstitial fibrosis) were the only effects that persisted throughout the recovery period. In addition, a significant increase in alveolar mean and median cord length in saline/diammonium sulphate treated rats was observed.
No significant pathological changes were observed in the nasal cavities of any group at any time point. The pulmonary function of rats exposed for 4 months to diammonium sulphate alone was significantly decreased compared to air control for a few measured parameters (residual volume/total lung capacity, quasistatic compliance, nitrogen-slope) in either saline or elastase treated rats. No significant changes in the pulmonary function were observed in rats exposed to both diammonium sulphate and sulphur dioxide. Immunological studies of peripheral lymphocytes and spleen cells of rats exposed to diammonium sulphate for 4 months revealed no significant depressive effects on the immune system.
The authors concluded that changes related to exposure to sulphur dioxide and/or diammonium sulphate were minimal and transient. No additive effect appeared to occur between diammonium sulphate and sulphur dioxide. The authors were mainly concerned about the increased alveolar fibrosis (observed after 8 months of exposure but not after 4 months of exposure or after 3 months of recovery) associated with diammonium sulphate exposure and the implications it might have in progressive fibrotic disease in human lungs. (Smith et al. 1989).

Male Sprague-Dawley rats and Hartley strain guinea pigs were instilled intratracheally with either sterile saline or porcine pancreatic elastase dissolved in saline. Groups of 15-29 animals from each pre-treatment group were exposed to filtered air (control), to diammonium sulphate aerosols in concentrations of 1.0 mg/m³ (MMAD = 0.4 mm), or to ammonium nitrate for 6 hours/day, 5 days/week for 5 or 20 days. Pulmonary function evaluations conducted in guinea pigs showed no significant effects of diammonium sulphate (or ammonium nitrate) exposure. Compared with air-exposed animals, rats exposed to diammonium sulphate aerosols had increased values of residual volume and functional residual capacity and a decreased nitrogen-slope in saline as well as elastase treated rats.
Diammonium sulphate exposure aggravated elastase-induced emphysema in the rat. Guinea pigs (but not rats) exposed to diammonium sulphate had hypertrophy and hyperplasia of non-ciliated epithelial cells in the alveoli and bronchioles. Alveolar septa of saline/diammonium sulphate exposed animals were affected by interstitial thickening caused by an increased collagen content and an increased number of interstitial cells. The alveolar cord length was increased in saline/diammonium sulphate exposed animals. (Loscutoff et al. 1985, Busch et al. 1984).

Groups of 6 male Sprague-Dawley rats were exposed to diammonium sulphate aerosols in concentrations of 0, 1 or 5 mg/m³ (MMAD = 0.5-0.6 mm), ozone in concentrations of 0, 0.20, 0.64 or 0.96 ppm or mixtures of both for 23.5 hours per day for 2-7 days. Exposure to diammonium sulphate alone showed no effect in several biochemical and morphometric assays for lung injury (e.g. protein content of lung lavage fluid, collagen synthesis rates, soluble proline content, fibroblast content, lung lesions). Ozone alone caused lung injury as evidenced by significantly differences between the control rats and the rats exposed to ozone in the assays for lung injury. A synergistic effect of the combination of ozone and diammonium sulphate (at a dose of 5 mg/m³, but not at 1 mg/m³) was observed in the form of significantly greater increases in the measured parameters as compared with results observed in rats exposed to ozone alone. Based on these and other studies with combinations of ozone or nitrogen dioxide with different aerosols, the authors conclude that acidity and size of the particles in the aerosol is responsible for the synergy. (Last 1991).

Groups of 10 adult male rats were exposed to diammonium sulphate aerosols in concentrations of 0 or 300 mg/m³ (particle size averaged 1-2 mm) for 8 hours/day for 1, 3, 7 or 14 days. No significant differences could be detected between control and exposed rats in arterial blood gases, pH and standard bicarbonate, in body weight and wet lung weight, in vital capacity and residual volume of the lungs, or in histological examination of the trachea, bronchial lymph nodes and lungs. (Pepelko et al. 1980).

Groups of 7-12 male guinea pigs were exposed to diammonium sulphate aerosols in concentrations of 0, 0.2, 0.4 or 2.0 mg/m³ (MMAD = 0.7-0.9 mm) for 2 hours/day, 5 days/week for 7½ weeks. The animals were successively 3 times a week for 2½-3 weeks exposed to aerosols for 2 hours, and after 30 minutes, to an albumin spray for 30 minutes. Breathing curves were continuously recorded during the sensitisation periods (periods with exposure to albumin) and for 5 minutes before exposure to albumin. The experiments showed that the degree of asthmatic dyspnoea (immediate type induced by inhalation of albumin) was dose-dependently increased by exposure to diammonium sulphate aerosols (only significant at 2 mg/m³). The aerosols alone did not alter the breathing curves at the concentrations studied. (Kitabatake et al. 1991).

11.2.2   Oral intake

Groups of 10 F344 rats of each sex were fed 0, 0.38, 0.75, 1.5 or 3% of diammonium sulphate in their CRF-1 powder diet for 13 weeks. The only effect observed was diarrhoea during the administration period in the males dosed with 3% diammonium sulphate. No changes indicating toxicity were observed in body and organ weights, in blood and biochemical parameters, or in the histopathological examination. According to the authors, the NOEL in this study was 1.5% (equal to 886 mg/kg b.w. per day) in males but 3% (equal to 1975 mg/kg b.w. per day) in females. (Takagi H et al. 1999 – quoted from Toxline Plus).

11.2.3   Dermal contact

No data have been found.

11.2.4   Other routes

No data have been found.

11.3   Toxicity to reproduction

No data have been found.

11.4   Mutagenic and genotoxic effects

11.4.1   In vitro studies

Diammonium sulphate was not mutagenic when tested (in accordance with OECD guideline 471) in 4 strains (TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium in a concentration of 20 – 5000 mg/plate in Ames tests with and without metabolic activation systems (BASF 1983 – quoted from IUCLID 2000).

In a cytogenetic assay without metabolic activation systems, an isotonic solution of diammonium sulphate did not cause chromosomal aberrations in V79 hamster cells. However, cells treated with a hypotonic diammonium sulphate solution (or other hypotonic solutions) had an increase in aberration frequencies. Post-treatment with a hypertonic solution of diammonium sulphate increased the mutagenicity of ethylmethanesulfonate, a known mutagen. (Nowak 1987, 1988 - both quoted from IUCLID 2000).

In two other cytogenetic assays performed in human lymphocytes and CHO cells, respectively, diammonium sulphate did not cause chromosomal aberrations (Obe & Kamra 1986, Obe et al. 1986 - both quoted from IUCLID 2000).

Diammonium sulphate tested negative in the yeast gene mutation assay in Saccharomyces cerevisiae D4 with and without metabolic activation systems (US Department of Commerce 1975 - quoted from IUCLID 2000).

11.4.2   In vivo studies

No data have been found.

11.5   Carcinogenic effects

Groups of Syrian hamsters received 5 mg of a known carcinogen (benzo(a)pyrene) by intratracheal intubation once a week for 15 weeks. One group of the animals was simultaneously exposed to 0.2 mg/m³ of diammonium sulphate 6 hours/day for 5 days/week for the 15 weeks. Other groups received benzo(a)pyrene or diammonium sulphate alone. There was no effect of diammonium sulphate inhalation on benzo(a)pyrene carcinogenesis. (US Department of Commerce - quoted from IUCLID 2000).

 12   Regulations

12.1   Ambient air

-

12.2   Drinking water

Denmark:                               0.5 mg ammonium/l

                                             250 mg sulphate/l            (MM 2001)

12.3   Soil

-

12.4   Occupational Exposure Limits

-

12.5   Classification

-

12.6   IARC

-

12.7   US-EPA

-

 13   Summary

13.1   Description

Diammonium sulphate occurs as colourless orthorhombic crystals or white granules with no odour. The water solubility is high (754-770 g/l).

13.2   Toxicokinetics

When hamsters inhaled diammonium sulphate at a concentration of 0.2 mg/m³, a substantial proportion of the compound was found in the nose. The clearance from the lungs was determined to be about 20 minutes. The results of clearance studies with guinea pigs and rabbits suggested that there was no species difference.

Systemic effects and death following oral exposure to diammonium sulphate indicate that it may be absorbed from the gastrointestinal tract.

Acute intoxication with ammonium may affect the cerebral nervous system by inhibition of the energy metabolism or by inhibition of glutamic acid metabolism.

13.3   Human toxicity

13.3.1   Single dose toxicity

In several studies healthy and asthmatic non-smoking volunteers aged 18-76 have been exposed by inhalation to diammonium sulphate aerosols in concentrations between 0.07 and 1.0 mg/m³ (and with MMAD of 0.5-1 mm) for up to 4 hours. In some of the studies diammonium sulphate was administered in combination with ozone, sulphur dioxide or carbachol (a known bronchoconstrictor). In all studies different measurements of pulmonary function was performed. In doses up to 0.5 mg/m³ consistent changes in pulmonary function or in symptoms occurred in neither healthy nor asthmatic volunteers. At a concentration of 1 mg/m³, diammonium sulphate produced a small but significant decrease in flow rates on maximum and partial expiratory flow-volume curves and potentiated the bronchoconstrictor action of carbachol both in asthmatic and healthy volunteers.
The effect of exposure to a combination of ozone and diammonium sulphate seemed slightly greater (although not statistically significant) than exposure to ozone alone. The combination of diammonium sulphate and sulphur dioxide produced no significant changes on the lung function at the doses studied. Upper respiratory and eye irritation was reported in one of the studies in some of the volunteers exposed to 0.5 mg/m³ of diammonium sulphate.

One case-report of death caused by acute intoxication due to ingestion of diammonium sulphate exists.

13.3.2   Repeated dose toxicity

No consistent changes in pulmonary function or in symptoms occurred in healthy or asthmatic men when exposed to diammonium sulphate aerosols at a concentration of 0.1 mg/m³ (MMAD = 0.3 mm) for 2 hours a day for 2-3 days.

13.3.3   Toxicity to reproduction

No data have been found.

13.3.4   Mutagenic and genotoxic effects

No data have been found.

13.3.5   Carcinogenic effects

No data have been found.

13.4   Animal toxicity

13.4.1   Single dose toxicity

The reported LC₅₀-value (exposure duration: 8 hours) of diammonium sulphate is higher than 1200 mg/m³ (particle size averaged 2-3 mm) for rats and higher than 900 mg/m³ (particle size not stated) for guinea pigs. The LD₅₀-value following oral exposure is reported to be 2840-4250 mg/kg b.w. for rats and 610-640 mg/kg b.w. for mice. Three rabbits dosed with 1500 mg/kg b.w. of diammonium sulphate through a gastric probe all died.

The airway responsiveness to inhaled carbachol (a known bronchoconstrictor) was enhanced in sheep exposed to 0.1 or 4 mg/m³ of diammonium sulphate with a MMAD of 1.5 mm (but not of 0.5 mm) for 4 hours.

The bronchiolar or tracheal mucociliary clearance was not affected either in donkeys, rats, rabbits, or sheep inhaling diammonium sulphate aerosols for 1-4 hours in doses of 0.3-3.6 mg/m³.

Diammonium sulphate was not irritating to intact or abraded rabbit skin or to rabbit eyes.

13.4.2   Repeated dose toxicity

Pulmonary histology

In rats exposed to 0.5 mg/m³ (MMAD = 0.44 mm), the authors were mainly concerned about the increased alveolar fibrosis observed after 8 months of exposure (but not after 4 months of exposure or after 3 months of recovery). Increased alveolar collagen content was also observed in rats and guinea pigs exposed to 1 mg/m³ (MMAD = 0.4 mm) for 20 days.
A significant increase in alveolar cord length was observed in rats exposed to 0.5 mg/m³ (MMAD = 0.44 mm) for 4 months (but not after 8 months of exposure) and after 3 months of recovery. Increased alveolar cord length was also observed in rats and guinea pigs exposed to 1 mg/m³ (MMAD = 0.4 mm) for 20 days.The number of non-ciliated epithelial cells in the bronchioles increased in rats exposed to 0.5 mg/m³ (MMAD = 0.44 mm) for 4 and 8 months (but not after recovery). Guinea pigs (but not rats) exposed to 1 mg/m³ (MMAD = 0.4 mm) for 20 days, had hypertrophy and hyperplasia of non-ciliated epithelial cells in the alveoli and bronchioles.

No significant changes could be detected in histological examination of the trachea, bronchial lymph nodes and lungs of rats, which had been exposed to 300 mg/m³ of diammonium sulphate aerosols (particle size averaged 1-2 mm) for 1-14 days. Rats exposed to 1 or 5 mg/m³ (MMAD = 0.5-0.6 mm) of diammonium sulphate aerosols for 2-7 days did not show any histological changes of the lungs either.

Pulmonary function

The pulmonary function of rats exposed for 4 months to 0.5 mg/m³ (MMAD = 0.44 mm) of diammonium sulphate was significantly decreased for a few measured parameters (residual volume/total lung capacity, quasistatic compliance, nitrogen-slope).
Rats exposed to 1.0 mg/m³ (MMAD = 0.4 mm) of diammonium sulphate aerosols for 20 days had increased values of residual volume and functional residual capacity and a decreased nitrogen-slope.
No significant changes could be detected in pulmonary function of rats, which had been exposed to 300 mg/m³ of diammonium sulphate aerosols (particle size averaged 1-2 mm) for 1-14 days or in guinea pigs exposed for 20 days to 1.0 mg/m³ (MMAD = 0.4 mm).

Interactions

Diammonium sulphate exposure at 0.5-1.0 mg/m³ aggravated the effects induced by elastase pre-treatment in the rats exposed for 20 days, 4 months and 8 months. A synergistic effect of the combination of ozone and diammonium sulphate was observed in rats exposed to diammonium sulphate for 2-7 days at a concentration of 5 mg/m³ (but not at 1 mg/m³) (MMAD = 0.5-0.6 mm). The degree of asthmatic dyspnoea (immediate type induced by inhalation of albumin) was dose-dependently increased in guinea pigs exposed to 0.2-2.0 mg/m³ (MMAD = 0.7-0.9 mm) diammonium sulphate aerosols (only significant at 2 mg/m³) for 7½ weeks.

Immunological studies of peripheral lymphocytes and spleen cells of rats exposed to 0.5 mg/m³ (MMAD = 0.44 mm) of diammonium sulphate for 4 months revealed no significant depressive effects on the immune system.

Diarrhoea in the males at the highest dose was the only observed effect in rats fed diammonium sulphate in their diet in doses up to 1975 mg/kg b.w. per day for 13 weeks.  

13.4.3   Toxicity to reproduction

No data have been found.

13.4.4   Mutagenic and genotoxic effects

Diammonium sulphate tested negative in Ames tests and in a yeast gene mutation assay with and without metabolic activation systems, and in cytogenetic assays in V79 hamster cells, in human lymphocytes, and in CHO cells.

Addition of diammonium sulphate increased the mutagenicity of a known mutagen (ethylmethanesulphonate).

13.4.5   Carcinogenic effects

Diammonium sulphate inhaled by hamsters at a concentration of 0.2 mg/m³ for 15 weeks did not affect the carcinogenic potential of the known carcinogen benzo(a)pyrene.

14   Evaluation

The critical effects of diammonium sulphate are the local effects observed in the lungs:
A lot of studies in humans and animals have evaluated the pulmonary effects of exposure to diammonium sulphate aerosols. Statistically small changes in the pulmonary function have been measured in some (but not all of the studies) in both humans and animals. Histological examination of the lungs of exposed rats and guinea pigs revealed transient increased alveolar fibrosis, alveolar cord length, and hypertrophy and hyperplasia of non-ciliated epithelial cells in the alveoli and bronchioles in some (but not all of the studies). In addition, diammonium sulphate seems to potentiate the effect of the bronchoconstrictor carbachol (studied in humans), ozone (studied in humans and rats), albumin used to induce asthmatic dyspnoea (studied in guinea pigs), and elastase used to model chronic pulmonary impairment (studied in rats).
All of the observed effects occurred at doses around 1 mg/m³ of diammonium sulphate with a MMAD of about 0.5 mm. However, in rats exposed to 300 mg/m³ of diammonium sulphate with an average particle size of 1-2 mm, no pulmonary changes were observed. The deposition of aerosols in the lungs is dependent on several factors (e.g. particle size and ventilation rate), which might have been different in the different studies and possibly can explain the apparent inconsistency in results.

Upper respiratory irritation was reported in one of the human studies in some of the volunteers exposed to 0.5 mg/m³ of diammonium sulphate.

Diammonium sulphate was, according to the citations in IUCLID (2000), not irritating to rabbit skin and eyes in the two studies reported. However, the description of the studies is limited (e.g., information on the concentration of the applied chemical is not stated); therefore, the studies are considered inadequate with respect to an evaluation of the irritant potential of diammonium sulphate.

Death following acute exposure to high oral doses of diammonium sulphate has been reported in a human case report and in animal studies.

The only effect observed in rats fed diammonium sulphate in their diet for 13 weeks was diarrhoea in the males in the highest dose group exposed to 1975 mg/kg b.w. per day.  

None of the tests for mutagenicity and genotoxicity indicate any potential for diammonium sulphate to possess such properties No adequate carcinogenic studies with diammonium sulphate have been found.

No data have been found for reproductive and developmental effects.

15   References

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Busch RH, Buschbom RL, Cannon WC, Lauhala KE, Miller FJ, Graham JA and Smith LG (1984). Effects of ammonium sulfate aerosol exposure on lung structure of normal and elastase-impaired rats and guinea pigs. Environ Res 33, 454-472.

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Kitabatake M, Imai M, Nakano H and Yoshika K (1991). Effects of exposure to sulfate aerosols and antigen on breathing curve patterns of guinea pigs. J Toxicol Environ Health 33, 157-170.

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Kulle TJ, Sauder LR, Shanty F, Kerr HD, Farrell BP, Miller WR and Milman JH (1984). Sulfur dioxide and ammonium sulfate effects on pulmonary function and bronchial reactivity in human subjects. Am Ind Hyg Assoc J 45, 156-161.

Last JA (1991). Synergistic effects of air pollutants: Ozone plus a respirable aerosol. Res Rep Health Eff Inst 38, 1-43.

Loscutoff SM, Cannon WC, Buschbom RL, Busch RH and Killand BW (1985). Pulmonary function in elastase-treated guinea pigs and rats exposed to ammonium sulfate or ammonium nitrate aerosols. Environ Res 36, 170-180.

Merck Index (1996).  Ammonium Sulfate. In: 12th. ed., Rahway, New Jersey, Merck & Co., Inc., 94.

MM (2001). Bekendtgørelse om vandkvalitet og tilsyn med vandforsyningsanlæg. Miljøministeriets bekendtgørelse nr. 871 af 21. september 2001. 

Pepelko WE, Mattox JK and Cohen AL (1980). Toxicology of ammonium sulfate in the lung. Bull Environ Contam Toxicol 24, 156-160.

Sato A, Gonmori K and Yoshioka N (1999). A case of fatal intoxication with ammonium sulfate and a toxicological study using rabbits. Forensic Sci Int 101, 141-149.

Schlesinger RB (1984). Comparative irritant potency of inhaled sulfate aerosols – effects on bronchial mucociliary clearance. Environ Res 34, 268-279.

Smith LG, Busch RH, Buschbom RL, Cannon WC, Loscutoff SM and Morris JE (1989). Effects of sulfur dioxide or ammonium sulfate exposure, alone or combined, for 4 or 8 months on normal and elastase-impaired rats. Environ Res 49 (1), 60-78.

Stacy RW, Seal E, House DE, Green J, Roger LJ and Raggio L (1983). A survey of gaseous and aerosol pollutants on pulmonary function of normal males. Arch Environ Health 38, 104-115.

Toxline Plus. Database with references to toxicological articles published between 1985 and 2001.

Utell MJ, Morrow PE and Hyde RW (1982). Comparison of normal and asthmatic subjects‘ responses to sulphate pollutant aerosols. Ann Occup Hyg 26, 691-697.

Appendix 3: Dimethyl ether (DME)  
16   General description

16.1   Identity

Molecular formula:          C₂H₆O

Structural formula:           H₃C-O-CH₃

Molecular weight:            46.07

CAS-no.:                        115-10-6

Synonyms:                      Methyl ether

                                      Oxybismethane

                                      Methoxymethane

                                      Methyloxide

                                      Dimethyl –13C2 (gas)

                                      Wood ether

16.2   Physical / chemical properties

Description:                    Colourless, flammable gas with a slight ethereal odour.

Melting point:                  -138.5 °C

Boiling point:                   - 23.7 °C

Density:                          0.669 g/ml (at 20°C)

Vapour pressure:            3982 mmHg (530.8 kPa) (at 20°C)

Concentration of

saturated vapours:           -

Conversion factor:          1 ppm = 1.91 mg/m³        (at 20°C and 760 mmHg)

                                      1 mg/m³ = 0.523 ppm

Solubility:                       Water:  328 g/l (at 20°C)

LogP_o/w:                        - 0,18

References:                    A&H (1995), Berkhout (1997), Kirwin & Galvin (1993), IUCLID (2000), Merck Index (1996). 

17   Toxicokinetics

Dimethyl ether (DME) is rapidly taken up via the lungs after inhalation or intratracheal administration (Eckard & Kemper 1979 - quoted from A&H 1995).

Pharmacokinetic studies in rats have shown that after inhalation DME is rapidly distributed to various organs and tissues. The steady state level is reached within 30 minutes. No tissue storage of DME was seen. (Daly & Kennedy 1987).

The DME content in various tissues after exposure to 1000 ppm (1900 mg/m³) in rats is reported to be 14-22 µg/g tissue. The DME concentrations in organs and tissues were reported to fall rapidly after termination of exposure, though somewhat more slowly in fat and muscle than in blood and other tissues. (Bohnenn 1979 - quoted from A&H 1995).

Elimination was described as a two-phase process. The half times in blood were reported to be 10 minutes for the a phase and 90 minutes for the b phase (Daly & Kennedy 1987).

No data on absorption, distribution or elimination of DME after oral intake or dermal contact were found.

17.1   Mode of action

No information is available on the mechanisms of DME in causing CNS effects and other physiological effects in humans or animals. 

18   Human toxicity

18.1   Single dose toxicity

18.1.1   Inhalation

Only two very old studies describing the effects of DME to humans have been found.

DME concentrations of 50 % were inhaled by humans in the laboratory (around 500000 ppm (960000 mg/m³) according to the citation in IUCLID). The gas was most unpleasant to inhale (being distinctly suffocating, even when taken with a high percentage of oxygen). (Brown 1924 - quoted from Kirwin & Galvin 1993, IUCLID 2000).

In human subjects, 50000 and 75000 ppm (96000 and 143000 mg/m³) of DME caused feelings of mild intoxication after 12 minutes exposure. At 82000 ppm (157000 mg/m³) some incoordination developed after 21.5 minutes, and a complaint was made of indistinct vision. At 100000 ppm (191000 mg/m³), no objective symptoms occurred during the first 15 minutes. Distinct signs of incoordination developed after 21 minutes of exposure. The experiment continued for 64 minutes, with the subject being unable to do simple tasks. At 144000 ppm (275000 mg/m³), symptoms first occurred after 7 minutes with the subject loosing consciousness after 26 minutes. Inhalation of 200000 ppm (382000 mg/m³) caused unconsciousness within 17 minutes. (Davidson 1925 - quoted from IUCLID 2000).

18.1.2   Oral intake

No data have been found.

18.1.3   Dermal contact

Liquid DME will cause severe frostbite if spilled on the skin (IUCLID 2000).

18.2   Repeated dose toxicity

No data have been found.

18.3   Toxicity to reproduction

No data on reproductive or developmental effects in humans related to DME exposure have been found.

18.4   Mutagenic and genotoxic effects

No data on mutagenic or genotoxic effects in humans related to DME exposure have been found. 

18.5   Carcinogenic effects

No data on carcinogenic effects in humans related to DME exposure have been found.

19   Animal toxicity

19.1   Single dose toxicity

19.1.1   Inhalation

The LC₅₀-value in rats was reported to be 164000 ppm (313000 mg/m³) after 4 hours of exposure (Daly & Kennedy 1987).

At exposure levels of 10000 ppm (19000 mg/m³), rats showed slight sedation and at 50000 ppm (96000 mg/m³) rats were asleep most of the time for exposures longer than 30 minutes (Daly & Kennedy 1987).

The LC₅₀-value in mice was reported to be 490000 ppm (936000 mg/m³) after 15 minutes of exposure and 380000 ppm (~726000 mg/m³) after 30 minutes exposure (Daly & Kennedy 1987).

The exposure to 120000 ppm (229000 mg/m³) for 20-25 minutes was reported to cause light narcosis in mice (Meyer & Gottlieb-Billroth 1921 - quoted from A&H 1995).

Besides effects on the central nervous system (CNS) and the blood profile, DME was reported to cause weak cardiac sensitisation in dogs exposed for 5 minutes to 200000 and 300000 ppm (382000 and 573000 mg/m³) DME, but not at 100000 ppm (~191000 mg/m³) (Daly & Kennedy 1987).

19.1.2   Oral intake

No data have been found.

19.1.3   Dermal contact

No data have been found.

19.1.4   Other routes

Intraperitoneal doses of 5 mg/kg of DME produced reversible anaesthesia in mice (no further details are given) (Gosselin et al. 1984).

19.2   Repeated dose toxicity

19.2.1   Inhalation

Rats exposed to up to 50000 ppm (96000 mg/m³) of DME (6 hours/day, 5 days/week, 2 weeks) showed the following effects: At 10000 ppm (19000 mg/m³), slight sedation. At 50000 ppm (96000 mg/m³), sedation, body weight gain suppression, minor haematological changes (a small increase in the number of leucocytes and perhaps a decrease in the number of red blood cells), and organ weight changes (in relative liver and kidney weights), but no histopathological organ changes. All changes seen during the two-week exposure period were completely reversed following a two-week recovery period. (Daly & Kennedy 1987).
In the same study, 4-week inhalation exposures of DME to rats and hamsters were conducted. Rats were exposed to up to 10000 ppm (19000 mg/m³) and hamsters to up to 20000 ppm (38000 mg/m³). There were no toxicological changes seen and particularly no signs of sedation in the hamsters exposed to 20000 ppm (38000 mg/m³) (Daly & Kennedy 1987).

In a 4-week study, rats were exposed to DME at concentrations of 1, 100, 1000, or 10000 ppm (1.9, 190, 1900, or 19000 mg/m³) for 6 hours/day, 5 days/week. Observations were made of behaviour, growth, food intake, haematology, urine composition, organ weights, and gross as well as microscopic pathology. None of the parameters investigated revealed any distinct treatment related effects. (Kruysse 1976 - quoted from IUCLID 2000).

In a study in which rats were exposed to DME (0, 2000, 10000, or 20,000 ppm - 0, 3800, 19000, or 38000 mg/m³) for up to 13 weeks, 6 hours/day, 5 days/week, altered blood profile (reduced percentage of lymphocytes and increase of neutrophilic white blood cells) was noted at the highest dose level (20000 ppm- 38000 mg/m³). Male rats of the top-level group showed a minimal, though statistically significant, increase in serum glutamate pyruvate transaminase (SGPT) activity. Total serum protein was slightly, but statistically significantly lower in females of the top-level group than in females of the control group. The reporters did not consider these changes biologically significant and thus, it was concluded that exposure of rats to 20000 ppm (38000 mg/m³) under the conditions of this study produced no effects of obvious toxicological significance. This conclusion was supported by the absence of chemically related significant gross toxic signs or abnormal histopathology. (Reuzel et al. 1981)

In a second 13-week study by Reuzel & Woutersen (1983 - quoted from IUCLID 2000), rats were exposed to DME at concentrations of 0, 1000, 5000, 10000 or 20000 ppm (0, 1900, 9600, 19000, or 38000 mg/m³). Health condition, behaviour and body weight did not change due to DME exposure. No toxicologically relevant significance was ascribed to a temporary difference in white blood cell counts in males. Neutrophil counts were higher in male rats at all test levels. A dose-effect relation could not be found. In view of the fact that increased neutrophil counts were also found in previous sub-chronic study, it was stated in the final conclusion that the no-effect level in rats was 10000 ppm (19000 mg/m³).

In a 13 week study in hamsters exposed to 0, 1000, 5000, 10000, or 20000 ppm (0, 1900, 9600, 19000, or 38000 mg/m³) for 6 hours/day, 5 days/week, the following observations were made: Exposures did not affect health condition, behaviour and body weights of hamsters. White blood cell counts and in particular absolute lymphocyte counts had decreased both in male and female hamsters, which had been exposed to 20000 ppm (38000 mg/m³). The difference concerning the absolute white blood cell counts was not statistically significant. The difference in absolute lymphocyte counts was only significant on day 56. A number of other measured haematological values (no further information given) showed a significant difference compared with values found in the animals of the control group. For several reasons the investigators considered these differences to be of no importance.The investigators concluded that the no-effect level for DME in hamsters was 5000 ppm (9600 mg/m³), as at concentrations of 20000 and 10000 ppm (38000 and 19000 mg/m³) a decrease in white blood cell counts and lymphocyte counts occurred. (Reuzel & Woutersen 1983 - quoted from IUCLID 2000).

In a 30-week study, rats were exposed to 0.02%, 0.2%, or 2% v/v (0, 200, 2000, or 20000 ppm – 0, 380, 3800, or 38000 mg/m³) of DME in air, 6 hours/day, 5 days/week. There was no evidence that exposure to DME at any dose level had a toxicologically significant effect on body weight or food consumption. There were no toxicologically significant clinical signs in animals from any of the groups during the 30 weeks of the experiment nor were there any significant ophthalmoscopic findings. (Collins et al. 1978).
The only abnormalities in blood chemistry concerned the SGPT and SGOT (serum glutamate oxaloacetate transaminase) levels. At 24 weeks, there were abnormally high SGPT values in a few rats in both the male and female high-dose groups, suggesting the possible onset of a hepatotoxic effect of DME. At week 27, SGPT levels showed no differences between groups for either sex. However, at the end of the study there was a statistically significant increase in SGPT for both males and females of the high dose group (20000 ppm - 38000 mg/m³) when compared with the control group. There was a statistically significant increase in SGOT levels in the male medium dose group (2000 ppm- 3800 mg/m³).
The only organ weight measurement that indicated a toxicological effect was that of the liver of the high dose male rats. There was a statistically significant reduction in liver weight relative to body weight in this group compared with the control group (P< 0.05). The unusual event, that decreased liver weights are accompanied with increased GPT levels, was explained as a possible hepatic fibrosis, which was insufficiently severe to be detected by routine histopathological examination. There were no histopathological changes seen that were considered to be related to exposure of the rats to DME.

Rats were exposed to DME by inhalation for 104 weeks, 6 hours/day, 5 days/week. The concentrations were 2000, 10000, or 25000 ppm (3800, 19000, or 48000 mg/m³). There was no specific tissue damage, as indicated by either clinical function studies or morphologic studies of the tissues examined at the end of the study. Haematology and clinical pathology studies conducted at three-month intervals during the two years showed no evidence of change. Female animals in both the 10000 and 25000 ppm (19000 and 48000 mg/m³) groups showed a slight decrease in mean survival time. The decrease was not statistically significant, but it was different from that seen either at the low level or in the control subjects. According to the authors, the no-effect level was 20000 ppm (38000 mg/m³). (Daly & Kennedy 1987).
It should be noted that the no-effect level concluded by the authors is not one of the concentrations used in the study.

19.2.2   Other routes

No information on repeated dose toxicity following oral, dermal or other routes of exposure were found.

19.3   Toxicity to reproduction

19.3.1   Inhalation

Two developmental studies have been reported for DME.

In the first study, female rats were exposed to concentrations of 0, 1250, 5000, 20000, or 40000 ppm (0, 2400, 9600, 38000, or 76000 mg/m³) of DME, 6 hours/day, from days 6 to 15 of gestation. Animals exposed to 5000 ppm (9600 mg/m³) or more showed some evidence of narcotic effect, somewhat proportional to dose. At a concentration of 40000 ppm (76000 mg/m³), these female animals showed a suppression of body weight gain. There was no evidence of any teratogenic effect on the foetuses. There was no increase in foetal resorption. At concentrations of 20000 and 40000 ppm (38000 and 76000 mg/m³), there was some evidence that the foetuses were somewhat smaller, and that foetal variations such as ossification of the rib bones and some of the phalangeal bones in the extremities of these animals was somewhat retarded. These findings the reporters considered as variations reflecting developmental delay rather than a specific effect on the foetus. (Daly & Kennedy 1987).

In the second study, female rats were exposed to concentrations of 0, 20000, or 28000 ppm (0, 38000, or 53000 mg/m³) of DME from days 6 to 15 of gestation. The researchers did not find any signs of narcosis in the maternal animals. Maternal body weight gain was normal. There was a slight increase in the number of extra ribs in the foetuses at both levels tested; the increase was statistically significant, yet there was no dose-response. No gross malformations were noted in these offspring, and embryo mortality was not reflected by increased resorptions. (Daly & Kennedy 1987).

19.3.2   Other routes

No information on reproductive and developmental effects following oral, dermal or other route of exposure were found.

19.4   Mutagenic and genotoxic effects

19.4.1   In vitro studies

DME was not mutagenic when tested in the Ames test with and without metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 at a concentration of 119000 ppm (Willems 1978 - quoted from IUCLID 2000).

Mutagenic effect of DME could not be shown in V79 Chinese hamster cells in suspension with 230, 460, 1150 and 3450 mg/l DME (Kramers et al 1981 - quoted from IUCLID 2000).

No induction of DNA-repair synthesis was observed at any dose level when primary rat liver cells were exposed to 230, 460, 1150, 2300, or 3450 mg/l DME (Kramers et al. 1981 - quoted from IUCLID 2000).

19.4.2   In vivo studies

Progeny examination led to the conclusion that DME was not mutagenic under the test conditions when Drosophila melanogaster was exposed to 8000 or 28000 ppm (15000 or 53000 mg/m³) for 3 days or 28000 ppm (53000 mg/m³) for 14 days (Kramers et al. 1981 - quoted from IUCLID 2000).
In the host mediated assay in mice, exposure to 10000 or 20000 ppm (19000 or 38000 mg/m³) for 3 hours gave negative results (DGF 1992  - quoted from A&H 1995).

19.5   Carcinogenic effects

In a 104-week study in rats exposed to 2000, 10000, or 25000 ppm (3800, 19000, or48000 mg/m³) of DME, there was no evidence of increased tumour formation in any of the tissues or organs of the animals (Daly & Kennedy 1987).

No data on carcinogenic effects following oral, dermal or other route of exposure were found. 

20   Regulations

20.1   Ambient air

Denmark (C-value):        1 mg/m³  (MST 2002).

20.2   Drinking water

Denmark:                       -

20.3   Soil

Denmark:                       -

20.4   Occupational Exposure Limits

Denmark:                       1000 ppm (1885 mg/m³) (At 2002).

Germany:                        1000 ppm (1910 mg/m³) (MAK 1993).

EU-SEG:                        1000 ppm (1910 mg/m³) (SEG 1991).

20.5   Classification

DME is classified as extremely flammable (Fx;R12) (MM 2002).

20.6   IARC                  

-

20.7   US-EPA

-

21   Summary

21.1   Description

Dimethyl ether (DME) is a colourless gas at room temperature and with a slight ethereal odour. The substance has a very high vapour pressure, and is soluble in water.

21.2   Toxicokinetics

DME is rapidly taken up after inhalation and distributed to various organs and tissues, where a steady state level is reached within 30 minutes. After end of exposure, the concentration of DME in organs and tissues falls very rapidly again.
The elimination is described as a two-phase process. No tissue storage is seen.No data on absorption, distribution or elimination of DME after oral intake or dermal contact were found.

21.3   Human toxicity

The only effects described after DME exposure to humans originate from very old studies, where human subjects have been exposed to very high acute doses of DME. The target organ after exposure to high concentrations of DME is the central nervous system, covering effects from incoordination, indistinct vision, and inability to do simple tasks and to unconsciousness (exposure levels from 75000 to 200000 ppm - 143000 to 382000 mg/m³).

No information on toxic effects in humans following repeated dose exposure was found. Likewise no information on reproductive or developmental effects, as well as on mutagenic, genotoxic or carcinogenic effects in humans was found.

21.4   Animal toxicity

21.4.1   Single dose toxicity

The LC₅₀-values for DME in mice have been reported to be 490000 ppm (936000 mg/m³) after exposure in 15 minutes and 380000 ppm (726000 mg/m³) after exposure in 30 minutes.
In rats, the LC₅₀-value has been reported to be 164000 ppm (313000 mg/m³) after 4 hours exposure. The effects of DME in rats exposed to sub-lethal doses range from sedation to narcosis.
In dogs exposed to 200000 and 300000 ppm (382000 and 573000 mg/m³), DME has been reported to be a light cardiac sensitiser.

21.4.2   Repeated dose toxicity

Short-term studies (2 weeks) in which rats were exposed to concentrations of DME of 50000 ppm (96000 mg/m³) caused sedation, body weight gain suppression, haematology and organ weight changes, but no histopathological organ changes. All changes were completely reversed after cessation of exposure.In sub-chronic studies (13 or 30 weeks) with exposure of DME up to 20000 ppm (38000 mg/m³) in rats and hamsters, the reported effects focus on changes in haematological parameters (white blood cells) for both rats and hamsters, and an increase of serum SGPT and SGOT values in rats, the last suggesting a possible onset of a hepatotoxic effect of DME. By histopathological examinations, no effects were observed in the liver.
The no-effect level for haematological effects in the 13-week studies was reported to be 10000 ppm (19000 mg/m³) for rats and 5000 ppm (9600 mg/m³) for hamsters. In the 30-week study on rats, the no-effect level for increased levels of SGPT was reported to be 2000 ppm (3800 mg/m³) and for increased levels of SGOT 200 ppm (380 mg/m³).

In a lifetime study in rats exposed to concentrations up to 25000 ppm (48000 mg/m³) of DME, the only effect reported was a decrease in mean survival time for female rats from 10000 ppm (19000 mg/m³) . The decrease was not statistically significant, but different from that seen in the low dose group (2000 ppm - 3800 mg/m³) and the control group. The no-effect level was reported to be 20000 ppm (38000 mg/m³).

21.4.3   Toxicity to reproduction

In a developmental study, female rats were exposed to concentrations of 20000 or 28000 ppm (38000 or 53000 mg/m³) of DME from days 6 to 15 of gestation. The researchers found a statistically significant increase in the number of extra ribs in the foetuses at both dose levels, these findings was not accompanied by maternal toxicity at any of the dose groups. There were no signs of teratogenicity or embryo mortality.

In another developmental study, with exposures of 0, 1250, 5000, 20000, or 40000 ppm (0, 2400, 9600, 38000, or 76000 mg/m³), to female rats from day 6 to 15 of gestation, the female animals showed evidence of narcotic effect from 5000 ppm (9600 mg/m³) and suppressed body weight gain at 40000 ppm (76000 mg/m³). There was no evidence of teratogenic effects or increase in foetal resorptions. Retarded ossification of the rib bones and some of the phalangeal bones in the extremities of the foetuses was considered as variations reflecting developmental delay rather than a specific effect on the foetuses.

21.4.4   Mutagenic and genotoxic effects

DME showed no signs of a mutagenic or genotoxic potential in 3 in vitro and 2 in vivo test systems.

21.4.5   Carcinogenic effects

In a lifetime study in rats exposed to DME at concentrations up to 25000 ppm (48000 mg/m³), there was no increase in cancer in any of the tissues or organs of the animals. 

22   Evaluation

As DME is a gas at ambient temperature, the main exposure routes for humans seem to be by inhalation and accidental dermal contact occurring from the use of DME as a carrier of active substances in aerosols.

The critical target organ at acute high concentrations is the CNS resulting in a narcotic effect. No contemporary human data are available in relation to CNS changes such as neurobehavioral disturbances.

Based on the limited and old data on human exposure to dimethyl ether it is not  possible to estimate levels for the narcotic effects in humans.

The structurally related substance – diethyl ether – has been more extensively investigated according to narcotic effects, where exposure to concentrations of about 100000 ppm (308000 mg/m³) induces anaesthesia, and about 15000 ppm (46200 mg/m³) is the lowest anaesthetic concentration. (Strube 1995).

Available animal studies show a low order of acute and chronic toxicity, and any capability of dimethyl ether in being a genotoxic, carcinogenic or developmental toxicant has not been demonstrated. 

Repeated dose toxicity studies have shown some effects of high concentrations of dimethyl ether on the liver (higher SGPT (= alanine amino transferase ALAT) and SGOT (= aspartate amino transferase (ASAT) values suggesting a possible onset of a hepatotoxic effect) and changes in white blood cell counts. The no-effect level in subchronic studies for haematological effects was reported to be 10000 ppm (19000 mg/m³) for rats and 5000 ppm (9600 mg/m³) for hamsters. In a 30-week study on rats, the no-effect level for increased levels of SGPT was reported to be 2000 ppm (3800 mg/m³) and for increased levels of SGOT 200 ppm (380 mg/m³). In a life-time study in rats, the no-effect level was stated to be 20000 ppm (38000 mg/m³); haematological investigations showed no evidence of changes, whereas no information is given whether biochemical parameters (as e.g., SPGT and SGOT) were investigated.

Overall, the no-effect level for effects of dimethyl ether in repeated dose toxicity studies is considered to be 2000 ppm (3800 mg/m³) based on the increased levels of SPGT observed at higher concentrations in the subchronic studies.

 23   References

A&H (1995). Consensus Report for Dimethyl ether. Arbete och Hälsa 19. Nordiska expertgruppen för gränsvärdesdokumentation. Arbetarskyddsverket, 20-24.

At (2002). Grænseværdier for stoffer og materialer. At-vejledning C.0.1, oktober 2002.

Berkhout H (1997). Dimethyl ether. The (almost) universal propellant. Aerosol Spray Rep 36, 23-27.

ChemFinder (2001). Methyl Ether. Http://www.chemfinder.com

Collins CJ, Cobb LM & Purser DA (1978). Effects of chronic inhalation of Dimethyl ether in the rat. Toxicology 11, 65-71.

Daly KK and Kennedy GL (1987). Dimethyl ether: A safety evaluation. Chemical Times & Trends 10, 40-54.

European Commission (1994). Occupational exposure limits. Recommendations of the Scientific Expert Group 1991-92. Comm Eur Communities, EUR 15091, 95 p.

Gosselin RE, et al., Clinical Toxicology of Commercial Products, 5^th Edition, 1984.

HSDB (2000). Dimethyl ether. In: Hazardous Substances Data Base. Last revised: 10/2000.

IUCLID (2000). In: International Uniform Chemical Information Database. Year 2000 CD-ROM edition, European Commission.

Kirwin CJ and Galvin JB (1993). Dimethyl ether. In: Patty’s Industrial Hygiene and Toxicology, Fourth Edition, Vol. 2, Part A. John Wiley & Sons, New York, 458-459.

Merck Index (1996). Methyl Ether. In: 12th. ed., Rahway, New Jersey, Merck & Co., Inc., 1037.

MM (2002). The Statutory Order from the Ministry of the Environment No. 439 of June 3, 2002, on the List of Chemical Substances.

MST (2002). B-værdivejledningen. Vejledning Nr. 2 2002, Miljøstyrelsen, Miljøministeriet.

MST (1990). Begrænsning af luftforurening fra virksomheder. Vejled­ning fra Miljøstyrelsen nr. 6 1990.

Reuzel PGJ, Bruyntjes JP and Beems RB (1981). Sub-chronic (13 weeks) inhalation toxicity study with dimethyl ether in rats. Report No. R 5717. Aerosol Report 20, 23-28.

RTECS (2000). Methyl ether. In: Registry of Toxic Effects of Chemical Substances. Database quest, last revised: 10/2000.

SEG 1991, European Commission (1994). Occupational exposure limits. Recommendations of the Scientific Expert Group 1991-1992. Comm Eur Communities, EUR 15091. Dimethyl ether.

Strube M (1995). Evaluation of health hazards by exposure to diethyl ether and estimation of a limit value in ambient air. Institute of Toxicology, Danish National Food Agency. Baggrundsrapport udarbejdet for Miljøstyrelsen, 1995.

Appendix 4: Hexamethylenetetramine  
24   General description

24.1   Identity

Molecular formula:          C₆H₁₂N₄

Structural formula:          

Molecular weight:            140.19

CAS-no.:                        100-97-0

Synonyms:                      Ammonioformaldehyde

                                      Formamine

                                      Hexamethyleneamine

                                      Hexamine

                                      Hexilmethylenamine

                                      HMT

                                      HMTA

                                      Methenamine

                                      1,3,5,7-Tetraazaadamantane

                                      1,3,5,7-Tetraazatricyclo(3.3.1.1(3,7))decane

24.2   Physical / chemical properties

Description:                    Hexamethylenetetramine occurs as hygroscopic and colourless crystals or as a white crystalline powder with no or mild ammonia odour.

Melting point:                  Sublimates at about 263°C without melting and with partial decomposition to mainly formaldehyde and ammonia.

Boiling point:                   Sublimates.

Density:                          1.27 g/ml (at 25°C).

Vapour pressure:            0.004 mmHg (0.53 Pa) (at 25°C).

Concentration of
saturated vapours:           5.3 ppm (calculated) at 25°C and 760 mmHg.

Conversion factor:           1 ppm = 5.83 mg/m³   20°C

                                      1 mg/m³ = 0.17 ppm   1 atm

Solubility:                        Water:  449 g/l (at 12°C).

References:                    ChemFinder (2001), CIR (1992), Dreyfors et al. (1989), HSDB (2000), ICSC (1993), Loeper & Berzins (1995), Merck Index (1996).

25   Toxicokinetics

25.1   Absorption

Hexamethylenetetramine is rapidly absorbed in humans following oral administration. However, from 10 to 30 % of an orally administered dose of hexamethylenetetramine is hydrolysed in the gastric fluid to yield formaldehyde and ammonia. (CIR 1992, Loeper & Berzins 1995).

25.2   Distribution

Hexamethylenetetramine is distributed to blood and organs (not further specified) (Loeper & Berzins 1995).

It can pass the placenta and is detectable in the amniotic fluid and in milk (CIR 1992).

25.3   Elimination

The half-life for hexamethylenetetramine in 10 healthy volunteers administered 2 g per day for 8 days of two different formulations of tablets was about 4 hours. Following a single dose, approximately 82 % of the hexamethylenetetramine was recovered in the urine in 24 hours as the parent compound. During administration twice daily for 8 days, approximately 88 % of the dose was excreted as the parent compound within a 12-hour dosing period. (Klinge et al. 1982 – quoted from CIR 1992).

Part of hexamethylenetetramine is decomposed to formaldehyde and ammonia in the urine. The half-life of conversion of hexamethylenetetramine to formaldehyde is pH-dependent (about 20 hours at pH 5.0 and about 400 hours at pH 6.5). (Grady Strom & Won Jun 1993 – quoted from Loeper & Berzins 1995).

In slightly acidic environments, such as in sweat, hexamethylenetetramine partially degrades to formaldehyde and ammonia. (Loeper & Berzins 1995).
In addition, hexamethylenetetramine has been reported to produce formic acid upon contact with the skin (Dreyfors et al. 1989).

25.4   Mode of action

No relevant data have been found.

26   Human toxicity

26.1   Single dose toxicity

An accidental overdose of 8 g of hexamethylenetetramine-mandelate (corresponding to a dose of 384 mg/kg b.w. of hexamethylenetetramine in a 10 kg child), which was ingested by a 2½-year-old boy, caused haemorrhagic cystitis (inflammation of the bladder) and mild azotaemia (increased concentration of nitrogen in the blood). The boy recovered without specific treatment. (Ross & Conway 1970 – quoted from Loeper & Berzins 1995).

26.2   Repeated dose toxicity

26.2.1   Inhalation

A cross-sectional study was performed with 33 employees of a hexamethylenetetramine producing plant to assess the health effects of hexamethylenetetramine on the airways and the skin of the workers. Sixteen of the employees (blue and white collar workers) with no or occasional low exposure to hexamethylenetetramine served as controls while 17 employees (baggers, shiftleaders, and executive staff) with exposure to far higher concentrations served as the exposed group. In addition, 4 out of 5 employees that had left the production for medical reasons during the last 10 years were included in the study. For each worker, anamnestic data were recorded and a physical examination of the skin and lungs was performed. In addition, total and specific IgE to four environmental allergens was measured, lung function and bronchial hyperresponsiveness was assessed, and skin prick tests and patch tests with known sensitising substances as well as with hexamethylenetetramine were performed. Measurements were made at different sites in the plant and/or in personal air space of inhalable dust, respirable dust, hexamethylenetetramine, formaldehyde, and ammonia to assess the exposure level.

All measured concentrations of dust and chemicals were in the range of 0.2 – 2.6 mg/m³. Geometric mean hexamethylenetetramine concentrations as assessed by personal sampling were 0.3 mg/m³ in shiftleaders and 0.6 mg/m³ in baggers.
Irritant dermatitis of the hands, predominantly on the palmar parts, was observed in all highly exposed subjects, but also (to a lower extent) in two controls. No other differences were found between the exposed and the control group. Two workers in the exposed group and one in the control group had work-related symptoms. Of the two exposed workers, one person reported shortness of breath, rhinoconjunctivitis and dermatitis of the hands, face and neck that occurred after “accidents with high formaldehyde exposure”. The second worker had pre-existing hay fever and seasonal asthma and gave a history of work-related shortness of breath and rhinitis but he had a normal lung function and no bronchial hyperresponsiveness. One control worker reported work-related conjunctivitis, probably due to long-lasting computer work. No sensitisation to hexamethylenetetramine as assessed by skin prick and patch tests were found in exposed or control workers.
Two out of the four ex-workers (both former baggers) had a positive patch test (but a negative skin prick test) to hexamethylenetetramine. These two former workers had a negative patch test to formaldehyde and no airway symptoms. They reported eczema (located at exposed skin areas in one person and generalised in the other) and conjunctivitis during exposure after 2 weeks or 7 months but were free of symptoms after removal from exposure. The two former workers that tested negative in the patch tests, experienced eczema of the neck “shortly” after exposure and recurrent swelling of eyelids and wrists after one year, respectively. At the time of the study they both presented with eczema, although improved. (Merget et al. 1999).

Seven workers in the lacquer or plastics industries that had worked around epoxy resins, plastics, or paint developed asthma and other allergic symptoms (allergic coryza (nasal catarrh), contact dermatitis, allergic conjunctivitis). An intracutaneous skin test with 0.02 ml of a 1:100 dilution of hexamethylenetetramine gave positive reactions in all workers. The positive reaction was characterised by immediate wheal formation. In a provocative test the workers were inhaling the lacquer product in aerosol form and showed either 1) wheezing and heaviness on the chest or severe asthma, 2) allergic coryza, or 3) skin manifestations of allergy. Some of the workers also had positive reactions to ethylenediamine in both tests. It is unclear from the article which other chemicals the workers might have been exposed to. (Gelfand 1963).

Workers in a tire manufacturing plant were exposed daily to a hexamethylenetetramine-resorcinol mixture (which comprised 2-3 % of the total rubber mix) as well as to their reaction products which were thought to include formaldehyde, ammonia, cyanides and curatives. Fifty-two exposed workers completed a questionnaire and had their lung function measured. The workers suffered from acute symptoms such as itching, skin rashes, coughing, chest tightness, burning eyes and impaired breathing. The excess of symptoms (in comparison with a non-exposed group of workers) persisted when the effects of smoking and drinking were accounted for. Significant reductions in expiratory flow rates at low lung volumes were reported, indicating increased resistance in the small airways. (Gamble et al. 1976).

Workers in foundry where a moulding process utilizes a phenol-formaldehyde resin and hexamethylenetetramine as catalyst, suffered from nasal symptoms such as sneezing, rhinorrhoea, and obstruction as well as wheezy breathing. Twenty of 46 workers in the core shop or casting areas reported asthma or wheezy breathing. Of these, 11 developed the symptoms after starting work at the foundry. Workers involved in other working processes less frequently reported these symptoms. Most environmental measurements of chemical contaminant were below the threshold limits. However, the concentration of furfuryl alcohol (50 ppm in air) and formaldehyde (4 ppm in air) was above the limits in the general foundry. The authors suggest that both irritant and hypersensitivity mechanisms are present because of the onset of symptoms in relation to exposure to fumes and vapours. (Low & Mitchell 1985 – quoted from Loeper & Berzins 1995).

26.2.2   Oral intake

When hexamethylenetetramine is given orally at doses greater than 500 mg four times per day, gastrointestinal distress occur. Bladder irritation, painful and frequent micturition (urination), albuminuria, haematuria, and various rashes may result from doses of 4-8 g a day for longer than 3-4 weeks. (Goodman & Gilman 1975 – quoted from Loeper & Berzins 1995).Adverse effects have been reported in less than 3.5 % of patients receiving hexamethylenetetramine and its salts as a drug. The most frequent adverse effect is gastrointestinal disturbances, including nausea, vomiting, diarrhoea, abdominal cramps, and anorexia. Rarely, hypersensitivity reactions including rash, pruritus, urticaria, and stomatitis have occurred. Other adverse effects reported rarely are headache, dyspnoea, generalized oedema, tinnitus, muscle cramps, dysuria, and microscopic or gross haematuria. (McEvoy 1997 – quoted from HSDB 2000).

26.2.3   Dermal contact

A maximization test has been performed using 25 adults, 8 men and 17 women, to assess the potential for contact sensitisation of a mascara, which contained 0.1 % hexamethylenetetramine. A pre-test using 25 adults who got an occlusive patch of test material applied to the volar aspect of the forearm for 48 hours showed that the test material was non-irritating. For induction, 0.3 g of test material was applied under an occlusive patch to the volar aspect of the forearm and was covered with a 15 mm aluminium chamber for five 48-hour periods. Sodium lauryl sulphate, 1 %, was used during the induction because the test material was found to be non-irritating during the pre-test. Following a 10-day non-treatment period a challenge was performed by applying an occlusive patch to a new site for 48 hours using the same procedure as for the induction. However, this time a 5 % aqueous solution of sodium lauryl sulphate was used. Observations were made upon removal of the patch and after 24 hours. No signs of sensitisation were observed following the challenge. (Ivy Research Laboratories Inc. 1980 – quoted from CIR 1992).

Hexamethylenetetramine has been reported to cause allergic eczema in rubber workers. After exposure to hot rubber containing 0.1 % hexamethylenetetramine, 60 workers suffered from acute dermatitis with itching and redness of exposed skin. Removal of the chemical from all rubber stock prevented further cases. (Cronin 1924 – quoted from IUCLID 2000).

Seven (2.0 %) out of 357 tested patients remitted to an occupational dermatology clinic in Finland during a 6-year period had a positive allergic patch test reaction to 2 % hexamethylenetetramine. In the same period 5.8 % (82/1414) were positive to 1 % formaldehyde. The study is not stating whether any cross-reaction between hexamethylenetetramine and formaldehyde was observed. (Kanerva et al. 1999).

A similar result was reported in another study where 1.9 % of 309 tested patients had a positive reaction to 2 % hexamethylenetetramine and 6.2-8.7 % of tested patients had a positive reaction to 1-2 % formaldehyde (Holness & Nethercott 1997).

One case report exists of a worker who had a positive patch test to hexamethylenetetramine but who tested negative to formaldehyde. The worker developed itchy eruptions, which were aggravated by sweating, on his hands, neck, and shoulders. When tested, negative results were obtained for a range of chemicals tested in open patch tests on the upper arm for 20 minutes. In closed patch tests, chemicals were applied for 24 hours and readings were taken 1 and 24 hours after removal. Only 1 % hexamethylenetetramine in petrolatum produced a positive reaction with erythema and papules. For 2 % aqueous formaldehyde a questionable positive result was observed at the 48-hour reading but at the 72-hour reading the result was negative. Eight months later a retest was performed using hexamethylenetetramine and formaldehyde. At this test a negative result was obtained for formaldehyde. As a negative control, hexamethylenetetramine tested negative in 9 colleagues. Positive controls for formaldehyde were performed using formaldehyde-sensitised patients. (Hayakawa et al. 1988 – quoted from CIR 1992, and Loeper & Berzins 1995).

Hexamethylenetetramine has been reported as allergenic not only in occupational use but also in contact with finished rubber objects (Fregert 1981 – quoted from Loeper & Berzins 1995).

Patients sensitised by external exposure to formaldehyde have experienced dermatitis when exposed dermally to hexamethylenetetramine in rubber dress shields or when ingesting it as a drug (Fisher 1978, Sulzberger 1940 – both quoted from CIR 1992).
Positive patch test reactions have occurred in persons who were sensitised to formaldehyde when they were tested with hexamethylenetetramine (Fisher 1986 – quoted from Loeper and Berzins 1995).
Contact dermatitis was also observed when ethylenediamine sensitive persons were patch tested with hexamethylenetetramine (Balato et al. 1986 – quoted from Loeper & Berzins 1995).

See also the study performed by Merget et al. (1999) described in section 3.2.1 Inhalation.

26.2.4   Other routes

No data have been found.

26.3   Toxicity to reproduction

In a surveillance study conducted between 1985 and 1992 of Michigan Medicaid recipients involving 229,101 completed pregnancies, 209 newborns had been exposed to hexamethylenetetramine during the first trimester. Eight (3.8 %) major birth defects were observed. Nine major birth defects were expected. (Briggs et al. 1994 – quoted from HSDB 2000).

No congenital abnormalities were observed in the children of 3 women who had taken hexamethylenetetramine as well as 5 other drugs (choleinic sodium, phenolphthalein, papaverine HCL, methylhomatropine, and menthol) during the first two weeks of pregnancy (Siffel & Czeizel 1995).

26.4   Mutagenic and genotoxic effects

Urine samples were collected from 72 men, 44 of whom smoked, who worked in a tire plant where hexamethylenetetramine was a workplace pollutant. Twenty-three clerks, 16 of whom smoked, were used as controls. Urine concentrates equivalent to 10 ml of urine were tested for mutagenicity in the Ames test with metabolic activation in Salmonella typhimurium strains TA 98, TA100, and TA1535, and in a microtitre fluctuation test. A smoking related increase in mutagenicity was observed in strain TA98. Hexamethylenetetramine was not mutagenic in this study. (Crebelli et al. 1984, 1985 – quoted from CIR 1992 and Loeper & Berzins 1995).

26.5   Carcinogenic effects

An increase in intestinal, lung, bladder, and skin cancer as well as leukaemia has been reported among 13570 men who had worked for at least 5 years at one company in rubber making, especially in areas where the rubber ingredients were mixed and compounded. Hexamethylenetetramine was used as an accelerator in the process in this plant. However, the mixture consisted of several antioxidants and other accelerators with suspected carcinogenic properties. (Monson & Fine 1978 – quoted from IUCLID 2000 and Loeper & Berzins 1995).

The mortality from cancer was increased in a cohort of 632 Danish moulders, mainly because of excess deaths from bladder cancer. Hexamethylenetetramine is added as a catalyst to some synthetic resin moulds. However, the workers have been exposed to a mixture of chemicals including carbon monoxide, nitrogen oxides, hydrogen cyanide, ammonia, amines, aldehydes, phenols, benzene, benzoic acid, toluene, cresols, methane, ethylene, acetylene, and various polycyclic aromatic hydrocarbons (of which some are carcinogenic). (Hansen 1991).

27   Animal toxicity

27.1   Single dose toxicity

27.1.1   Inhalation

No data have been found.

27.1.2   Oral intake

The oral LD₅₀-values reported for hexamethylenetetramine range from 9200 to higher than 20000 mg/kg b.w. for rats (3 values reported) and 1853 mg/kg b.w. for mice (CIR 1992, IUCLID 2000, JECFA 1974, Loeper & Berzins 1995).

27.1.3   Dermal contact

No data have been found.

27.1.4   Other routes

The LD₅₀-values reported for hexamethylenetetramine range from 215 mg/kg b.w. for mice injected subcutaneously and from 9200 to higher than 10000 mg/kg b.w. for rats injected intravenously. In mice, toxicity symptoms observed after intraperitoneal and subcutaneous administrations were trembling, weakness of the hind quarters, and terminal convulsions. (CIR 1992, IUCLID 2000, JECFA 1974, Loeper & Berzins 1995).

27.2   Repeated dose toxicity

27.2.1   Inhalation

No data have been found.

27.2.2   Oral intake

Groups of 27-102 of each sex of three different strains of mice (CTM, C3Hf/Dp and SWR/Dp) and groups of 48 of each sex of Wistar rats received 0 or 1.0 % hexamethylenetetramine in their drinking water. The calculated daily intake equal to 1 % was 2500 mg/kg b.w. for mice and 1500-2500 mg/kg b.w. for rats according to Loeper & Berzins 1995. The mice were treated for 60 weeks and the rats for 104 weeks. One group of 50 CTM mice per sex was given 0.5 % hexamethylenetetramine for 60 weeks, and another group of 29-50 of each sex received 5 % hexamethylenetetramine for 30 weeks. One group of 12 rats of each sex were given 5 % hexamethylenetetramine for 2 weeks. After the termination of treatment, the animals were observed for the remainder of their lifetimes. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. A yellow discoloration of the furs of treated rats (but not of treated mice) was observed. The SWR mice that received 1 % hexamethylenetetramine and the CTM mice that received 5 % hexamethylenetetramine had a slight retardation of growth. A slight reduction in survival was observed for the CTM mice dosed with 5 % hexamethylenetetramine. The same dose caused 50 % mortality in the rats. Rats from the 5 % group that did not die recovered rapidly and did not have any lasting ill effects. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 5 or 15 BD II rats of each sex were administered 400 mg per day (equivalent to 1000 mg/kg b.w. per day assuming a rat weight of 400 g) of hexamethylenetetramine by gavage for 90 or 333 days. The only substance related effect was a yellow discolouration of the fur. No macroscopic changes in organ histology or in body weights could be detected. (Brendel 1964 – quoted from CIR 1992, IUCLID 2000, JECFA 1974 and Loeper & Berzins 1995).

Groups of 16 Wistar rats of each sex were fed a standard diet containing either 0 or 0.16 % (equal to an average intake of 0 or 100 mg/kg b.w. per day according to CIR 1992) of hexamethylenetetramine for life. A yellow staining of the hairs of the perineum was observed in one male and three females in the test group. The average life span was 6-9 % longer for controls than for the dosed animals. This difference was associated with slightly lower terminal body weights in the test group, especially for males. No significant differences in body weight, voluntary muscular activity, and relative organ weights of the liver, kidneys, adrenal glands or gonads were observed between the control and treatment group. The majority of deaths in controls as well as dosed rats were attributed to pneumonia. (Natvig et al. 1971 – quoted from CIR 1992and JECFA 1974).

The yellow discolouration of the fur of rats exposed to hexamethylenetetramine orally has been explained as a result of a reaction between formaldehyde present in the urine from treated rats and kynurenine which is a normal constituent of rat hair (Kewitz & Welsch 1966 – quoted from CIR 1992 and IUCLID 2000).

Groups of 2-3 cats of each sex were fed hexamethylenetetramine in the diet at doses of 0 or 50000 ppm (equal to 1250 mg/kg b.w. per day) for 2 years. One female in the dosed group died of a pyrogenic infection of the nasal cavity and paranasal sinuses in the twenty-third month. No differences between control and dosed cats were found in feed consumption, weight gain, appearance, or histology of tissues. (Kewitz 1966 – quoted from CIR 1992 and JECFA 1974).

27.2.3   Dermal contact

Groups of 6 male rabbits had 0 or 2 ml of a 0.20 % solution of hexamethylenetetramine in distilled water applied to the skin for 5 days a week for 6 weeks. The application was not under occlusive patches. General behaviour, hair growth, and weight gain of the rabbits was the same in the control and the test group. No skin irritation was observed. (COLIPA 1989 – quoted from CIR 1992).

Six male rabbits had 0.5 ml of a 0.20 % solution of hexamethylenetetramine in distilled water topically applied to both intact and abraded sites on the flanks for 24 hours under occlusive patches. The rabbits were observed for 72 hours. Slight skin irritation was observed. (COLIPA 1989 – quoted from CIR 1992).

Mild irritation was seen when a 5 % solution of a mixture (ingredients not specified) containing 40 % hexamethylenetetramine was placed on the skin of guinea pigs (DuPont Company 1976 - quoted from Trochimowicz et al. 1993).

27.2.3.1     Sensitisation

The sensitisation potential of hexamethylenetetramine was studied in groups of 5 Dunkin-Hartley albino guinea pigs per sex by performing a guinea pig maximisation test of Magnusson and Kligman. One group was a control group and another group was induced and challenged with hexamethylenetetramine in distilled water at a concentration of 0.20 %. No erythema or oedema was observed in treated animals. (COLIPA 1989 – quoted from CIR 1992).

The sensitisation potential of hexamethylenetetramine was studied in 20 guinea pigs by performing a guinea pig maximisation test. The guinea pigs were induced by intradermal application of 0.1 ml of a 30 % solution of hexamethylenetetramine on the first day followed by epidermal application on day 8 of 0.5 g, which were covered for 48 hours. The guinea pigs were challenged on day 22 by epidermal application of 0.2 ml of a 50 % solution of hexamethylenetetramine, which was covered for 24 hours. Reactions were scored on day 24 and 25. Seventeen of the guinea pigs reacted positive with erythema and swelling. (Degussa AG 1985 – quoted from IUCLID 2000).

The sensitisation potential of AH26 (a root canal filling material containing 25 % hexamethylenetetramine, 10 % silver powder, 60 % bismuth oxide and 5 % titanium dioxide) was studied in groups of 10 female Dunkin-Hartley albino guinea pigs by performing a guinea pig maximisation test of Magnusson and Kligman. One group was a control group and another group was induced intracutaneously with AH26 in saline at a powder/saline ratio of 1.75/1 and percutaneously with AH26 in petrolatum. The animals were challenged with AH26 in petrolatum, 1/10 (w/w). Nine of ten animals were sensitised to AH26. (Kallus et al. 1983 – quoted from CIR 1992 and Loeper & Berzins 1995).

27.2.4   Other routes

27.2.4.1     Intramuscular injection

Groups of 5 BD II rats of each sex were administered 200 mg per day (equivalent to 500 mg/kg b.w. per day assuming a rat weight of 400 g) of hexamethylenetetramine by intramuscular injection for 90 days. The only substance related effect was a yellow discolouration of the fur. No macroscopic changes in organ histology or in body weights could be detected. (Brendel 1964 – quoted from CIR 1992, IUCLID 2000, JECFA 1974 and Loeper & Berzins 1995).

27.2.4.2     Ocular irritation

Six male rabbits had 0.1 ml of a 0.20 % solution of hexamethylenetetramine in distilled water applied once to the conjunctival sac of the eyes. The solution was not rinsed from the eyes. Conjunctival irritancy, iris alterations, or corneal lesions were not observed following dosing. (COLIPA 1989 – quoted from CIR 1992).

Nine New Zealand White albino rabbits had 100 mg of mascara containing 0.1 % hexamethylenetetramine applied to the conjunctival sac of one eye. The eyes of 3 rabbits of each sex were not rinsed after application of the test material while the eyes of 3 male rabbits were rinsed with deionised water 30 seconds after application. The treated eyes of all nine animals were examined 1, 2, 3, 4, and 7 days after application.  The mascara was judged as mildly irritant for unrinsed eyes and non-irritant for rinsed eyes. (Stillmeadow, Inc., 1980 – quoted from CIR 1992).

27.3   Toxicity to reproduction

27.3.1   Inhalation

No data have been found.

27.3.2   Oral intake

Groups of 9-11 female Beagle dogs were fed 0, 600 or 1250 ppm (equal to about 0, 15 or 31 mg/kg b.w. per day based on an average body weight of 12 kg according to JECFA 1974) hexamethylenetetramine on days 4 to 56 after mating. Pregnancy rate, weight gain during pregnancy, length of gestation and litter size was not affected by treatment. In the 1250 ppm group, the percentage of stillborn pups was slightly increased, and the weight gain and the survival to weaning of the pups were slightly impaired. No congenital malformations were observed in any of the 264 live-born and 20 stillborn pups. Some of the pups were kept for observation and a few of them were being used for breeding. After 2 years, all were normal in behaviour, motility, and muscular coordination. Neither the bitches nor the pups had any abnormalities or reproductive disorders. (Hurni & Ohder 1973 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 16 Wistar rats of each sex were fed a standard diet containing either 0 or 0.16 % (equal to an average intake of 0 or 100 mg/kg b.w. per day according to CIR 1992) of hexamethylenetetramine for life. After 3 months of administration, the males and females of the control and test groups were mated. No significant difference was observed in fertility. In both groups, 16 male and 16 female offspring were chosen and fed the same diet as their parents from the time of weaning to natural death. No difference was observed in the relative organ or body weights or in voluntary muscular activity between test and control animals. A yellow staining of the hairs of the perineum was observed in some of the dosed rats. (Natvig et al. 1971 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 12 female and 6 male Wistar rats were given drinking water containing either 0 or 1 % (equal to about 0 or 2000 mg/kg b.w. per day according to Loeper & Berzins 1995) of hexamethylenetetramine starting two weeks before mating. The females were treated during both pregnancy and lactation. No difference were observed between the treated and control groups in regard to fertility, litter size, and malformations. From the pups that were born, 24 of each sex were given 0 or 1 % hexamethylenetetramine in the drinking water until 20 weeks of age. Up to week 9 of age for males and week 20 of age for females, the body weights of the treated animals were significantly lower than the controls. At necropsy, no differences were observed in respect to organ weight, and gross and microscopic changes. (Della Porta et al. 1966, 1970 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 80, 80, or 245 rats were given 0, 5, or 50 mg/kg b.w. per day of hexamethylenetetramine in their drinking water in a five generation study that lasted 3.5 years. At half-yearly intervals starting at 1.5 years, animals (including pregnant dams) were selected from each group to be used for study of lesions. From that time on, no lesions due to hexamethylenetetramine were found in test animals, foetuses, or placenta. No further details were given. (Malorny 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 10 rats of each sex were fed 0, 400, 800, or 1600 ppm (equivalent to 0, 20, 40, or 80 mg/kg b.w. per day) of hexamethylenetetramine for 2 years. The ten pairs were mated at the age of 20, 28, and 35 weeks. No differences were observed in growth rate, survival, reproduction, offspring viability, or lesions between treated and control groups. No further details were given. (Berglund 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 1-4 mongrel dogs of each sex were fed 0 or 1250-1875 ppm (equal to about 0 or 94-141 mg/kg b.w. per day according to IUCLID 2000) of hexamethylenetetramine for 32 months. Groups of 2-3 pups of each sex from litters of these dogs were fed 0 or 1250 ppm of hexamethylenetetramine for 22 months. Subsequently, the test animals were fed control diet and the controls were fed the 1250 ppm diet for 1 year. No differences were observed in feed consumption, growth, reproduction, litter number, or litter weight between treated and control groups. Of the 30 litters in the dosed group, approximately 20 had a few stillborn and cannibalised pups, and 5 pups were born with abnormalities. Of the 16 litters in the control group, 1 had stillborn pups, and no pups had malformations. No further details were given. (Kewitz 1966 – quoted from CIR 1992, IUCLID 2000 and JECFA 1974).

27.3.3   Dermal contact

No data have been found.

27.3.4   Other routes

No data have been found.

27.4   Mutagenic and genotoxic effects

27.4.1   In vitro studies

In four studies, hexamethylenetetramine was not mutagenic when tested in 2-5 strains (TA98, TA100, TA1535, TA1537 and TA1538) of Salmonella typhimurium in Ames tests with and without metabolic activation systems (Crebelli et al. 1984, 1985, Orstavik & Hongslo 1984, Andrews et al. 1980 – all quoted from CIR 1992).
In one study, hexamethylenetetramine was reported to be mutagenic towards strain TA98 and TA100 (but not towards strain TA1535, TA1537 and TA1538) without metabolic activation systems (Shimuzu et al. 1985 – quoted from Loeper & Berzins 1995).
After in vitro nitrosation hexamethylenetetramine has been reported to be mutagenic toward strain TA98 and TA100 with and without metabolic activation systems (Crebelli et al. 1984, Andrews et al. 1980 – both quoted from CIR 1992).

Hexamethylenetetramine induced a DNA-damaging effect in the recombination assay using the spores of Bacillus subtilis strains H17 and M45 when tested in the absence of a metabolic activation system. The DNA-damaging effect was decreased in the presence of a metabolic activation system. (Ueno & Ishizaki 1984 – quoted from Loeper & Berzins 1995).Hexamethylenetetramine inhibited the growth of a mutant of Escherichia coli, which is deficient in DNA polymerase. The inhibition of cultures was concluded to be dependent on the degradation of hexamethylenetetramine to formaldehyde. (Fluck et al. 1976, Gillner 1987 – both quoted from Loeper & Berzins 1995).

The number of transformations was dose-dependently and significantly increased when hexamethylenetetramine was tested in the Styles‘ cell transformation assay using baby hamster kidney BHK-21/cl.13 cells in the concentration range of 1 to 10000 mg/ml. The transformation activity was observed at a non-toxic or very weak toxic concentration and it was not dependent on a metabolic activation system. As a comparison, an equal number of transformations and equivalent toxicity was observed with 20 mg formaldehyde/ml and 1000 mg hexamethylenetetramine/ml. (Plesner & Hansen 1983 – quoted from CIR 1992 and Loeper & Berzins 1995).

Hexamethylenetetramine was negative in the mouse lymphoma assay with L5178Y TK+/- cells (Dooley et al. 1985 – quoted from IUCLID 2000).

Hexamethylenetetramine was negative for chromosomal aberrations in human leucocytes but positive in HeLa cells (Roehrborn & Vogel 1967, Balderman & Roehrborn 1967 – both quoted from IUCLID 2000).

27.4.2   In vivo studies

Hexamethylenetetramine caused mutations in larval spermatocytes of Drosophila melanogaster at concentrations, which were higher than those used in medical therapy (concentrations were not stated but for medical therapy doses of 1 g twice daily is recommended according to Lægemiddelkataloget (2001)). The causative agent of the mutagenic effect was formic acid, which was present as an impurity in formaldehyde. (Auerbach 1951, 1977, Stumm-Tegethoff 1964, Nafei & Auerbach 1964 – all quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Hexamethylenetetramine induced dominant lethal mutations in C3H mice which were administered the compound intraperitoneally or orally at a dose of 25000 mg/kg b.w. Doses of 800 to 10000 mg/kg b.w. did not cause dominant lethal mutations. (Balderman et al. 1967, Röhrborn & Vogel 1967 – both quoted from Loeper & Berzins 1995).

In a micronucleus assay in C3H mouse, no clastogenic activity was observed in bone marrow after oral administration of approximately 69, 206 or 618 mg/kg b.w. of hexamethylenetetramine for 1 or 5 days. Bone marrow samples were collected 6, 12, and 24 hours after application of the acute dose and 6 hours after the last exposure in the 5-day study. (Vujosevic et al. 1986 – quoted from IUCLID 2000).

27.5   Carcinogenic effects

27.5.1   Inhalation

No data have been found.

27.5.2   Oral intake

Groups of 7-15 Wistar rats of each sex in three successive generations received 1% hexamethylenetetramine (equal to about 2000 mg/kg b.w. per day according to Loeper & Berzins 1995) in the drinking water up to the age of 40 weeks in the F1 and F2 generations and up to 20 weeks in the F3 generation. Another group of rats received 2 % hexamethylenetetramine in the drinking water, and 16 offspring of each sex were treated for 59 weeks with the same dose. A group of 48 rats of each sex served as a control group. All groups were observed for 2 years after exposure. No evidence of carcinogenicity was found in any of the hexamethylenetetramine treated groups of rats. (Della Porta et al. 1970 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 27-102 of each sex of three different strains of mice (CTM, C3Hf/Dp and SWR/Dp) and groups of 48 of each sex of Wistar rats received 0 or 1.0 % hexamethylenetetramine in their drinking water. The calculated daily intake equal to 1 % was 2500 mg/kg b.w. for mice and 1500-2500 mg/kg b.w. for rats according to Loeper & Berzins 1995. The mice were treated for 60 weeks and the rats for 104 weeks. One group of 50 CTM mice per sex was given 0.5 % hexamethylenetetramine for 60 weeks, and another group of 29-50 of each sex received 5 % hexamethylenetetramine for 30 weeks. One group of 12 rats of each sex were given 5 % hexamethylenetetramine for 2 weeks. After the termination of treatment, the animals were observed for the remainder of their lifetimes. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. No evidence of substance related carcinogenicity was found in any of the hexamethylenetetramine treated groups of animals. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

A total of 80, 80, or 245 rats were given 0, 5, or 50 mg/kg b.w. per day of hexamethylenetetramine in their drinking water in a five generation study that lasted 3.5 years. At half-yearly intervals starting at 1.5 years, animals (including pregnant dams) were selected from each group to be used for pathological studies. From that time on, no changes due to hexamethylenetetramine were found in test animals, foetuses, or placenta. Tumours were observed in three of 48 animals in the high dose group. No further details were given. (Malorny 1966 – quoted from CIR 1992 and JECFA 1974).

Groups of 30 NMRI/Han albino mice of each sex were fed 0 or 1 % of hexamethylenetetramine (equivalent to 1500 mg/kg b.w. per day) in the feed for 2 years. Twenty neoplasms were found in the dosed group and 11 in the control group. With the exception of one control male and two dosed males, all neoplasms occurred in females. Most of the malignant tumours were subcutaneous carcinomas and adenocarcinomas. The author concluded that the possibility of an increased tumour incidence effect by hexamethylenetetramine could not be ruled out. A further study was performed using groups of 50 female mice, which were administered hexamethylenetetramine at concentrations of 0, 0.1, 0.5, or 1 % in the diet. After 31 weeks, no difference in neoplasm incidence was observed between the groups. (Kewitz 1966 – quoted from CIR 1992 and JECFA 1974).

27.5.3   Dermal contact

Groups of 13 mice were treated daily by cutaneous application of chloroform or 10 % hexamethylenetetramine in chloroform for 300 days. No malignant tumours were found in any group. (Kewitz 1966 – quoted from CIR and JECFA 1974).

39-44 CTM mice of each sex and 20 Wistar rats of each sex were injected subcutaneously 5 times every other day with 5 g/kg b.w. of a 30 % solution of hexamethylenetetramine in water. The animals were observed for 100-104 weeks. Necropsy was performed on all animals, and tissues and lesions taken at necropsy were evaluated microscopically. No evidence of substance related carcinogenicity was found in any of the hexamethylenetetramine treated groups of animals. (Della Porta et al. 1968 – quoted from CIR 1992, JECFA 1974 and Loeper & Berzins 1995).

Groups of 7-15 albino rats of each sex were injected subcutaneously with 1 ml of a 40 % solution of hexamethylenetetramine or with sodium chloride or sucrose (negative controls) weekly for 1½ year. In the dosed rats, 1 injection site spindle-cell sarcoma, 1 distal spindle-cell sarcoma, 1 alveolar mammary carcinoma, 1 fibrosarcoma, and 2 benign tumours were seen. In the control group, 1 blastoma was found in a sodium chloride injected animal. (Kewitz 1966 – quoted from JECFA 1974).

27.5.4   Other routes

No data have been found.

28   Regulations

28.1   Ambient air

-

28.2   Drinking water

-

28.3   Food

JECFA has set an ADI (acceptable daily intake) of 0-0.15 mg/kg b.w. for hexamethylenetetramine (JECFA 1974).

Hexamethylenetetramine may be used as a preservative (E239) in Provolone cheese in some countries.
However, on the Danish market it may not be used as a food additive  (Positivlisten 2000).

28.4   Cosmetics

Hexamethylenetetramine may legally be used as a preservative in cosmetic products in Europe at a maximum concentration of 0.15%. It may also be used in other concentrations for other purposes in cosmetics. (MM 2000).

28.5   Drugs

Hexamethylenetetramine may be used as a drug for urinary infections (Lægemiddelkataloget 2001).

28.6   Soil

-

28.7   Occupational Exposure Limits

The occupational exposure limits in Norway, Sweden and Poland varies between 3 and 5 mg/m³ (RTECS 2000).

28.8   Classification

Hexamethylenetetramine is classified for flammability (F;R11 – highly flammable) and for sensitising properties (R42/43 – may cause sensitisation by inhalation and skin contact). (MM 2002).

28.9   IARC

-

28.10   US-EPA

-

29   Summary

29.1   Description

Hexamethylenetetramine occurs as hygroscopic and colourless crystals or as a white crystalline powder with no or mild ammonia odour. It has a low vapour pressure (0.004 mmHg) and a high water solubility (449 g/l).

29.2   Toxicokinetics

Hexamethylenetetramine is rapidly absorbed in humans following oral administration. It is distributed to various tissues and is detectable in e.g. amniotic fluid and milk. In slightly acidic environments such as in sweat, gastric fluid or urine, hexamethylenetetramine partially degrades to formaldehyde and ammonia. Upon contact with the skin, hexamethylenetetramine has also been reported to produce formic acid. In the gastric fluid, about 10-30 % of an orally administered dose of hexamethylenetetramine is degraded. The half-life for a formulation of hexamethylenetetramine in humans is about 4 hours. In the same study, more than 80 % of the oral dose of hexamethylenetetramine was excreted unchanged in the urine during the first 24 hours following administration.

29.3   Human toxicity

29.3.1   Single dose toxicity

Inflammation of the bladder and an increased concentration of nitrogen in the blood have been reported following an accidental ingestion of an overdose of hexamethylenetetramine-mandelate.

29.3.2   Repeated dose toxicity

Pulmonary or nasal symptoms (e.g. wheezy breathing, chest tightness, shortness of breath, couching, sneezing, rhinorrhoea, obstruction) have been reported in workers exposed to hexamethylenetetramine in a production plant, in the lacquer and plastics industries, in a tire manufacturing plant and in a foundry. In all of the workplaces, the workers were exposed to other chemicals (e.g. formaldehyde, ammonia, resorcinol, phenol, furfuryl alcohol, cyanides, epoxy resins, curatives) as well. Lung function measurements in one of the studies revealed significant reductions in expiratory flow rates at low lung volumes. In another study, an intracutaneous skin test with hexamethylenetetramine gave positive reactions in all workers and a provocative inhalation test with an aerosol of a lacquer product revealed allergic reactions from either the lungs, the nose or the skin.

Hexamethylenetetramine has been reported to cause allergic eczema and irritation of the skin and eyes in e.g. workers in the rubber, lacquer and plastics industry but also in people in contact with finished rubber objects. No signs of sensitisation were observed in a maximization test, which was performed in 25 adults with mascara, which contained 0.1 % hexamethylenetetramine. However, several patients have tested positive in allergic patch tests to 2 % hexamethylenetetramine. In the same studies three to four times as many patients tested positive to 1-2 % formaldehyde. Positive patch test reactions for hexamethylenetetramine have occurred in persons who were sensitised to formaldehyde or ethylenediamine. Case reports also exist of workers who had positive patch tests to hexamethylenetetramine but who tested negative to formaldehyde.

Adverse effects have been reported in less than 3.5 % of patients receiving hexamethylenetetramine and its salts orally as a drug. The most frequent adverse effect is gastrointestinal disturbances. Rarely, hypersensitivity reactions have occurred. Other adverse effects reported rarely are headache, dyspnoea, generalized oedema, tinnitus, muscle cramps, dysuria, and microscopic or gross haematuria.

29.3.3   Toxicity to reproduction

Only the expected number of congenital abnormalities was observed in more than 200 newborns that had been exposed to hexamethylenetetramine during the first trimester.

29.3.4   Mutagenic and genotoxic effects

Hexamethylenetetramine was not mutagenic in Ames test and in a microtitre fluctuation test with urine concentrates from 72 men who worked in a tire plant where hexamethylenetetramine was a workplace pollutant.

29.3.5   Carcinogenic effects

An increase in certain types of cancer has been reported among workers in a rubber making company and among Danish moulders. Both groups of workers have been exposed to hexamethylenetetramine but also to a mixture of other chemicals including some with suspected and/or confirmed carcinogenic properties.

29.4   Animal toxicity

29.4.1   Single dose toxicity

The oral LD₅₀-values reported for hexamethylenetetramine range from 9200 to higher than 20000 mg/kg b.w. for rats and 1853 mg/kg b.w. for mice.

29.4.2   Repeated dose toxicity

No adverse effects were noted in mice (CTM and C3Hf/Dp), rats (Wistar and BD II) and cats dosed with up to 2500, 1500 or 1250 mg/kg b.w., respectively, of hexamethylenetetramine in their drinking water, in their feed or by gavage for 13-104 weeks. A yellow discoloration of the fur of treated rats (but not of treated mice and cats) was observed. The discolouration has been explained as a result of a reaction between formaldehyde present in the urine from treated rats and kynurenine, which is a normal constituent of rat hair. SWR/Dp mice that received 2500 mg/kg b.w. and CTM mice that received 12500 mg/kg b.w. had a slight retardation of growth. A slight reduction in survival was also observed for CTM mice dosed with 12500 mg/kg b.w. A dose of 7500 mg/kg b.w. caused 50 % mortality in the rats.

No to mild skin irritation was observed in rabbits and guinea pigs exposed dermally to hexamethylenetetramine in concentrations of 0.2-2 %. A 0.2 % solution of hexamethylenetetramine was non-irritant to the rabbit eye while a mascara containing 0.1 % hexamethylenetetramine was mildly irritant for unrinsed rabbit eyes.

Hexamethylenetetramine caused skin sensitisation in 17/20 of guinea pigs induced with a 30 % solution and challenged with a 50 % solution in a maximisation test. No skin sensitisation was observed in another maximisation test where hexamethylenetetramine was used in a concentration of 0.2 %.

29.4.3   Toxicity to reproduction

No substance-related reproductive or developmental effects were seen in 4 studies where rats were fed or given hexamethylenetetramine in their drinking water in doses up to about 2000 mg/kg b.w. However, a significant lower body weight was found in pups, which were born of dams treated during pregnancy and lactation with 2000 mg/kg b.w. of hexamethylenetetramine and, which in addition were treated with the same dose of the chemical for the first 20 weeks of age.

In dogs fed about 15 mg/kg b.w. of hexamethylenetetramine, no substance-related reproductive or developmental effects were noted. In dogs fed about 31 mg/kg b.w. of hexamethylenetetramine, the percentage of stillborn pups was slightly increased, and the weight gain and the survival to weaning of the pups were slightly impaired. In another study where dogs were fed about 94 mg/kg b.w. of hexamethylenetetramine, a few pups were born with abnormalities.

29.4.4   Mutagenic and genotoxic effects

In most studies, hexamethylenetetramine was not mutagenic in Ames test with and without metabolic activation systems. It was negative in a mouse lymphoma assay and for chromosomal aberrations in human leucocytes. Hexamethylenetetramine was positive for chromosomal aberrations in HeLa cells, for DNA-damage in a recombination assay with spores of Bacillus subtilis, for inhibition of growth of a mutant of Escherichia coli, and for an increased number of transformations in the Styles‘ cell transformation assay with baby hamster kidney cells. In the cell transformation assay an equal number of transformations and equivalent toxicity was observed with a 50 times smaller dose of formaldehyde.

Hexamethylenetetramine was negative in vivo for clastogenic activity in the bone marrow in a micronucleus assay in C3H mouse. It was positive for dominant lethal mutations in C3H mice orally administered a very high dose but negative at doses below 10000 mg/kg b.w. Hexamethylenetetramine caused mutations in larval spermatocytes of Drosophila melanogaster at concentrations, which were higher than those used in medical therapy.

29.4.5   Carcinogenic effects

No evidence of substance-related carcinogenicity was found in 4 different studies where mice or rats were fed or given hexamethylenetetramine in their drinking water in doses up to about 2500 mg/kg b.w. for up to 2 years or in 2 studies where mice or rats were exposed dermally to hexamethylenetetramine in a concentration of up to 30 % for up to 2 years. In rats injected subcutaneously with a 40 % solution of hexamethylenetetramine for 1½ year, more tumours were found than in the control group. 

30   Evaluation

The critical effect of hexamethylenetetramine is the sensitisation it may cause following exposure by inhalation or skin contact. Skin sensitisation has been observed in a guinea pig maximisation test as well as in humans in several patch tests. Several workers with mixed exposures, including exposure to hexamethylenetetramine, have reported allergic eczema and/or asthma-like symptoms. In one study, reductions in expiratory flow rates at low lung volumes was reported. In another study, intracutaneous skin tests with hexamethylenetetramine gave positive reactions in all workers and a provocative inhalation test with an aerosol of a lacquer product showed allergic reactions from either the lungs, the nose or the skin.
It is a cause of concern that no inhalation studies with hexamethylenetetramine have been performed in animals since it because of the mixed exposures is unclear from the human studies whether the symptoms from the lungs are caused by hexamethylenetetramine.

No systemic effects following inhalation of hexamethylenetetramine have been reported in humans. No toxicokinetic inhalation studies have been performed. However, due to the high water solubility of hexamethylenetetramine it is most likely absorbed following inhalation.

A low order of acute oral toxicity has been observed in experimental animals. 

Irritation of the skin and eyes has been reported in workers with mixed exposures, including exposure to hexamethylenetetramine, as well as in laboratory animals.

Only few adverse effects (of which the most frequent is gastrointestinal disturbances) have been reported in patients receiving hexamethylenetetramine and its salts orally as a drug.
In laboratory animals exposed orally to high doses of hexamethylenetetramine, only slight growth retardation and survival was observed.

No substance-related reproductive or developmental effects were observed in rats exposed to high doses (up to 2000 mg/kg b.w.) of hexamethylenetetramine in their drinking water. Dogs seemed more sensitive since some developmental effects (increased percentage of stillborn pups, decreased weight gain and survival to weaning of pups) were observed in dogs fed about 31 mg/kg b.w. of hexamethylenetetramine. However, at 15 mg/kg b.w. no developmental effects were noted.
In humans, only the expected number of congenital abnormalities was observed in more than 200 newborns that had been exposed to hexamethylenetetramine during the first trimester.

Both positive and negative results were obtained in assays for mutagenicity and genotoxicity of hexamethylenetetramine. However, in the in vivo assays, positive results were only observed when the administered doses were very high.

Oral and dermal carcinogenicity studies in rats and mice exposed to high doses of hexamethylenetetramine revealed no evidence of substance-related tumours. 

Some of the effects observed following exposure to hexamethylenetetramine may be due to its degradation products, e.g. formaldehyde, ammonia, and formic acid, which may be formed in slightly acidic environments. Formaldehyde is known to be a strong sensitising agent to the skin and is, according to the EU classification criteria, classified as such (R43).

31   References

ChemFinder (2001). Hexamethylenetetramine. Http://www.chemfinder.com

CIR (1992). Final report on the safety assessment of methenamine. Twentieth report of the Cosmetic Ingredient Review expert panel. J Am Coll Toxicol 11, 531-558.

Dreyfors JM, Jones SB and Sayed Y (1989). Hexamethylenetetramine: A review. Am Ind Hyg Assoc J 50, 579-585.

Gamble JF, McMichael AJ, Williams T and Battigelli M (1976). Respiratory function and symptoms: an environmental-epidemiological study of rubber workers exposed to a phenolformaldehyde type resin. Am Ind Hyg Assoc J 37, 499-513.

Gelfand HH (1963). Respiratory allergy due to chemical compounds encountered in the rubber, lacquer, shellac, and beauty culture industries. J Allergy 34, 374-381.

Hansen ES (1991). Cancer mortality among Danish molders. Am J Industrial Med 20, 401-410. 

HSDB (2000). Methenamine. In: Hazardous Substances Data Base. Last revised: 10/2000.

Holness DL and Nethercott JR (1997). Results of patch testing with a special series of rubber allergens. Contact Dermatitis 36, 207-211.

ICSC (1993). Hexamethylenetetramine. International Chemical Safety Card. Http://www.inchem.org/documents/icsc/icsc/eics1228.htm

IUCLID (2000). Methenamine. In: International Uniform Chemical Information Database. Existing Chemicals 2000. ECB, JRC, Ispra.

JECFA (1974). Hexamethylenetetramine. In: Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers and thickening agents. WHO Food Additives Series 5.  Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). World Health Organization, Geneva, 63-74. Http://www.inchem.org/documents/jecfa/jecmono/v05je10.htm

Kanerva L, Jolanki R , Alanko K and Estlander T (1999). Patch test reactions to plastic and glue allergens. Acta Dermatol Venereologia 79, 296-300.

Loeper I and Berzins T (1995). Health effects of selected chemicals - volume 3. Hexamethylenetetraamine. Nord 28, 93-114.

Lægemiddelkataloget (2001). Methenamin. Http://www.lk-online.dk/

Merck Index (1996). Methenamine. 12^th. Ed., Rahway, new jersey, Merck & Co., Inc., 1021.

Merget R, Topcu M, Friese K, Vormberg R, Fuchs T, Raulf HM and Breitstadt R (1999). A cross-sectional study of workers in the chemical industry with occupational exposure to hexamethylenetetramine. Int Arch Occup Environ Health 72, 533-538.

MM (2000). Miljø- og Energiministeriets bekendtgørelse nr. 594 af 6. juni 2000 om kosmetiske produkter.

MM (2002). The Statutory Order from the Ministry of the Environment no. 439 of June 3, 2002, on the List of Chemical Substances.

Positivlisten (2000). Http://www.foedevaredirektoratet.dk/publikationer/publikationer/publikationer/positiv2000/index.html

RTECS (2000). Hexamethylenetetramine. In: Registry of Toxic Effects of Chemical Substances. Database quest, last revised: 10/2000.

Siffel C and Czeizel AE (1995). Study of developmental abnormalities and deaths after human zygote exposure. Mutat Res 334, 293-300.

Trochimowicz HJ et al. (1993). Hexamethylenetetramine. In: Heterocyclic and miscellaneous nitrogen compounds. Patty’s Industrial Hygiene and Toxicology. Vol. 2E, 4^th ed., eds. Clayton GD and Clayton FE, John Wiley & Sons Inc. NY.

Appendix 5: 1-Methyl-1,2-ethanediyl dioleate  
32   General description

32.1   Identity

Molecular formula:          C₃₉H₇₂O₄

Structural formula:          

CH₃-(CH₂)₇-CH:CH-(CH₂)₇-COOCH₂-(CH-CH₃)-COO-(CH₂)₇-CH:CH-(CH₂)₇-CH₃

Molecular weight:            605.0

CAS-no.:                        105-62-4

Synonyms:                       1,2-Bisoleoyloxypropane
                                       1,2-Dioleoylooxypropane
                                       1-Methyl-1,2-ethanediyl 9-octadecenoate
                                       9-Octadecenoic acid, 1-methyl-1,2-ethanediyl ester
                                       Octadec-9-enoic acid, 1-methyl-2-octadec-9-enoyloxyethyl ester
                                       9-Octadecenoic acid, 1,3-propanediyl ester
                                       Oleic acid, propylene ester
                                       1,2-Propanediol dioleate
                                       1,2-Propylene glycol dioleate
                                        Propylene glycol dioleate        

32.2   Physical / chemical properties

Description:                     1-Methyl-1,2-ethanediyl dioleate is a liquid. It is a diester of propylene glycol and the fatty acid, oleic acid.

Melting point:                  -

Boiling point:                   -

Density:                          -

Vapour pressure:             -

Concentration of

saturated vapours:           -

Conversion factor:           1 ppm =  25.2 mg/m³       (at 20°C and 760 mmHg)

                                       1 mg/m³ =  0.040 ppm

Solubility:                        -

References:                     IUCLID (2000), Johnson (1999), TSCA (2000).

33   Toxicokinetics

No data have been found.

34   Human toxicity

Vapour of heated 1-methyl-1,2-ethanediyl dioleate can cause irritation when inhaled (Unichema International – quoted from IUCLID 2000).

No other data have been found.

 35   Animal toxicity

No data have been found.

36   Regulations

Propylene glycol esters have been used as emulsifiers in foods and pharmaceuticals. The Federal Drug Administration (FDA) in the US has determined that propylene glycol mono- and diesters of edible fats or fatty acids or oleic acid can be used safely in food in amounts not in excess of that reasonably required to produce their intended effect. The esters can also be used in food contact materials. (Rosen 1978 – quoted from Johnson 1999).

1-Methyl-1,2-ethanediyl dioleate is used in cosmetics (Johnson 1999). It is not mentioned in the statutory order on cosmetics, which means that it legally can be used in cosmetics in Europe without any limitations except as a colouring agent, a preservative, or a UV-filter (MM 2000).

37   Summary

1-Methyl-1,2-ethanediyl dioleate is a diester of propylene glycol and the fatty acid, oleic acid. It is a liquid that is used e.g., in foods and pharmaceuticals as an emulsifier and in cosmetics.

Vapour of heated 1-methyl-1,2-ethanediyl dioleate can cause irritation in humans when inhaled.

38   Evaluation

No toxicological studies regarding effects following exposure to 1-methyl-1,2-ethanediyl dioleate have been found.

Only one reference to 1-methyl-1,2-ethanediyl dioleate has been found in which is was stated that vapour of heated 1-methyl-1,2-ethanediyl dioleate can cause irritation in humans when inhaled. This study is of limited value because of the lack of information on exposure levels and duration. In addition, the irritation might be caused by degradation products of 1-methyl-1,2-ethanediyl dioleate formed by the heating of the substance.

No useful toxicological data regarding 1-methyl-1,2-ethanediyl dioleate are therefore available for evaluation.

39   References

IUCLID (2000).1-Methyl-1,2-ethanediyl dioleate. In: International Uniform Chemical Information Database. Existing Chemicals 2000. ECB, JRC, Ispra.

Johnson W (1999). Final report on the safety assessment of propylene glycol (PG) dicaprylate, PG diaprylate/dicaprate, PG dicocoate, PG dipelargonate, PG isostearate, PG laurate, PG myristate, PG oleate, PG oleate SE, PG diolate, PG dicaprate, PG diisostearate, and PG dilaurate. Int J Toxicol 18 (Suppl 2), 35-52.

MM (2000). Miljø- og Energiministeriets bekendtgørelse nr. 594 af 6 . juni 2000 om kosmetiske produkter.

TSCA (2000). 9-Octadecenoic acid (9Z)-, 1-methyl-1,2-ethandiyl ester. In: Toxic Substances Control Act. Database quest, last revised: 8/2000.

Appendix 6: Isopropyl myristate   
40   General description

40.1   Identity

Molecular formula:          C₁₇H₃₄O₂

Structural formula:          

Molecular weight:            270.5

CAS-no.:                        110-27-0

Synonyms:                      Isopropyl tetradecanoate

                                      Myristic acid, isopropyl ester

                                      Tetradecanoic acid, isopropyl ester

                                      Tetradecanoic acid 1-methylethyl ester

40.2    Physical / chemical properties

Description:                    Colourless and practically odourless liquid with a low viscosity, good penetration and spreading properties, and a non-oily feel.

                                      The commercial product usually contains small amounts of esters of palmitic and other saturated fatty acids.

Melting point:                  Approximately 0-3°C

Boiling point:                   Decomposes at 208°C

Density:                          0.85 g/ml (at 20°C)

Vapour pressure:             9.35 x 10^-5 mmHg (1.25 x 10^-2 Pa) (at 25°C)

Concentration of

saturated vapours:           0.12 ppm (= 1.3 mg/m³)(at 25°C and 760 mmHg)

Conversion factor:           1 ppm = 11.2 mg/m³        (at 20°C and 760 mmHg)

                                      1 mg/m³ =  8.9 x 10^-2 ppm

Solubility:                        Practically insoluble in water, glycerol, sorbitan, and propylene glycol. Soluble in castor oil, cottonseed oil, acetone, chloroform, ethyl acetate, ethanol, toluene, and mineral oil.

References:                    BIBRA (1988), ChemFinder (2001), CIR (1982), HSDB (2000), IUCLID (2000), Merck Index (1996).

41  Toxicokinetics

41.1   Absorption, distribution and elimination

Four monkeys were exposed for 5 seconds to the aerosols of an antiperspirant spray containing C¹⁴-labelled isopropyl myristate (the dose was not stated). Labelled material was mainly found in the lungs. Very little systemic absorption (0.25 %) occurred. About 85 % of the absorbed dose was eliminated in 24 hours, mainly as exhaled carbon dioxide. (Finkelstein & Wulf 1974 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

Whole body auto-radiography of hairless mice showed no visible penetration into the skin or organs of isopropyl myristate, whereas microautoradiography of angora rabbits and guinea pigs showed local penetration following dermal application. Whole body auto-radiography also revealed that isopropyl myristate injected subcutaneously into mice was distributed into almost all organs. (Suzuki et al. 1978 – quoted from IUCLID 2000).

41.2   Mode of action

No information has been found.

41.3   Influence on skin permeability of other chemicals

Isopropyl myristate enhanced the in vitro penetration rate of the nitrosamine N-nitrosodiethanolamine through human abdominal skin at 200 times the rate for water and 344 times the rate for propylene glycol (Bronaugh et al. 1981 – quoted from BIBRA 1988 and IUCLID 2000). It also increased the rate of benzyl alcohol penetration (Barry et al. 1985 – quoted from BIBRA 1988). Betamethasone 17-benzoate penetration of intact human forearm skin was increased by isopropyl myristate (Pepler et al. 1971 – quoted from CIR 1982). In an in vivo study, dexamethasone penetrated the stripped forearm skin (but not the intact skin) of nine humans seven times better when in gelled isopropyl myristate as compared with petrolatum (Dempski et al. 1969 – quoted from BIBRA 1988 and CIR 1982).  

The dermal absorption of ethanol and butanol through hairless mouse skin in vitro was 35 or 3 times, respectively, greater when isopropyl myristate was used as a vehicle compared to water (Garcia et al. 1980 – quoted from BIBRA 1988 and IUCLID 2000).

 42   Human toxicity

42.1   Single dose toxicity

42.1.1   Inhalation

No relevant data have been found.

42.1.2   Oral intake

No data have been found.

42.1.3   Dermal contact

See section 3.2 Repeated dose toxicity.

No evidence of phototoxicity was observed in 10 volunteers when a bath oil containing 43% isopropyl myristate was applied dermally at 4.25 mg/cm² under an occlusive patch for 24 hours. The application site was exposed to ultraviolet irradiation (UV-range A) from a solar simulator after 6 and 24 hours. (CTFA 1978 – quoted from BIBRA 1988 and CIR 1982).

42.1.4   Other routes

No data have been found.

42.2   Repeated dose toxicity

42.2.1   Inhalation

No data have been found.

42.2.2   Oral intake

No data have been found.

42.2.3   Dermal contact

42.2.3.1     Local effects

In studies with single or repeated dermal administration of undiluted isopropyl myristate, no or minimal skin irritation was seen. The studies lasted up to 21 days and were performed on groups of 5-200 subjects and included both covered and uncovered application. (Anon 1953, Avon 1971, 1975, Campbell & Bruce 1981, Hill top research 1976, Motoyoshi et al. 1979 – all quoted from BIBRA 1988, CIR 1982 and/or IUCLID 2000). In one study the skin was scratched (scarified) beforehand (Frosch & Kligman 1976 – quoted from BIBRA 1988).

When applied in petrolatum under cover for 48 hours to the backs of 50 subjects, the highest non-irritant concentration of isopropyl myristate was 10%. No details were given of effects seen at higher concentrations. (Meneghini et al. 1971 – quoted from BIBRA 1988 and IUCLID 2000). 

42.2.3.2     Skin sensitisation

Negative results were obtained in patch tests with 5% isopropyl myristate in petrolatum on 290 patients with eczema (Meneghini et al. 1971 – quoted from BIBRA 1988), and with 30% isopropyl myristate in petrolatum in an unspecified number of patients at a dermatological clinic (Hjort & Trolle-Lassen 1963 – quoted from BIBRA 1988).

Several experimental studies with healthy volunteers have failed to detect any skin sensitising potential (Anon 1953, CTFA 1976, 1978a, b, Kligman 1974 – all quoted from BIBRA 1988, CIR 1982, and/or IUCLID 2000).
In one such study, flannel impregnated with 170 mg undiluted isopropyl myristate under cellophane was allowed to remain on the backs of 200 volunteers for 5 days, and in another study, material saturated with 1.28-1.70 g undiluted isopropyl myristate was applied for 24 hours every other day for 15 applications to 50 volunteers. In these two studies a 48-hour challenge application was given 3 weeks later. (Anon 1953 – quoted from BIBRA 1988 and IUCLID 2000).
In a maximization test on 25 volunteers, pre-treatment of the forearm with an irritant (sodium lauryl sulphate) was followed by five 48-hour applications of 20% isopropyl myristate in petrolatum with 24 hours between each application, and a challenge application was given 10 days later (Kligman 1974 – quoted from BIBRA 1988, CIR 1982, and IUCLID 2000).
Other studies involving repeated contact with various cosmetic preparations containing 15-58% isopropyl myristate also gave negative skin sensitisation results in groups of 25-320 volunteers (CTFA 1976, 1978a, b – quoted from BIBRA 1988 and CIR 1982).

No photo-allergic reactions were induced in 25 volunteers when a bath oil containing 43% isopropyl myristate was applied dermally at 4.25 mg/cm² under an occlusive patch for 24 hours, twice weekly for 3 weeks. Each application was followed by three exposures to ultraviolet irradiation (UV-range A) from a solar simulator sufficient to produce slight redness, and a challenge patch followed by irradiation was applied 10 days later. (CTFA 1978 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

A woman developed a mild dermatitis after using a feminine hygiene spray containing isopropyl myristate at an unspecified concentration for 6 months. On closed patch testing she had a strong positive reaction to undiluted or 10% isopropyl myristate. (Fisher 1973 – quoted from BIBRA 1988 and CIR 1982).

Three of 41 hospital workers with hand eczema had positive skin-prick test reactions to 20% isopropyl myristate. It was not stated what isopropyl myristate was diluted in. None of 55 patch-tested with the same concentration under cover for 48 hours showed a positive response. It is unclear whether these 55 included any of the 41 tested by skin-prick. (Nielsson 1985 – quoted from BIBRA 1988).

42.2.4   Other routes

No data have been found.

42.3   Toxicity to reproduction

No data have been found.

42.4   Mutagenic and genotoxic effects

No data have been found.

42.5   Carcinogenic effects

No data have been found.

 43   Animal toxicity

43.1   Single dose toxicity

43.1.1   Inhalation

Twenty rats were exposed for 6.5 seconds/minute for an hour to an aerosol antiperspirant containing 16-20% isopropyl myristate at a nominal concentration of 33-41 mg/l. During exposure all rats exhibited lethargy, and slight muscle and eye discharge. No deaths occurred, and no evidence of systemic toxicity was found at necropsy or during the 14-day post-exposure observation period. (Hazleton Laboratories 1978 – quoted from BIBRA 1988 and CIR 1982).

Groups of 6 rats served as controls or were exposed for four 15-minute periods each separated by 5-minute fresh air periods to an aerosol antiperspirant containing 4.7% isopropyl myristate at a nominal concentration of 9.7 mg/l. No deaths occurred and no significant differences were found between control and exposed rats during exposure or at necropsy. (CTFA 1974 – quoted from BIBRA 1988 and CIR 1982).

43.1.2   Oral intake

The oral LD₅₀-value of undiluted isopropyl myristate has been reported to be higher than 13700 mg/kg b.w. for rats (4 studies) and from 42400 to higher than 85000 mg/kg b.w. for mice (2 studies) (Anon 1953, Avon 1971, Inolex 1975, Kolmar Research Center 1972, Lebarco Laboratories 1973, Platcow & Voss 1954 – all quoted from BIBRA 1988 and CIR 1982).

43.1.3   Dermal contact

43.1.3.1     Acute toxicity

The dermal LD₅₀-value of undiluted isopropyl myristate has been reported to be higher than 5000 mg/kg b.w. for rabbits (1 study) (Moreno 1974 – quoted from BIBRA 1988 and CIR 1982).

In rabbits exposed dermally to a single dose of 7600 mg/kg b.w. of isopropyl myristate experienced no overt toxic symptoms or significant haematological changes occurred. Urinary analysis revealed no evidence of metabolic disturbance or kidney damage. (Anon 1953 – quoted from BIBRA 1988).

No deaths or abnormalities at autopsy were reported in a study where 6 guinea pigs were dosed dermally (with an antiperspirant) with about 1000 mg/kg b.w. of isopropyl myristate (CTFA 1972 – quoted from BIBRA 1988 and CIR 1982). 

43.1.3.2     Skin irritation

The Draize primary skin irritation test or a slight modification of it was used in rabbits to evaluate undiluted isopropyl myristate in 5 studies and four product formulations containing the ester in 5 studies. Isopropyl myristate caused no to minimal irritation. (Avon 1974, 1976, 1977, Bio/dynamics 1978, CFTA 1972, 1978, Consumer Product Testing 1978, Inolex 1975, Leberco Laboratories 1973, MB Research Laboratories 1974 – all quoted from CIR 1982).

Isopropyl myristate was slightly irritating in one of the rabbit tests, which was performed according to OECD-guideline 404 and following the principles of GLP (ECETOC 1995 – quoted from Bagley et al. 1996 and IUCLID 2000).

43.1.4   Other routes

43.1.4.1     Eye irritation

The Draize rabbit eye irritation test or a modification of it was used to evaluate undiluted isopropyl myristate in more than 10 separate studies and four product formulations containing the ester in 4 studies. No or minimal irritation generally followed a single application of the undiluted material or the product formulations (Avon 1972a, b, c, 1976, 1977, Bio/dynamics 1978, CFTA 1972, 1978, Guillot et al. 1977, Kolmar Research Center 1967, Leberco Laboratories 1973, Platcow & Voss 1954, Weil & Scala 1971 – all quoted from BIBRA 1988 and/or CIR 1982; Ohno et al. 1999), although in one study slight corneal opacity was reported in a minority of rabbits (Guillot et al. 1982 – quoted from BIBRA 1988).

43.2   Repeated dose toxicity

43.2.1   Inhalation

Two groups of twenty guinea pigs were exposed for an hour three times a day, seven days a week for 4 (half of the animals) or 13 (the other half of the animals) weeks to an aerosol antiperspirant containing 16-20% isopropyl myristate. Test groups were exposed to mean isopropyl myristate concentrations of 10-13 or 36-45 mg/m³. A control group of 40 guinea pigs were exposed to air. One female guinea pig died for unknown causes. Both absolute and relative lung weights increased in the exposed animals but no histological changes were found. The organs examined were not specified. (Bio/dynamics 1979 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

Four groups of nine cynomolgus monkeys were exposed for an hour three times a day, seven days a week for 13 weeks to an aerosol antiperspirant containing 16-20% isopropyl myristate. Test groups were exposed to isopropyl myristate concentrations of about 0.95, 1.5, 6.0 or 6.7 mg/m³. A control group of 9 monkeys were exposed to air. During the study, the treated monkeys wheezed and coughed and two of the monkeys exposed to about 1.5 mg/m³ of isopropyl myristate developed nosebleeds. Lung function tests after 6 and 13 weeks were normal, as were the results of haematology, blood chemistry and urinalysis. No gross lesions were seen at necropsy, and organ weights were unaffected by treatment. Histological examinations revealed a dose-related accumulation of macrophages within the alveolar and bronchiolar walls of the lungs of treated monkeys. (Hazleton Laboratories 1978 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

Groups of 12 female CD rats and of 12 female hamsters were exposed by whole body inhalation to aerosols of a diluted complex fragrance mixture at 0 or 50 mg/m³ (aerodynamic mean diameter = 1.4 mm) for 4 hours per day, 5 days per week for 13 weeks. The fragrance mixture consisted of approximately 200 ingredients with close to one-half of the ingredients present at a level of 1% or more. The animals were exposed to isopropyl myristate at a maximal concentration of 0.16 mg/m³. No toxicological significant effects were observed on survival, behaviour, body weights, organ weights, haematology, clinical chemistry, gross pathology, or histopathology. (Fukayama et al. 1999).

43.2.2   Oral intake

Groups of 50 rats were fed 2.5, 5, or 10% of isopropyl myristate (equal to about 2000, 3700 or 7900 mg/kg b.w. per day) in their diet for up to 16 weeks. Histopathological examinations revealed no damage to any organs or tissues at any dose. At the highest dose, the liver weight and the blood levels of two liver enzymes were increased. In addition the males had increased spleen, small intestine and kidney weights, and in both sexes the proportion of neutrophilic leucocytes was increased. At the mid-dose only transient changes in some of the organ weights (spleen, liver, small intestine) were observed. (Gaunt et al. 1972 – quoted from BIBRA 1988 and IUCLID 2000).

Groups of Wistar rats were given 0, 100, 500 or 1000 mg/kg b.w. per day of isopropyl myristate by gavage once a day, 5 days per week for 28 days. No mortality, symptoms of intoxication, or substance-related injury of organs were recorded. Histologically, a reversible hyperplasia of the forestomach mucosa was observed in all test groups including the negative control group indicating local irritating effects of the olive oil carrier. (Henkel KgaA – quoted from IUCLID 2000).

43.2.3     Dermal contact

43.2.3.1   Systemic toxicity

Rabbits had dermal applications of 5100 mg/kg b.w. of isopropyl myristate daily for 20 days. Skin inflammation, liver damage (mild cloudy swelling), and leucocytosis were observed. No changes were found in the kidneys, testes, or spinal cord. (Anon 1953 – quoted from BIBRA 1988 and IUCLID 2000).

Groups of 10 rabbits were dosed for five days a week for 4 weeks on abraded skin of their back with either distilled water or 320-400 mg/kg b.w. of isopropyl myristate from an aerosol antiperspirant containing 16-20% of the chemical. None of the animals died and no changes considered to be related to the product were seen in general behaviour, body weights, or haematological studies. See also section 4.2.3.2 Local effects regarding local dermal effects. (IRDC 1978 – quoted from BIBRA 1988, CIR 1982, and IUCLID 2000).

Groups of 6 rabbits either remained untreated or were dosed for five days a week for a total of 21 dermal applications with 730-800 mg/kg b.w. of isopropyl myristate from an aerosol antiperspirant containing 43-47% of the chemical. One of the dosed animals died from causes thought to be unrelated to treatment. No significant pathological changes were discovered at necropsy other than local skin damage – see also section 4.2.3.2 Local effects for more details. (CFTA 1972 – quoted from BIBRA 1988 and CIR 1982).

43.2.3.2   Local effects

A 0.5 ml (425 mg) sample of undiluted isopropyl myristate was applied for three consecutive days to a 2 inch² area of clipped skin on a total of 42 rabbits (7 studies). Isopropyl myristate was moderately to severely irritating under the conditions of the study as evidenced by the occurrence of oedema, severe erythema, drying, cracking, and scaling. No further details were given. (Avon 1970, 1971a, b, c, 1972a, b, c – all quoted from CIR 1982).

New Zealand white rabbits or Albino Spartan mice had daily dermal applications of undiluted isopropyl myristate for up to 14 or 28 days, respectively. Erythema, lichenification and fissure formation occurred at the application site. Microscopically, the treated skin of exposed animals showed acanthosis, parakeratosis, hyperkeratosis, focal erosion, and focal haemorrhage. In mice, the skin lesions tended to regress during continued treatment while the lesions in rabbits regressed slowly after cessation of treatment. No further details were given.  (Fitzgerald et al.  1968 – quoted from CIR 1982 and IUCLID 2000).

Groups of 10 rabbits were dosed for five days a week for 4 weeks on their abraded back with either distilled water or 320-400 mg/kg b.w. of isopropyl myristate from an aerosol antiperspirant containing 16-20% of the chemical. Moderate to severe erythema, desquamation, slight coriaceousness, moderate fissuring, and atonia occurred at the application site. Microscopically, the treated skin of all exposed rabbits showed marked to severe acanthosis and hyperkeratosis with varying degrees of parakeratosis and mixed inflammatory cell infiltration. No further details were given. (IRDC 1978 – quoted from CIR 1982).

Groups of 6 rabbits either remained untreated or were dosed for five days a week for a total of 21 dermal applications with 730-800 mg/kg b.w. of isopropyl myristate from an aerosol antiperspirant containing 43-47% of the chemical. Signs of product-related changes in the skin included erythema, oedema, drying, cracking, and fissuring. No further details were given. (CFTA 1972 – quoted from CIR 1982).

White albino rabbits, which had 1, 5 or 100% of isopropyl myristate in propylene glycol applied to the ears twice daily 5 days a week for 2 weeks developed significant comedones formation even at the lowest concentration (Fulton et al. 1976 – quoted from BIBRA 1988 and IUCLID 2000).

43.2.3.3   Skin sensitisation

In 2 guinea pig sensitisation tests with 2 or 10 animals, respectively, isopropyl myristate suspended at 0.1% in physiologic saline showed no evidence of a sensitisation potential (Inolex 1975, Platcow & Voss 1954 – both quoted from CIR 1982). 

Isopropyl myristate was found to be weakly positive for skin sensitisation in the local lymph node assay (LLNA) in mice. In this assay, groups of 5 female CBA/J mice were treated once a day for 3 consecutive days on the dorsum of both ears with 25 ml of 25, 50 or 100% of isopropyl myristate in acetone/olive oil (4:1) or with vehicle alone. Five days after the initial treatment, all mice were injected intravenously via the tail vein with marked methylthymidine. The incorporation of methylthymidine in the cells of the draining auricular lymph nodes was determined. It is generally taken as a measure for the lymphocyte proliferation provoked by sensitising chemicals during the induction phase. The authors are suggesting that the positive result might be a false-positive response since a) isopropyl myristate is not expected based on structure to be a skin sensitiser, b) it has been reported that some irritants can produce a positive response in LLNA and isopropyl myristate is known to be a skin irritant at high concentrations, c) and isopropyl myristate was only weakly positive at the highest concentration tested. (Ryan et al. 2000).

43.2.4   Other routes

43.2.4.1   Eye irritation

Applications of isopropyl myristate to the eyes of rabbits daily for 3 consecutive days apparently caused no irritation (Avon 1970, 1971a – all quoted from BIBRA 1988 and CIR 1982) or slight irritation that had vanished after 7 days (Avon 1971b – quoted from BIBRA 1988 and CIR 1982).

43.3   Toxicity to reproduction

Twenty Swiss albino mice (housed one male and one female per cage) had 0.1 ml of 1% isopropyl myristate (equivalent to 42.5-56.6 mg/kg b.w.) in acetone applied to the skin once weekly for 18 months. No abnormalities were found in litters, which were examined grossly. (Giles & Byron 1968 – quoted from BIBRA 1988 and IUCLID 2000).

43.4   Mutagenic and genotoxic effects

Isopropyl myristate tested negative in an Ames test with 5 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) both with and without metabolic activation (Blevins & Tayler 1982 – quoted from BIBRA 1988 and IUCLID 2000).

43.5   Carcinogenic effects

In a lifetime study, groups of 50 female Swiss mice were given skin applications from the age of 7 weeks on their backs of 0.02 ml of 10, 50 or 100% isopropyl myristate (equivalent to 85, 425 or 850 mg/kg b.w.) in acetone twice a week. Control groups consisted of 135 untreated animals (negative), 50 treated with acetone (negative), and 50 treated with 7,12-dimethylbenzanthrancene (positive). Two benign skin tumours remote from the site of application developed in exposed mice. Internal tumours (including lymphomas, lung adenomas, liver haemangiomas and thymomas) were also found. However, no significant differences were observed between exposed or negative control animals in the incidence of skin or internal tumours. (Stenbäck & Shubik 1974 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

No skin tumours were found when 0.1 ml of a 1% solution of isopropyl myristate (equivalent to 42.5-56.6 mg/kg b.w.) in acetone was applied once a week to the clipped skin of 20 Swiss albino mice for 18 weeks. Acetone was used as a negative control and 9,10-dimethyl-1,2-benzanthracene as a positive control. Five mice escaped and 4 died before the end of the study in the exposed group. (Giles & Byron 1968 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

In a lifetime study, groups of 5 New Zealand white rabbits were given skin applications on the interior left ear from the age of 8 weeks of 0.02 ml of 10, 50 or100% isopropyl myristate (equivalent to 0.85, 4.25 or 8.5 mg/kg b.w.) in acetone twice a week. No tumours of the skin or internal organs developed. (Stenbäck 1977 – quoted from BIBRA 1988 and IUCLID 2000).

A 50% solution of isopropyl myristate (in ethanol according to BIBRA and IUCLID but in isopropyl alcohol according to CIR) significantly accelerated the carcinogenic activity of 0.15% benzo[a]pyrene (known skin carcinogen) on the skin of mice (Horton et al. 1966 – quoted from BIBRA 1988, CIR 1982 and IUCLID 2000).

44   Regulations

44.1   Ambient air

-

44.2   Drinking water

-

44.3   Food

JECFA has concluded that isopropyl myristate will not present safety concerns at the current level of intake as a flavouring agent. Isopropyl myristate has not been allocated an ADI owing to lack of data. (JECFA 2000).

44.4   Cosmetics

A major use of isopropyl myristate is in cosmetics and topical medicinal preparations (HSDB 2000). It is used as a solubilising, spreading, and penetrating agent in anhydrous skin lubricating lotions with high lanolin content (CIR 1982).

Isopropyl myristate is not mentioned in the statutory order on cosmetics, which means that it legally can be used in cosmetics in Europe without any limitations except as a colouring agent, a preservative, or a UV-filter (MM 2000).

44.5     Soil

-

44.6     Occupational Exposure Limits

-

44.7     Classification

-

44.8     IARC

-

44.9     US-EPA

-

45     Summary

45.1     Description

Isopropyl myristate is a colourless and practically odourless liquid with a low vapour pressure (9.35 x 10^-5 mmHg). It is practically insoluble in water.

45.2     Toxicokinetics

Very little systemic absorption (0.25 %) of isopropyl myristate occurred when four monkeys were exposed for 5 seconds to the aerosols of an antiperspirant spray containing the chemical. Following dermal application, isopropyl myristate showed local penetration in rabbits and guinea pigs but no visible penetration into the skin or organs in hairless mice.

A study with subcutaneous injection in mice indicates that if absorbed, isopropyl myristate will be distributed into almost all organs.

Isopropyl myristate has been shown to enhance the penetration rate of several other chemicals through human skin. It also has been shown to enhance the in vitro dermal absorption of ethanol and butanol through hairless mouse skin.

45.3     Human toxicity

In studies with single or repeated dermal administration of undiluted isopropyl myristate, no or minimal skin irritation was seen. When applied in petrolatum under cover for 48 hours to the backs of 50 subjects, the highest non-irritant concentration of isopropyl myristate was 10%.

No evidence of phototoxicity or photo-allergic reactions was observed in two studies with human volunteers exposed dermally to isopropyl myristate and UV-light.

Several experimental studies with healthy volunteers have failed to detect any skin sensitising potential of isopropyl myristate. That was the case in e.g. a maximization test on 25 volunteers, where a pre-treatment of the forearm with an irritant was followed by five 48-hour applications of 20% isopropyl myristate in petrolatum with 24 hours between each application, and a challenge application was given 10 days later. However, one case report exists of strong positive patch test reaction to isopropyl myristate in a woman that for 6 months had used a feminine hygiene spray containing the chemical. A case report also exist of three hospital workers with hand eczema who had positive skin-prick test reactions to 20% isopropyl myristate.

No data have been found for reproductive and developmental effects, for mutagenic and genotoxic effects, or for carcinogenic effects.

45.4     Animal toxicity

45.4.1     Single dose toxicity

The acute toxicity of undiluted isopropyl myristate following oral or dermal application is low (oral LD₅₀-value >13.7 g/kg b.w. (rats), 42.4 - > 85 g/kg b.w. (mice); dermal LD₅₀-value > 5 g/kg b.w. (rabbits)).

No deaths or evidence of systemic toxicity occurred in rats exposed to aerosol antiperspirants containing 5-20% isopropyl myristate at nominal concentrations of 10-41 mg/l for 6.5 seconds/minute for an hour or for four 15-minute periods separated by 5 minutes.

Undiluted isopropyl myristate or product formulations containing it caused no to minimal irritation of rabbit skin and eyes in several studies performed according to the Draize primary skin irritation test, the Draize rabbit eye irritation test or slight modifications of them.

45.4.2     Repeated dose toxicity

Lung weights increased (but no histological changes were found) in guinea pigs exposed for an hour three times a day, seven days a week for 4 or 13 weeks to isopropyl myristate (in an aerosol antiperspirant) in concentrations from 10 mg/m³.

In monkeys exposed for 13 weeks to isopropyl myristate (in an aerosol antiperspirant) in concentrations between 0.95 and 6.7 mg/m³, histological examinations revealed a dose-related accumulation of macrophages within the alveolar and bronchiolar walls of the lungs. During the study, the treated monkeys wheezed and coughed. However, lung function tests were normal, as were the results of haematology, blood chemistry and urinalysis. No gross lesions were seen at necropsy, and organ weights were unaffected by treatment.

No toxicological significant effects were observed in rats and hamsters exposed to isopropyl myristate aerosols (in a complex fragrance mixture) at a maximal concentration of 0.16 mg/m³ for 4 hours per day for 13 weeks.

No mortality, symptoms of intoxication, or substance-related injury of organs were recorded in rats gavaged with isopropyl myristate in doses up to 1000 mg/kg b.w. per day for 28 days or in rats fed isopropyl myristate in doses up to 2000 mg/kg b.w. per day for up to 16 weeks. Transient changes in some of the organ weights were observed in rats fed 3700 mg/kg b.w., and the blood levels of two liver enzymes and the proportion of neutrophilic leucocytes were increased in rats fed 7900 mg/kg b.w. per day of isopropyl myristate.

No systemic toxicity was observed in rabbits dosed dermally with up to 800 mg/kg b.w. of isopropyl myristate for about 20 days. Skin inflammation, mild cloudy swelling of the liver and leucocytosis were observed in rabbits dosed dermally with 5100 mg/kg b.w. of isopropyl myristate for the same period.

Isopropyl myristate was moderately to severely irritating (erythema, oedema, drying, cracking, scaling and fissuring) to rabbit and mice skin in studies lasting from 3 to 28 days. The animals were exposed dermally to 16-100% of isopropyl myristate. Microscopically, the treated skin of exposed animals showed acanthosis, parakeratosis, hyperkeratosis, and mixed inflammatory cell infiltration. In mice, the skin lesions tended to regress during continued treatment while the lesions in rabbits regressed slowly after cessation of treatment.   

Comedones were observed in rabbits, which had 1% or more of isopropyl myristate in propylene glycol applied to the ears twice daily for 2 weeks.

Applications of isopropyl myristate to the eyes of rabbits daily for 3 consecutive days apparently caused no irritation or slight irritation that had vanished after 7 days.

In 2 guinea pig sensitisation tests, isopropyl myristate suspended at 0.1% in physiologic saline showed no evidence of a sensitisation potential. However, isopropyl myristate was found to be weakly positive for skin sensitisation in the local lymph node assay in mice. The authors suggested that the positive result might be a false-positive response.

45.4.3     Toxicity to reproduction

No abnormalities were found in litters (examined grossly) of mice, which had 0.1 ml of 1% isopropyl myristate in acetone applied to the skin once weekly for 18 months.

45.4.4     Mutagenic and genotoxic effects

Isopropyl myristate tested negative in an Ames test with 5 strains of Salmonella typhimurium both with and without metabolic activation.

45.4.5     Carcinogenic effects

No significant differences were observed in the incidence of skin or internal tumours between negative control animals (mice and rabbits) and animals, which were given dermal applications of undiluted (or diluted) isopropyl myristate twice a week in lifetime (or shorter) studies.

A 50% solution of isopropyl myristate in ethanol (or isopropyl alcohol?) significantly accelerated the carcinogenic activity of 0.15% of the known skin carcinogen, benzo[a]pyrene, on the skin of mice.

46     Evaluation

The critical effect of isopropyl myristate is the local effects (mainly irritation) it might cause. In animals, undiluted isopropyl myristate was moderately to severely irritating to the skin following repeated exposure and at most slightly irritating to the eyes. However, in the majority of studies with human volunteers no or minimal skin irritation has been observed following repeated dermal administration of undiluted isopropyl myristate. In one human study the highest non-irritant concentration of isopropyl myristate was 10%. After application to rabbit skin comedones formation was observed. The wheezing and coughing of monkeys exposed by inhalation to a formulation containing isopropyl myristate is probably a result of respiratory tract irritation since the monkeys had normal lung function tests and the only histological observation was a dose-related accumulation of macrophages within the lungs. In addition, results of haematology, blood chemistry and urinalysis were normal as were organ weights. The only observed effect in guinea pigs exposed by inhalation to a formulation containing isopropyl myristate was increased lung weights but no histological changes were found. It should be noted, that in the inhalation studies, isopropyl myristate has only been tested as part of a formulation and not as the pure substance.

Experimental studies with healthy volunteers and the guinea pig sensitisation test have failed to show any potential for skin sensitisation. The local lymph node assay in mice was weakly positive for skin sensitisation. One human case report of a positive patch test to isopropyl myristate indicates that the chemical might sensitise humans via the skin. A few human case reports of positive skin-prick tests to isopropyl myristate also exist.

Isopropyl myristate was of low acute toxicity to laboratory animals by the oral and dermal routes. Low systemic toxicity was observed in animals exposed by inhalation, dermally, or orally to isopropyl myristate. Only when applied in high doses in the oral or dermal studies, repeated administration produced some changes in organ weights, increased blood levels of two liver enzymes, and/or leucocytosis. Only few studies exist on the absorption of isopropyl myristate. Following inhalation, the absorption was very low (less than 1%) in monkeys exposed for 5 seconds. Autoradiography of animals exposed dermally to isopropyl myristate only showed local penetration but no penetration into the organs. The low systemic toxicity could therefore possibly be explained in a low absorption of isopropyl myristate.

Only one developmental study and one mutagenic study have been performed. None of them caused any concern. However, the value of the developmental study is limited because of the relatively low dose of isopropyl myristate and the dermal route of application. 

In three carcinogenic studies with dermal application of isopropyl myristate, no significant differences in the incidence of skin or internal tumours was observed between control and exposed animals. However, the chemical did accelerate the carcinogenic activity of a known skin carcinogen, benzo[a]pyrene.

It is a cause of concern that isopropyl myristate has the ability to enhance the dermal absorption of other chemicals since it, as an inert ingredient in pesticide formulations, might alter the absorption of the active substance or of other of the inert ingredients and thus possibly alter the toxicity of these chemicals.

47     References

Bagley DM, Gardner JR, Holland G, Lewis RW, Regnier JF, Stringer DA and Walker AP (1996). Skin irritation: reference chemicals data bank. Toxicol Vitro 10, 1-6.

BIBRA (1988). Isopropyl myristate. Toxicity profile. The British Industrial Biological Research Association, 8p.

ChemFinder (2001). Isopropyl myristate. Http://www.chemfinder.com

CIR (1982). Final report on the safety assessment of myristyl myristate and isopropyl myristate. Cosmetic Ingredient Review. J Am Coll Toxicol 1, 55-80.

Fukayama MY, Easterday OD, Serafino PA, Renskers KJ, North RH and Schrankel KR (1999). Subchronic inhalation studies of complex fragrance mixtures in rats and hamsters. Toxicol Lett 111, 175-187.

HSDB (2000). Isopropyl myristate. In: Hazardous Substances Data Base. Last revised: 10/2000.

IUCLID (2000). Isopropyl myristate. In: International Uniform Chemical Information Database. Existing Chemicals 2000. ECB, JRC, Ispra.

JECFA (2000). Saturated aliphatic acyclic secondary alcohols, ketones and related saturated and unsaturated esters. In: Safety evaluation of certain food additives. WHO Food Additives Series 42. Prepared by the fifty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), International Programme on Chemical Safety (IPCS). World Health Organization, Geneva, 235-265.

Merck Index (1996). Isopropyl Myristate. In: 12th. ed., Rahway, New Jersey, Merck & Co., Inc., 890.

MM (2000). Miljø- og Energiministeriets bekendtgørelse nr. 594 af 6 . juni 2000 om kosmetiske produkter.

Ohno Y, Kaneko T, Inoue T, Morikawa T, Yoshida T, Fuji A, Masuda M, Ohno T, Hayashi M, Momma J, Uchiyama T, Chiba K, Ikeda N, Imanishi Y, Itakagaki H, Kakishima H, Kasai Y, Kurishita A, Kojima H, Matsukawa K, Nakamura T, Ohkoshi K, Okumura H, Saijo K, Sakamoto K, Suzuki T, Takano K, Tatsumi H, Tani N, Usami M and Watanabe R (1999). Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1). Overview of the validation study and Draize scores for the evaluation of the tests. Toxicol Vitro 13, 73-93.

Ryan CA, Gerberick GF, Cruse LW, Basketter DA, Lea L, Blaikie L, Dearman RJ, Warbrick EV and Kimber I (2000). Activity of human contact allergens in the murine local lymph node assay. Cont Derm 43, 95-102.

Appendix 7: Sodium ligninsulphonate 
48     General description

Sodium ligninsulphonate is a by-product of the sulphite pulping process for paper. Commercial formulations are mixtures of sodium ligninsulphonates and wood sugars.

48.1     Identity

Molecular formula:          -

Structural formula:          

Molecular weight:            250-100000

CAS-no.:                        8061-51-6

Synonyms:                             Lignosulphonic acid, sodium salt

                                             Sodium lignosulphonate

                                      Sodium lignosulphonic acid

                                      Sulphonated lignin sodium salt

48.2     Physical / chemical properties

Description:                            Sodium ligninsulphonate is a light tan to dark brown

                                      nonhygroscopic powder with no pronounced odour.

Melting point:                  No definite melting point.

Boiling point:                   Decompose above 200 °C.

Density:                          -

Vapour pressure:             -

Concentration of

saturated vapours:           -

Solubility:                        Sodium ligninsulphonate forms colloidal solutions or dispersions in water. It is practically insoluble in all organic solvents. The pH of a 1:100 solution is approximately between 3 and 8.

References:                    Aldrich Chemical Co., Inc. (2001), ChemFinder (2001), Committee on Food Chemicals Codex (1996), HSDB (2000), RTECS (2000), Ward & Tankersley (1980).

49     Toxicokinetics

No data have been found. Systemic effects following oral administration indicate that sodium ligninsulphonate is absorbed following administration by this route. However, because of the size of the molecule and its ionisation in solution, the absorption is probably limited.

50     Human toxicity

No data have been found.

51     Animal toxicity

51.1     Single dose toxicity

51.1.1     Inhalation

The LC₅₀-value reported for rats is greater than 480 mg/m³ (RTECS 2000).

Sodium ligninsulphonate may be irritating to mucous membranes and upper respiratory tract (Aldrich Chemical Co., Inc. 2001).

51.1.2     Oral intake

The oral LD₅₀-value reported for mice is 6 g/kg (RTECS 2000) and for rats greater than 40 g/kg (Luscombe & Nicholls 1973).

51.1.3     Dermal contact

Sodium ligninsulphonate may cause eye and skin irritation (Aldrich Chemical Co., Inc. 2001).

51.1.4     Other routes

The intraperitoneal LD₅₀-value reported for rats is 260 mg/kg (RTECS 2000).

Sodium ligninsulphonate has caused drowsiness and muscle weakness; species, route of exposure, and dose levels were not stated (Aldrich Chemical Co., Inc. 2001).

An anticoagulant effect of sodium ligninsulphonate has been demonstrated in mongrel dogs; route of exposure and dose levels were not stated. The anticoagulant activity of sulphonated lignins was related to their molecular weight with higher molecular weight fractions possessing greater anticoagulant activity than the lower molecular weight fractions. (Loomis & Beyer 1953 – quoted from Luscombe & Nicholls 1973 and Samson & Hollis 1992).

A European patent exists for a method of making a sodium ligninsulphonate with anti-thrombotic activity. Low doses (7.5-15 mg/kg b.w.) of this sodium ligninsulphonate administered by intravenous injection significantly decreased the incidence of thrombus formation and also the amount of thrombus formed in a White New Zealand rabbit thrombosis model despite the lack of anti-coagulant effect (as measured by lack of significant effect on prothrombin time, activated partial thromboplastin time, thrombin time, reptilase time, fibrinogen level and platelet count). At higher dosages (intravenous injection of 0.19 mg/ml blood), sodium ligninsulphonate caused significant changes in the blood coagulation with prolonged bleeding time in greyhound dogs. Within the first 15 minutes behavioural changes (increased salivation, unsteadiness and anxiety) were noted in the dogs. (Samson & Hollis 1992).

51.2     Repeated dose toxicity

51.2.1     Inhalation

No data have been found.

51.2.2     Oral intake

Groups of 20 male and 20 female Wistar rats were given AHR-2438B in their drinking water at levels of 0, 0.25, 2.5, 25 or 100 g/l (according to the authors equal to 0, 17, 168, 2830 or 10020 mg/kg b.w. per day in males and 0, 26, 283, 2420 or 9990 mg/kg b.w. per day in females) for 16 weeks. AHR-2438B is a purified sodium salt of a ligninsulphonate with an average molecular weight of 5000. No adverse effects on growth, organ weights, haematology, biochemistry, urine analysis and histopathology were observed at the three lowest dose levels. At the highest dose level, animals of both sexes showed skin lesions at the base of the tail. The lesions seemed to occur as a result of local irritation caused by adhering sticking faecal matter containing large quantities of the test material. Anaemia and raised leucocyte levels in the high dose animals was suggested to be a result of the presence of the tail lesions. In addition the high dose animals showed an increase in the absolute and relative weights of the liver, kidney and spleen, and male rats had a decreased weight gain. Histological changes were consistent with reticulo-endothelial activation in the liver and with vacuolar degeneration of the proximal convoluted tubules in the kidney. No ulcerative lesions were found in any section of the gastrointestinal tract. Blood coagulation times were unchanged throughout the period of dosing. (Luscombe & Nicholls 1973).

Groups of 5 male albino guinea pigs received as drinking fluid an aqueous solution of sodium ligninsulphonate at concentrations of 0, 10, 20, 50 or 100 g/l (according to the authors equal to 0, 1740, 2720, 1570 or 7780 mg/kg b.w. per day) for 1-5 weeks. At the end of the experiment, the stomach, duodenum and colon were examined histologically for ulcerations. Two animals in the 50 g/l dose group died after a week. All of the animals administered the two highest doses lost weight and some of them developed mild diarrhoea and had blood and mucus in the faeces. Animals receiving the two lowest doses gained less weight than controls but otherwise appeared clinically undisturbed. No ulcerations were found in the duodenum in any animal. However, ulcers of the upper part of the colon were found in 1-4 of the animals in each dosed group. The ulcers in the colon were more numerous (up to 100 or more) in the animals, which received the smaller concentrations of sodium ligninsulphonate. In the animals given higher concentrations, the ulcers numbered from 2 to 10. Ulcers of the upper two-thirds of the stomach developed in 4 animals in each of the two highest dosed groups. (Watt & Marcus 1976).

Groups of 8 male albino guinea pigs received as drinking fluid either water or a 1% aqueous solution of sodium ligninsulphonate (equivalent to 1700 mg/kg b.w. per day) for 2-6 weeks. None of the dosed animals developed diarrhoea. However, half of the dosed animals developed ulcers in the upper part of the colon. These animals had blood in the faeces and a lower weight gain than the rest of the group. One of the guinea pigs with ulcers died on the 13^th day of the experiment. (Marcus & Watt 1974).

Groups of 10 male Sprague-Dawley rats were fed either a control diet or a diet containing 3% of seven selected ligninsulphonate fractions for 14 days. In general, the diets containing ligninsulphonate were less digestible and caused a slightly lower pH value in the caecum and colon than the control diet. The caecum weighed more in rats fed ligninsulphonate than in control rats. Some of the ligninsulphonate fractions affected the short-chain fatty acid concentration and the concentration of two beneficial gastrointestinal tract bacteria. (Flickinger et al. 1998).

51.2.3     Dermal contact

A United States patent exist for a method of treating viral infections caused by Herpes simplex, types I and II, by topically applying a ligninsulphonate to the infected tissue. The inventors have examined different commercially available ligninsulphonates as well as fractions thereof. In general, the higher molecular weight fractions with the sugars removed are the most effective antiviral agents. The ligninsulphonates was shown to be effective against Herpes simplex virus in infected human cell lines in culture as well as in vaginal infections in female mice and guinea pigs. (Ward & Tankersley 1980).

51.2.4     Other routes

Female mongrel dogs with total gastric fistula were administered 500 mg of sodium ligninsulphonate (AHR-2438B) dissolved in 10 ml water twice during a two-hour period directly into the stomach through a cannula. Pepsin digestion was analysed using coagulated egg albumin as the substrate. AHR-2438B was an effective inhibitor of pepsin proteolysis. (Alphin et al 1971).

51.3     Toxicity to reproduction

No data have been found regarding reproductive and developmental effects following exposure by inhalation, oral administration, or dermal contact.

Sodium ligninsulphonate (as well as other surfactants) were tested for estrogenic activity in a recombinant yeast screen. The natural oestrogen 17b-oestradiol as well as other known chemicals with estrogenic activity was used as positive controls. Sodium ligninsulphonate did not possess estrogenic activity. (Routledge & Sumpter 1996).

51.4     Mutagenic and genotoxic effects

No data have been found.

51.5     Carcinogenic effects

No data have been found.

52     Regulations

No data have been found.

53     Summary

53.1     Description

Sodium ligninsulphonate is a light tan to dark brown nonhygroscopic powder with no pronounced odour. Commercial formulations are mixtures of sodium ligninsulphonates with different molecular weights and wood sugars.

53.2     Toxicokinetics

Systemic effects following oral administration indicate that sodium ligninsulphonate is absorbed following administration by this route.

53.3     Human toxicity

No data have been found.

53.4     Animal toxicity

53.4.1     Single dose toxicity

LD₅₀-values reported for oral administration of sodium ligninsulphonate ranged from 6 g/kg to greater than 40 g/kg for mice and rats, respectively, and the LC₅₀-value for inhalatory administration to rats was reported to be greater than 480 mg/m³.

Sodium ligninsulphonate may irritate eyes, skin, and the upper respiratory tract. It has caused drowsiness and muscle weakness.

At injections of 7.5-15 mg/kg b.w., sodium ligninsulphonate significantly decreased the incidence and amount of thrombus formation in a rabbit model despite a lack of anti-coagulant effect. At higher dosages, an anticoagulant effect of sodium ligninsulphonate was demonstrated in dogs. Within the first 15 minutes behavioural changes were noted in the dogs.

53.4.2     Repeated dose toxicity

Guinea pigs exposed to sodium ligninsulphonate in the drinking water in doses at or above 1700 mg/kg b.w. per day for up to 6 weeks developed ulcers in the upper part of the colon. At higher doses, stomach ulcers as well as weight loss, diarrhoea and deaths also occurred.

In a dog model, sodium ligninsulphonate was an effective inhibitor of pepsin proteolysis.

Rats exposed to sodium ligninsulphonate in the drinking water in doses at about 10000 mg/kg b.w. per day for 16 weeks had histological changes of the liver and kidneys, and an increased weight of the same organs as well as the spleen. In addition, these rats had skin lesions at the base of the tail, anaemia and raised leucocyte levels. Sodium ligninsulphonate caused no adverse effects at a dose level up to about 2500 mg/kg b.w. per day.

Ligninsulphonates may modify digestive physiology and gastrointestinal tract characteristics of rats.

In a United States patent, ligninsulphonates applied topically was shown to be effective against Herpes simplex virus in infected human cell lines in culture as well as in vaginal infections in female mice and guinea pigs.

53.4.3     Toxicity to reproduction

No data have been found regarding reproductive and developmental effects following exposure by inhalation, oral administration, or dermal contact.

Sodium ligninsulphonate did not possess estrogenic activity in a recombinant yeast screen.

53.4.4     Mutagenic and genotoxic effects

No data have been found.

53.4.5     Carcinogenic effects

No data have been found.

54     Evaluation

Based on the available knowledge, the critical effect of sodium ligninsulphonate in pesticide formulations is probably the irritative effects that it may cause on the eyes, the skin, and the upper respiratory tract. However, there is no information about the dose levels causing irritation, or whether the irritation was caused by the sodium ligninsulphonate powder or by the chemical in a solution.

Following oral intake of relatively high doses, sodium ligninsulphonate may affect the gastrointestinal tract.  Pepsin proteolysis may be inhibited but sodium ligninsulphonate has also been shown to cause ulcers of the colon and stomach in guinea pigs,  but not in rats at high doses. Guinea pigs are known to be highly susceptible to ulcerative disease of the colon (Marcus et al. 1974). This may explain the difference in effect on the gastrointestinal tract in rats and guinea pigs. At high doses, the liver and kidney of rats had histological changes, and sodium ligninsulphonate in the faeces irritated the skin at the base of the tail causing lesions, anaemia and an increased leucocyte level.

Following injections, sodium ligninsulphonate inhibited the blood coagulation in dogs and decreased the incidence and amount of thrombus formation in rabbits despite a lack of anti-coagulant effect. In rats, dosed orally with sodium ligninsulphonate, no anti-coagulant effect was noted. The differences in effect on blood coagulation in the different studies could have many explanations. The studies were performed with different species of which some may be more sensitive than others. In addition, different routes of administration were used. The bioavailability of sodium ligninsulphonate is most likely much higher following injection than following oral administration. However, no toxicokinetic data exist for sodium ligninsulphonate to be used for confirmation of this.

No carcinogenic or other chronic studies of sodium ligninsulphonate were found. Therefore it is not known whether sodium ligninsulphonate may cause cancer or other effects following chronic exposure.

Sodium ligninsulphonate did not possess estrogenic activity in a recombinant yeast screen. No data have been found regarding reproductive and developmental effects following exposure by inhalation, oral administration, or dermal contact.

In conclusion, it has been reported that sodium ligninsulphonate may irritate the eyes, skin and upper respiratory tract; however, there is no information about the dose levels causing irritation. Relatively high doses of sodium ligninsulphonate have to be ingested in order to cause toxic effects in short-term studies. In addition, oral intake and injections are not relevant routes of exposure for sodium lignosulphonate in pesticide formulations.

Based on the available data, the main concern regarding exposure to sodium ligninsulphonate in pesticide formulations is the irritation that it may cause. However, it should be noted that no data regarding effects following long-term exposure as well as of reproductive and developmental effects are available.

55     References

Aldrich Chemical Co., Inc. (2001). Material safety data sheet: Lignosulphonic acid, sodium salt. Milwaukee WI. http://www.sigma-aldrich.com/

Alphin RS, Vocac JA and Droppleman DA (1972). Effect of an inhibitor of pepsin proteolysis in total gastric fistula dogs. Experientia 28, 53-54.

ChemFinder (2001). Sodium ligninsulfonate. http://www.chemfinder.com/

Committee on Food Chemicals Codex (1996). New monograph – sodium lignosulphonate. http://www.nap.edu/html/fcc/soligno.pdf

Flickinger EA, Campbell JM, Schmitt LG and Fahey GC (1998). Selected lignosulphonate fractions affect growth performance, digestibility, and cecal and colonic properties in rats. J Anim Sci 76, 1626-1635.

HSDB (2000). Sodium ligninsulfonate. In: Hazardous Substances Data Base.  Last revised: 10/2000.

Luscombe DK and Nicholls PJ (1973). Acute and subacute oral toxicity of AHR-2438B, a purified sodium lignosulphonate, in rats. Fd Cosmet Toxicol 11, 229-237.

Marcus R and Watt J (1974). Ulcerative disease of the colon in laboratory animals induced by pepsin inhibitors. Gastroenterol 67, 473-483.

Routledge EJ and Sumpter JP (1996). Estrogenic activity of surfactants and some of their degradation products using a recombinant yeast screen. Environ Toxicol Chem 15, 241-248.

RTECS (2000). Lignosulphonic acid, sodium salt. In: Registry of Toxic Effects of Chemical Substances. Database quest, last revised: 10/2000.

Samson RH and Hollis JW (1992) Lignosulphonate based pharmacologic agent with antithrombotic activity. European patent specification no. 303236, Nov. 11, 1992.

Toxline (1985-2000).

Ward JW and Tankersley RW (1980). Method of combating herpes simplex viruses with lignosulfonates. United States patent no. 4185097, Jan. 22, 1980.

Watt J and Marcus R (1976). Experimental gastro-intestinal ulceration induced by polyanionic electrolytes. Progress Peptic Ulcer 287-298.

Appendix 8: Calciumdodecylbenzenesulphonate (CaDBS) 
56     General description

56.1     Identity

Calciumdodecylbenzenesulfonate, CaDBS, is an anionic surfactant used as dispersing agent for pesticide formulation. It is often produced and used in a mixture of linear alkylbenzene sulfonates, LAS. Only very few data were found specifically on CaDBS.

Molecular formula:          (C₁₈H₂₉ SO₃ ^- )₂ ^.  Ca⁺⁺

Structural formula:           æC₁₂H₂₅               ö

                                                         ê       ê           ÷       Ca⁺⁺          

                                                            è    C₆H₄ –SO₃^- ø 2

Molecular weight:            691.14

CAS-no.:                        26264-06-2

Synonyms:                      Calcium bis(dodecylbenzenesulfonate)

                                      Dodecyl-benzenesulfonic acid, calcium salt

56.2     Physical / chemical properties

Description:                            The substance is reported to be a yellowish-brown liquid with a solvent odour or a light to white granular solid.

Melting point:                  -

Boiling point:                   -

Density:                          -

Vapour pressure:             -

Concentration of

saturated vapours:           -

Conversion factor:           1 ppm = 28.74 mg/m³  (at 20°C and 760 mmHg)

                                      1 mg/m³ = 0.035 ppm 

Solubility:                        Water:  yes

References:                    RTECS (2001), HSDB (2001).

57     Toxicokinetics

Only one study was found on calciumdodecylbenzenesulfonate (CaDBS).

2.1       Absorption, distribution, metabolism and elimination 

After oral administration to Wistar rats of 2 mg radiolabelled CaDBS, the radiolabel was detected in plasma after 15 minutes, reaching the maximum of 0.8 mg/g CaDBS at 2 hours, and then decreasing with time, the mean biological half-live being calculated to 10.9 hours.

Four hours after administration, the concentration of the radiolabel was high in the digestive tract, large intestine and in the urinary bladder (22.56, 43.24 and 34.89 mg/g salt, respectively). Concentrations were also high in the liver, kidney, testis, spleen and lung.

At 168 hours after administration, 51 % of the radioactivity was excreted in faeces and 50 % in urine. The 2 metabolites in urine and 2 of the 4 faecal metabolites were identified by thin layer chromatography to probably be sulfophenyl butanoic and sulfophenyl pentanoic acids. (Sunakawa et al. 1979 – quoted from WHO 1996).

The urinary metabolites point at CaDBS being degraded by w-oxidation followed by b-oxidation of LAS (Michael 1968 – quoted from WHO 1996).

57.1     Mode of action

No data on CaDBS were found in the literature.

58     Human toxicity

No data were found in the literature on CaDBS.

59     Animal toxicity

Only data on acute oral toxicity were found on CaDBS.

59.1     Single dose toxicity

59.1.1     Oral intake

A rat LD₅₀  of 4 g/kg is listed in RTECS. However, no details are given. (Yakkyoku, 1950 – quoted from RTECS 2001).

An LD₅₀-value in mouse of 3680 mg/kg is listed in RTECS. However, no details are given (Yakkyoku 1950  – quoted from RTECS 2001).

60     Regulations

60.1     Ambient air

Denmark (C-value):        -

60.2     Drinking water

Denmark:                       -

60.3     Soil

Denmark:                       -

60.4     Occupational Exposure Limits

Denmark:                       -

ACGIH:                         -

Germany:                        -

60.5     EU Classification

-

60.6     IARC

-

60.7     US-EPA

-

61     Summary

Information on calciumdodecylbenzenesulfonate (CaDBS) is very scarce. Only one metabolism study and one report of acute oral toxicity in rats and mice were found in the literature.

61.1     Description

Calciumdodecylbenzenesulfonate is a yellowish liquid with a solvent odour or a white granular solid.

61.2     Toxicokinetics

CaDBS is readily absorbed from the gastrointestinal tract. No accumulation is seen in any organ. CaDBS is excreted equally via urine and faeces. From thin layer chromatography, the two urinary and two of the four faecal metabolites were identified to probably be sulfophenylbutanoic acid and sulfophenylpentanoic acid. The urinary metabolites point at CaDBS being degraded by w-oxidation followed by b-oxidation of linear alkylbezene sulfonate, LAS.

61.3     Human toxicity

No data were available on CaDBS.

61.4     Animal toxicity

Low acute oral toxicity is reported from rats and mice with LD₅₀ – values of 4000 mg/kg in rats and 3680 mg/kg in mice.

62     Evaluation

The database on calciumdodecylbenzenesulfonate is very limited. An evaluation of the toxicology of the substance is not possible on this basis.

The few available data show that the calciumdodecylbenzenesulfonate is readily absorbed from the gastroinstestinal tract and excreted equally through urine and faeces with no accumulation in the tissues.  

Calciumdodecylbenzenesulfonate is of low acute oral toxicity.

No further data are available on the substance.

The toxicology of calciumdodecylbenzenesulfonate is expected to be related to the anion part of the substance, the identity of the salt being of secondary importance. A larger database is available on sodiumdodecylbenzenesulfonate and on linear alkylbenzene sulfonates (LAS) in general. Analogy considerations with LAS indicate that the critical effects of calciumdodecylbenzenesulfonate are probably skin, eye and respiratory tract irritancy. However, differences in chain length in the compounds studied may bias such structure-activity comparisons.

63     References

HSDB (2001). Calciumdodecylbenzenesulfonate. In: Hazardous Substances Data Base.

RTECS (2001) Dodecylbenzenesulfonate. In: Registry of Toxic Effects of Chemicals Substances.

WHO (1996). Linear Alkylbenzene Sulfonates and Related Compounds. Environmental Health Criteria 169, IPCS, World Health Organisation, Geneva.

Appendix 9: Ethylene glycol (EG) 
64     General description

64.1     Identity

Molecular formula:          C₂H₆O₂

Structural formula:           HO-CH₂-CH₂-OH

Molecular weight:            62.07

CAS-no.:                        107-21-1

Synonyms:                                      1,2-Dihydroxyethane

                                      1,2-Ethanediol

                                      Ethane-1,2-diol

                                      2-Hydroxyethanol

                                      Glycol     

                                             MEG

                                      Monoethylene glycol

64.2     Physical / chemical properties

Description:                            Ethylene glycol is a clear, colourless, slightly viscous, hygroscopic liquid with a sweet taste.

Melting point:                  -13 °C

Boiling point:                   197.56 °C (at 760 mmHg)

Density:                          1.113 g/ml (at 20 °C)

Vapour pressure:             0.06 mmHg (8 Pa) (at 20 °C)

Concentration of

saturated vapours:           79 ppm (204 mg/m³) (at 20 °C and 760 mmHg) (calculated)

Conversion factor:           1 ppm = 2.58 mg/m³        (at 20 °C and 760 mmHg)

                                      1 mg/m³ = 0.388 ppm

Solubility:                               Water: Miscible.

Miscible with lower aliphatic alcohols, glycerol, acetic acid, acetone and similar ketones, aldehydes and pyridine. Practically insoluble in benzene, chlorinated hydrocarbons, petroleum ether and oils.

logP_{octanol/water}:                 -1.93 to -1.36 (at 25 ºC).

References:                    IUCLID (2000), ATSDR (1997), BUA (1991), ACGIH (2001).

65     Toxicokinetics

65.1     Absorption, distribution, and excretion

65.1.1     Inhalation

In nose-only exposure experiments, rats were exposed to ¹⁴C-ethylene glycol vapour (32 mg/m³) for 30 minutes or to an ethylene glycol aerosol (184 mg/m³) for 17 minutes. Estimates indicate that at least 60% of inhaled ethylene glycol from both exposures was deposited in the nasal cavity. Absorption and distribution from the site of deposition was rapid, since in animals, which were sacrificed immediately after exposure, 75-80% of the received dose had already been distributed in the animal’s body. The half-time for clearance of plasma ¹⁴C-activity (ethylene glycol plus metabolites) was 39 hours for ethylene glycol vapour and 34 hours for the aerosol (Marshall & Cheng 1983 – quoted from BUA 1991, NTP 1993).

65.1.2     Oral intake

The approximate serum half-life of ethylene glycol has been reported to be 2.5 hours in children (Rothman et al. 1986 – quoted from ATSDR 1997) and between 3.0 and 8.4 hours in adults (Jacobsen et al. 1988, Peterson et al. 1981 – both quoted from ATSDR 1997).

Reif (1950 – quoted from Ware 1988), on three separate occasions, drank pure ethylene glycol in 100 ml of water. The amounts consumed were 5.5, 11.0 and 13.2 g (corresponding to 78.5, 157, and 188.6 mg/kg, respectively, assuming a body weight of 70 kg). Ethylene glycol was recovered in the urine at 24 to 31% of the administered dose within 24 to 48 hours. Oxalic acid concentrations in the urine were higher than normal for 8 to 12 days, with a peak on the fourth day.

In rats, ingested ethylene glycol is rapidly absorbed and evenly distributed throughout the body reaching peak blood levels at 1 to 4 hours after ingestion of doses of 7-29 mg/kg (Winek et al. 1978 – quoted from ATSDR 1997).

The kinetics of orally administered ethylene glycol and its major metabolites, glycolic acid and oxalic acid, in pregnant rats were compared across doses, and between pregnant (P) and non-pregnant (NP) rats. Groups of rats were administered ¹³C-ethylene glycol by gavage at doses of 10 (P and NP), 150 (P), 500 (P), 1000 (P), or 2500 (P and NP) mg/kg b.w. (Pottenger et al. 2001).

Pregnancy status (gestation days 10-11) had no significant impact on the blood concentration-time profiles of ethylene glycol, glycolic acid, or oxalic acid. Thus, the kinetic parameters estimated (maximal concentration in blood (C_max), time to reach maximum blood levels (T_max), area-under-the curve (AUC), and half-life of elimination (bt_½)) did not differ significantly between the pregnant and non-pregnant groups.

The T_max for ethylene glycol for all dose groups occurred at one hour after dosing; the blood concentration decreased in a log-linear fashion thereafter and was no longer detectable for the low dose group (10 mg/kg) by 12 hours post-dosing and by 24 hours for the 150 and 500 mg/kg dose groups. The estimated half-time for elimination of ethylene glycol from blood was less than 2 hours for all dose levels. Blood levels of glycolic acid increased to a peak at 3 hours post-dosing, except at the lowest dose level (10 mg/kg) where this metabolite could not be detected in the blood. The blood levels decreased by 24 hours post-dosing to undetectable levels. The concentrations of oxalic acid in the blood varied between undetectable and about 2 times the limit of quantification over the 24-hour collection period.

There were no substantial differences in the urinary elimination profiles between the pregnant and non-pregnant dose groups, at comparable dose levels. Urinary elimination of ethylene glycol and its metabolites demonstrated dose-dependency, with the high dose groups (2500 mg/kg) eliminating almost 70% of the administered dose in urine, compared with about 16% in the low dose groups (10 mg/kg). The shift in urinary elimination was mainly due to increased urinary glycolic acid and ethylene glycol, and not to increased elimination of oxalic acid.

In rats, 10-20% of oral doses up to 1000 mg/kg of ethylene glycol were recovered from the body tissues and carcass 96 hours after dosing, whereas mice retained only a small percentage of the dose in their tissues. Total recovery of the oral doses in rats and mice was approximately 90-100%, indicating substantial absorption. The major excretory route of [¹⁴C] was via exhalation of carbon dioxide (42%), while 24% of the dose was excreted via the urine and 3% via the faeces. (Frantz et al. 1989, 1991 – quoted from ATSDR 1997).

The elimination half-life in plasma has been estimated at 1.7 hours in rats given 2000 mg/kg and 1.4 to 2.5 hours following administration of 10 to 1000 mg/kg (Frantz et al. 1989 – quoted from ATSDR 1997).

Mice showed a similar profile, exhaling 55% of the dose, and excreting 24% in the urine and up to 12% in the faeces. The elimination half-life in plasma in mice has been estimated at 0.3 to 1.1 hours following administration of 10 to 1000 mg/kg.  (Frantz et al. 1991 – quoted from ATSDR 1997).

In contrast, approximately 50% of an oral dose of ethylene glycol administered to dogs was excreted via the urine (Grauer et al. 1987 – quoted from ATSDR 1997).

65.1.3     Dermal contact

The in vitro permeability of human skin to ethylene glycol was determined by Loden (1986 – quoted from ATSDR 1997); the rate of resorption was 118 µg/cm²/hour, with a steady state concentration of 0.97 mg/cm².

¹⁴C-ethylene glycol (in acetone vehicle) was applied to the surface of three different fresh human skin samples at a dose of 8 µg/cm². After 24 hours of exposure, 18.3% of the applied dose was recovered in the receptor fluid (absorbed through the skin), 8.3% in the skin, and 12.5% in the skin surface wash (total accountability was approximately 39%). Individual differences existed for the three samples; average absorption was 26.6% relative to the 8 µg/cm² applied dose for a 24-hour exposure duration. This represented a flux of approximately 0.09 µg/cm²/hour for ethylene glycol. The maximum flux observed was 0.25 µg/cm²/hour. (Driver et al. 1993).

In dermal applications using an occlusion bandage, approximately 30% of doses of ethylene glycol up to 1000 mg/kg was absorbed through rat skin; 14% of the absorbed dose was expired, while 7% was excreted in the urine, and 1% was recovered from the faeces (Frantz et al. 1989 – quoted from ATSDR 1997).

Following administration of 100 mg/kg undiluted radiolabeled ethylene glycol to mice using an occlusive bandage, 99.5% of the dose was recovered, with tissues and excreta accounting for 76.5%. Most was recovered as volatile organic radioactivity (25-39%) or as radioactive carbon dioxide (8-12%). Urine and faeces each accounted for another 4.9% of the dose. Tissue recoveries were less than 1% of the dose, while the residual carcass contained about 10-18% of the dose. Following application of 1000 mg/kg undiluted ethylene glycol or as an 50% aqueous solution, the total recovery was 89% of the dose with 84% in tissues and excreta, and approximately 7% in faeces, cage wash water, and carcass. According to the authors, mice absorbed 85-100% of the administered dermal dose. (Frantz et al. 1991 – quoted from ATSDR 1997).

65.2     Metabolism

The metabolic pathway for ethylene glycol is shown in Figure 2.2. Solid arrows represent the steps that are quantitatively most important, while the broken arrows indicate minor metabolic conversions in humans. (ATSDR 1997).

Ethylene glycol is oxidised to glycolaldehyde by NAD-dependent alcohol dehydrogenase in the liver and kidney. Glycolaldehyde is further oxidised to glycolic acid by mitochondrial aldehyde dehydrogenase and cytosolic aldehyde oxidase. There is no evidence for an accumulation of glycolaldehyde and it appears to be rapidly metabolised to glycolic acid (Jacobsen & McMartin 1997).

A small amount of glycolaldehyde is converted to glyoxal which, in presence of lactate dehydrogenase and/or aldehyde oxidase, is further converted to glycolic acid or is directly metabolised via an oxidative mechanism to glyoxylic acid.

Further degradation of glycolic acid is oxidation to glyoxylic acid by glycolic acid oxidase or lactic dehydrogenase. This step occurs apparently at a slow rate since glycolate has been shown to accumulate in large amounts (Jacobsen & McMartin 1997).

Glyoxylic acid is further metabolised via a number of intermediate metabolic pathways, for example, to oxalic acid by the enzyme glycolic acid oxidase and, via formic acid, to carbon dioxide and water, or to glycine. The major metabolic route in terms of toxicological importance is the conversion to oxalate and the most important alternate pathway appears to be formation of glycine (Jacobsen & McMartin 1997). Glyoxylate can induce lactic acid formation via oxalomalate production and its inhibitory effects on the citric acid cycle.

(ATSDR 1997, Jacobsen & McMartin 1997, BUA 1991, Cavender & Sowinski 1994).

65.3     Mode of action

The toxicity of ethylene glycol is mainly a result of the effects of its metabolites

such as glycolaldehyde, glycolic acid, glyoxylic acid, and oxalic acid. (ATSDR 1997, Jacobsen & McMartin 1997, LaKind et al. 1999, BUA 1991, NTP 1993).

The clinical symptoms of acute ethylene glycol poisoning in humans can be divided into three (and occasionally four) fairly distinct stages (see also part 3.1.2) (LaKind et al. 1999, Cavender & Sowinski 1994, BUA 1991, ACGIH 2001).

The first stage, which usually begins within 30 minutes to 12 hours after ingestion,  consists primarily of central nervous system (CNS) toxicity and is usually attributed both to unmetabolised ethylene glycol and to the formation of aldehydes that peak 6 to 12 hours after ingestion (LaKind et al. 1999). The appearance of cerebral symptoms coincides with the highest concentration of the metabolite, glycolaldehyde (Balazs et al. 1982 – quoted from BUA 1991).

The second stage has been described as the cardio-pulmonary toxicity stage that occurs 12 to 72 hours after ingestion. This stage may also be characterised by severe metabolic acidosis. (LaKind et al. 1999).

Aldehyde intermediates are held responsible for the cytotoxic effects and glycolaldehyde seems to be responsible for the observed cardio-pulmonary symptoms (Balazs et al. 1982 – quoted from BUA 1991).

Figure 2.2. Metabolic pathway of oxidation of ethylene glycol. From ATSDR (1997).

Remark: Glycolaldehyde is oxidised to glycolic acid by aldehyde dehydrogenase, not alcohol dehydrogenase as stated in the figure.

The third stage is known as the “renal failure” stage and occurs 24 to 72 hours after ingestion. This stage ischaracterised by profound acidosis caused, according to LaKind et al. (1999), primarily by the accumulation of the metabolites glycolic acid and glyoxylic acid.

Recent studies of cases of human ethylene glycol poisoning have demonstrated that the major determinant of the metabolic acidosis is the degree of glycolic acid accumulation and glycolate accumulation correlates with the increase in anion gap or decrease in arterial bicarbonate concentrations in humans, as well as in animals. Several studies have suggested accumulation of glyoxylate, although at much lower levels (60 µM) than that of glycolate (15 mM). Glycolate is less toxic in vitro than either glycolaldehyde or glyoxylate. Whether the levels of glyoxylate that have been measured in human cases can contribute to the clinical features cannot yet be determined. (Jacobsen & McMartin 1997).

Calcium oxalate precipitation within the renal tubules has long been accepted as an important pathogenic factor for the development of renal toxicity; however, the mechanism of the renal toxicity is not yet known and there is no evidence directly linking oxalate precipitation with development of renal tubular necrosis. Furthermore, the renal damage can occur at levels of exposure where no or few crystals of oxalate are detected. Therefore, the renal toxicity has also been suggested to occur from a metabolite-induced cytotoxicity. (Jacobsen & McMartin 1997, LaKind et al. 1999).

Glycolic acid and glyoxylic acid react with bicarbonate causing a decrease in the pH in body fluids, particularly in blood. An elevated anion gap develops, and the serum osmolal gap across cells increases, resulting in renal oedema that compromises intrarenal blood flow and promotes renal failure. Blood phosphorous (inorganic) levels are increased due to uncoupling of oxidative phosphorylation, and, as a result, blood calcium levels are lowered. Acidosis results in oliguric or anuric renal failure with renal changes. (LaKind et al. 1999).

Generally, the role of oxalate accumulation in the toxicity of ethylene glycol has not been clarified. Urinary calcium oxalate crystals are an important hallmark of ethylene glycol poisoning. However, plasma oxalate determinations in human cases have showed low levels (< 0.4 mM) possibly because the oxalate in plasma rapidly precipitates as calcium oxalate, whose crystals have been noted in the kidney, brain and other tissues. Such precipitation probably leads to the hypocalcaemia that is characteristic of ethylene glycol poisonings. (Jacobsen & McMartin 1997).

Other neurological symptoms, effects on cranial nerves, have been identified 6 or more days after ingestion and constitute a possible fourth clinical stage of toxicity. The mechanism(s) of these delayed neurological effects is unknown, but is distinctly different from the pathological events of the first three stages. The cause may be related to oxalate crystal deposition or, perhaps, ethylene glycol may induce pyridoxine deficiency, resulting in peripheral neuropathy. (LaKind et al. 1999).

The mechanism(s) of ethylene glycol toxicity as related to developmental effects warrants further exploration. A link between maternal metabolic acidosis and developmental toxicity has been suggested. (LaKind et al. 1999).

Pottenger et al. (2001) has suggested that glycolic acid is the proximate developmental toxicant.

66     Human toxicity

66.1     Single and repeated dose toxicity

66.1.1     Inhalation

Twenty male volunteers (prisoners, age not reported) were exposed for 30 days, 20-22 hours a day, to ethylene glycol atmospheres (aerosol, diameters of droplets 1-5 µm) containing mean concentrations of 17 to 49 mg/m³ (lowest concentration: 0.8 mg/m³; highest concentration: 67 mg/m³). Fourteen other volunteers, as a control group, were treated as similarly as possible to the exposed group. Follow-up observations were made on both groups two weeks after the end of the exposure period. No subject experienced any serious signs of toxicity, but there were complaints of irritation of the throat and slight headache and low backache were also reported occasionally.

During the last two weeks of the experiment, the concentration of ethylene glycol was intentionally increased up to 308 mg/m³ during the absence of the volunteers and observation was made of their responses to these elevated concentrations of the aerosol when they re-entered the exposure chamber. When the volunteers returned to a concentration of ethylene glycol in the chamber air of 188 mg/m³, this concentration was irritating but could be tolerated for 15 minutes. A concentration of 244 mg/m³ could not be tolerated for more than a minute or two and a concentration of 308 mg/m³ could not be tolerated at all. Other similar trials revealed that concentrations of ethylene glycol in the air greater than about 200 mg/m³ were intolerable due to strong irritation of the upper respiratory tract, with a burning sensation along the trachea and a burning cough. The irritation became common when the concentration of ethylene glycol in the chamber air was raised to about 140 mg/m³.

Exposure to ethylene glycol under the conditions of this study produced no significant alterations of the haematological, clinically chemical, or clinically pathological parameters studied, including the concentrations of urea nitrogen and creatinine in the blood of the exposed volunteers.

(Wills et al 1974).

Troisi (1950 – quoted from LaKind et al. 1999 and from BUA 1991) examined 38 female workers at a condenser factory who were exposed for 1½ to 5 years to vapour from a mixture of ethylene glycol (40%), boric acid (55%), and ammonia (5%) kept at a temperature of 105 °C. Nine workers reportedly suffered from nystagmus and frequently lost consciousness; five other workers exhibited nystagmus only. Following changes to the production process which precluded further exposure, the reported symptoms ceased to occur.

66.1.2     Oral intake

There are numerous case reports in the literature of poisoning in humans due to accidental or intentional ingestion of ethylene glycol. In the United States, 6 to 60 deaths/year have been attributed to ethylene glycol ingestion. (LaKind et al. 1999, Jacobsen & McMartin 1997, ACGIH 2001, BUA 1991, ATSDR 1997).

The clinical symptoms of acute ethylene glycol poisoning can be divided into three (and occasionally four) fairly distinct stages. The severity of these stages and the advance from one stage to the next depends greatly on the amount of ethylene glycol entering the body. (LaKind et al. 1999, Jacobsen & McMartin 1997, Cavender & Sowinski 1994, BUA 1991, ACGIH 2001).

The first stage consists primarily of “central nervous system (CNS) effects”. This stage usually occurs shortly after ingestion of ethylene glycol, within 30 minutes to 12 hours. The CNS effects are characterised by signs of drunkenness (but without the characteristic breath odour of alcohol), nausea, vomiting, and at large doses coma followed by convulsions and death in some cases. Mild hypotension, tachycardia, low-grade fever, depressed reflexes, generalised or focal seizures, myoclonic jerks, and titanic contractions can occur. Ocular signs (nystagmus, ophthalmoplegia, papilledema, and subsequent optic atrophy) have also been reported.

The second stage has been described as the “cardiopulmonary effects” stage that occurs 12 to 72 hours after ingestion. This stage may also be characterised by severe metabolic acidosis. Symptoms that occur include tachypnoea, tachycardia, mild hypotension, and cyanosis. In severe cases, pulmonary oedema, bronchopneumonia, cardiac enlargement, and congestive failure are present. Hypocalcaemia may occur secondary to precipitation of metabolically formed oxalate and calcium deposits. Death in this stage usually occurs between 24 and 72 hours after exposure and is attributed to pulmonary oedema, cardiac dilation, and bronchopneumonia.

The third stage is known as the “renal failure” stage and occurs 24 to 72 hours after ingestion. This stage is characterised by profound acidosis. The renal damage may vary from mild increase in blood urea nitrogen and creatinine followed by recovery, to complete anuria with acute tubular necrosis that can lead to death. Histological changes include tubular epithelial degeneration and necrosis and oxalate crystal deposition in the kidney, lower urinary tract, and other organs (e.g., the brain).

The three clinical stages of acute oral ethylene glycol toxicity may overlap with the different latency periods before each stage, depending on the amount of ethylene glycol ingested. Often, Stage II never develops, yet the patient shows symptoms of the third stage of intoxication. (LaKind et al. 1999).

Other neurological symptoms, effects on cranial nerves, have been identified six or more days after ingestion and constitute a possible fourth clinical stage. These delayed neurological effects have resulted in facial paralysis, facial palsy, hearing loss, elevated protein levels in the cerebrospinal fluid, and bilateral cranial nerve dysfunction. These symptoms are uncommon and the mechanism of injury is unknown, but is distinctly different from the pathological events of the first three stages. (LaKind et al. 1999).

The oral dose of ethylene glycol required to cause death in humans is not well defined in the literature (ATSDR 1997).

The minimal lethal dose has been estimated, based on poisonings from accidental or intentional ingestion, at about 100 ml (about 111 g corresponding to about 1.6 g/kg b.w. for an adult) (LaKind et al. 1999, ACGIH 2001, BUA 1991, Cavender & Sowinski 1994, ATSDR 1997, NTP 1993).

However, early diagnosis and appropriate therapeutic measures can significantly reduce mortality, so that even doses of 970 ml (about 1080 g corresponding to 15 g/kg b.w. for an adult) have been survived (Gaultier et al. 1976 – quoted from BUA 1991).

66.1.3     Dermal contact

The potential for subchronic human exposure to ethylene glycol (as well as other substances) to induce effects in motor-servicing workers who performed various tasks that brought them into contact with motor vehicle antifreeze has been studied by Laitinen et al. (1995 – quoted from LaKind et al. 1999). Individual exposures were intermittent, but the workers had been performing their jobs for up to 23 years. Exposures resulted in enhanced urinary excretion of ethylene glycol by workers compared with office worker controls, as well as enhanced ammonia excretion (typical of chronic acidosis associated with ethylene glycol exposure). Because measured airborne levels of ethylene glycol were below the detection limits, the authors concluded that exposure occurred primarily via dermal contact with ethylene glycol during extended contact with the antifreeze.

66.1.4     Skin irritation

In order to study the irritation and sensitisation potential, ethylene glycol was applied to infrascapular skin of 401 human volunteers nine times over a 3-week period, in 24-hour occluded and semioccluded patch tests. Sites were evaluated after 24 hours for local irritancy. Initial and challenge applications resulted in marginal erythematous reactions in 9.3 and 12.2% of the individuals, respectively. Definite erythema, seen in a smaller group during the induction period, suggested potential cumulative irritation. (Union Carbide 1990 – quoted from LaKind et al. 1999).

One-inch square gauze pads completely wetted with 0.11, 1.1, or 10% (v/v) ethylene glycol were applied to the backs of 13 volunteers with no known previous exposure to ethylene glycol. No skin reactions were reported from any of the patches after 1, 2, 4, or 8 hours. (Shupack et al. 1981 – quoted from LaKind et al. 1999 and from ACGIH 2001).

Out of 1556 dermatitis patients subjected to 20/24-hour closed patch tests, 3.9% (61) experienced an irritant response (of unspecified severity) to neat ethylene glycol (Hannuksela et al. 1975 – quoted from IUCLID 2000).

After treatment with a concentration of 5% ethylene glycol in water, one woman showed strong skin reactions while 10 other subjects showed no reactions (Hindson & Ratcliffe 1975 – quoted from IUCLID 2000).

The application of 3% ethylene glycol in ethanol (patch test) resulted in a positive reaction in 1 out of 9 persons (Dawson 1976  – quoted from IUCLID 2000).

Ethylene glycol produces no significant irritant effect on the skin. A slight maceration of the skin may result from prolonged exposures. (Rowe & Wolf 1982 – quoted from Cavender & Sowinski 1994 and from IUCLID 2000).

66.1.5     Eye irritation

Exposure of human eyes to vapour or spray of ethylene glycol for 4 weeks at 17 mg/m³ resulted in no effects (Grant 1986 – quoted from Cavender & Sowinski 1994 and from ACGIH 2001).

A splash of neat ethylene glycol into the eye of a worker produced marked inflammation, but no permanent damage (BIBRA 1993 – quoted from IUCLID 2000).

66.1.6     Skin sensitisation

Ethylene glycol was applied to infrascapular skin of 401 human volunteers nine times over a 3-week period, in 24-hour occluded and semioccluded patch tests. Sensitisation was assessed after a 3-week rest, by 24-hour patch test challenges at distal sites. Sensitisation was suggested in fewer than 1% of the volunteers, but this response was not confirmed after rechallenge. (Union Carbide 1990 – quoted from LaKind et al. 1999).

In 15 (1%) out of 1556 dermatitis patients subjected to 20/24 hour closed patch tests, the skin reaction was described as allergic in character (Hannuksela et al. 1975 – quoted from IUCLID 2000).

Allergic dermatitis have been reported in two workers handling a fluid containing ethylene glycol (25 or 33%) in the preparation of contact lenses for periods of 3 to 4 months. The sensitisation was confirmed by their positive reactions to 48-hour closed patch tests. The application of 3% ethylene glycol in ethanol or 5% in water to the skin of the two workers induced a positive reaction. (Dawson 1975, Hindson & Ratcliffe 1975 – both quoted from IUCLID 2000 and from BUA 1991).

66.2     Toxicity to reproduction

No data have been found.

66.3     Mutagenic and genotoxic effects

No data have been found.

66.4     Carcinogenic effects

No data have been found.

67     Animal toxicity

67.1     Single dose toxicity

The acute toxicity of ethylene glycol in experimental animals closely mirrors the acute effects seen in humans (see part 3). One exception is the fourth clinical stage of delayed cranial neurological consequences observed in humans; no studies have been found that reported this stage in animals (LaKind et al 1999).

Susceptibility to ethylene glycol intoxication varies with species, sex, and individual (NTP 1993). Cats appear to share the susceptibility of humans; however, because cats are unusually sensitive to ethylene glycol due to their high baseline production of oxalic acid, they are not representative of experimental animal species predictive of human responses to ethylene glycol exposure (LaKind et al 1999).

67.1.1     Inhalation

According to Cavender & Sowinski (1994), the one-hour LC₅₀-value in rats is 10.9 g/m³.

In a study of rats, all animals survived an 8-hour exposure to saturated atmosphere (20 ºC: ca. 200 mg/m³ (calculated)) of ethylene glycol (BASF 1961 – quoted from IUCLID 2000).

67.1.2     Oral intake

The reported oral LD₅₀-values for ethylene glycol in rats ranged from >2.0 to 11.3 g/kg b.w. (IUCLID 2000, LaKind et al. 1999, NTP 1993, BUA 1991, Cavender & Sowinski 1994, A&H 1993).

In other experimental animals, the reported oral LD₅₀-values ranged from 5.89 to 15.4 g/kg b.w. in mice, from 7.0 to 9.3 g/kg b.w. in rabbits, and from 4.0 to 8.2 g/kg b.w. in guinea pigs (IUCLID 2000, LaKind et al. 1999, NTP 1993, BUA 1991, Cavender & Sowinski 1994, A&H 1993).

For the cat, oral LD₅₀-values of 1670 and 4700 mg/kg b.w. have been reported and for the dog, from 4000 to 8200 mg/kg b.w. (IUCLID 2000, A&H 1993).

A minimal lethal dose of 3800 mg/kg b.w. has been reported for rats, of 1000 mg/kg b.w. for cats, and of 6700 and 7300 mg/kg b.w. for dogs (LaKind et al. 1999).

Monkeys (Macaca fascicularis) received ethylene glycol orally at doses of 1000, 2000, or 4000 mg/kg b.w. and were observed 14 days after dosing. At the highest dose level, the two animals were found comatose 22 hours after dosing and died 26 and 28 hours after treatment. No further details are given. (ICI 1979 – quoted from IUCLID 2000).

67.1.3     Dermal contact

Dermal LD₅₀-values for ethylene glycol of 9.53 g/kg b.w. (Cavender & Sowinski 1994, A&H 1993) and of 10.6 g/kg b.w. have been reported for the rabbit (IUCLID 2000, BUA 1991).

67.1.4     Skin irritation

Guillot et al. (1982) have evaluated skin irritation in rabbits by determination of the primary cutaneous irritation index under patch-test and by determination of the cumulative irritation index after repeated exposure (6 weeks, MMII) according to official French methods but with some complements or modifications.

In the primary cutaneous irritation test (the experimental procedure is quoted from BUA 1991), 0.5 ml of neat ethylene glycol was applied to undamaged and scarified skin of 6 rabbits and covered with an occlusive dressing. The duration of exposure was 23 hours and assessments of the effects were made one hour and 48 hours after removal of the dressing. Ethylene glycol showed a primary irritation index of 0.08 (an index of up to 0.5 is considered non-irritant and an index of 0.5-2.0 as slightly irritant).

In the cumulative cutaneous irritation test (the experimental procedure is quoted from IUCLID 2000), three rabbits were treated daily with 2 ml of neat ethylene glycol or with a 10% aqueous solution for 6 weeks.  Ethylene glycol undiluted showed a mean maximum irritation index of 0.47 interpreted as ’well tolerated’ and the 10% solution an index of 0 interpreted as ’very well tolerated (the maximum index that could be scored is not stated; the highest index scored was 1.83 for polypropylene glycol and interpreted as ’relatively well tolerated).

Ethylene glycol was tested for skin irritative properties according to the Draize test in female New Zealand rabbits. Skin response was evaluated 24 and 72 hours after treatment. Ethylene glycol had a low potential for skin irritation (score 0.4, with a score of <2.0 meaning mild or no irritation). (Clark et al. 1979).

67.1.5     Eye irritation

Moderate to severe eye irritation was observed in rats and rabbits exposed continuously for 90 days to ethylene glycol (vapour) at a concentration of 12 mg/m³. Two rats developed corneal opacity after 8 days and appeared to be blind for the remainder of the exposure. Erythema, oedema, and discharge began in rabbits after 3 days of exposure, the oedema being severe enough to result in virtual closure of the eyes. Guinea pigs, dogs, and monkeys exposed similarly showed no effects on the eyes. See also part 4.2.1. (Coon et al. 1970).

Rats, guinea pigs, rabbits, dogs, and monkeys were exposed to a concentration of 57 mg/m³ of ethylene glycol (vapour) 8 hours a day, 5 days per week for 6 weeks; none of the animals showed any signs of ocular irritation. In rabbits exposed similarly to 10 mg/m³, mild conjunctivitis was noted in one eye of each of 2 rabbits during the 4^th and 5^th weeks, which persisted until the end of the exposure; each of these rabbits also developed a small lesion over the irritated eye. These signs were, according to the authors, probably brought on by accidental trauma, which may have been aggravated by the exposure. See also part 4.2.1. (Coon et al. 1970).

Diluted ethylene glycol (10, 20, or 50 % solution in water) caused only slight oedema and erythema under occlusive conditions in the eyes of rabbits. Instillation of neat ethylene glycol produced moderate to severe oedema and erythema. No effects were observed 48 hours post-treatment. According to IUCLID, ethylene glycol is evaluated as being moderately irritating to the eyes. (Star 1980 – quoted from IUCLID 2000 and from BUA 1991).

Toxicity and irritation of ethylene glycol were assessed in rabbit eyes following multiple topical or multiple intraocular (anterior chamber) administrations. The concentration of the test solutions was 0.04, 0.4, 4.0, and 40% (topical administration only) in balanced salt solution. The balanced salt solution and saline were used as negative controls. Only sterile test solutions were used. (McDonald et al. 1972).

Regarding multiple topical administration, one drop (approximately 0.05 ml) of the test solution was instilled into the cul-de-sac of the test eye (6 eyes per concentration) at 10-minute intervals for a total of 36 applications in a 6-hour period. All eyes were examined with a biomicroscope at 6 hours after the first application and the eyes were graded (iris, flare, cornea) according to an arbitrary numerical score. Eyes were also scored for palpebral and bulbar conjunctival irritation by the method of Draize at 2, 4, and 6 hours after treatment and daily thereafter, and scored. Based on the Draize scores, 0.4% was the highest concentration to be non-irritating. Irritation at higher concentrations consisted of chemosis, swelling, and conjunctival redness. No test concentration elicited significant toxic findings on biomicroscopic examination. All eyes were normal by 7 days.

Regarding multiple intraocular administration, 0.5 ml of the test solution was instilled once per day for 5 days into the anterior chamber. All eyes were examined with a biomicroscope before test and on days 2, 4, 7, and 14 after the first injection and the eyes were graded (iris, flare, cornea) according to an arbitrary numerical score. Based on the biomicroscopic scores, 0.4% was the highest concentration to be non-toxic.

No evidence of systemic toxicity was, according to the authors, observed following extraocular and intraocular administration, based on behaviour, appearance, and body weight.

Twenty-four hours after the application of 0.5 ml 80% ethylene glycol into the eye of rabbits, no eye irritation could be observed. The lowest non-irritating concentration of ethylene glycol was 20% when applied as 0.1 ml solution 5 times a day for 21 consecutive days. (McDonald et al. 1977 – quoted from IUCLID 2000 and from BUA 1991).

Guillot et al. (1982) have evaluated eye irritation in rabbits by determination of the ocular irritation index according to official French methods but with some complements or modifications. Ethylene glycol (0.1 ml) was instilled into the eyes of six rabbits. It is not stated whether the test substance was washed out or not. Assessments of the irritative effect were made 1, 24, 48, 72, 96, and 168 hours after treatment. Ethylene glycol showed an ocular irritation index of 11.33; a compound was not considered to provoke any significant injury to the eye mucous membrane when no opacity of the cornea occurred and when the ocular irritation index was less than 15. The experimental procedure is quoted from BUA (1991).

Ethylene glycol was tested for eye irritative properties according to the Draize test in female New Zealand rabbits. Eyes were inspected 1, 24, 48, 72 and 96 hours after instillation of 0.1 ml of the fluid (>99% ethylene glycol). A slight irritation was observed one hour after treatment (score: 3.0), but continually diminished with time and could not be noticed after 96 hours. The maximum score possible for a single evaluation was 110. (Clark et al. 1979).

67.1.6     Sensitisation

No data have been found.

67.2     Repeated dose toxicity

67.2.1     Inhalation

67.2.1.1     Rats

Rats (15 Sprague-Dawley and Long-Evans male and female animals per group, no information about distribution between species and sexes) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 123 animals. (Coon et al. 1970).

In the continuous study, 1 rat died during exposure and 4 rats in the control group. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. Moderate to severe eye irritation was observed and 2 animals developed corneal opacity after 8 days and appeared to be blind for the remainder of the exposure.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed fatty changes and focal necrosis in the liver in 1 of 8 rats. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

Rats (10 animals) were exposed to ethylene glycol at concentrations of 350 to 400 mg/m³ 8 hours a day, 5 days per week for 16 weeks. One rat died. The histological examination of brain, lung, heart, liver, pancreas, spleen, lymph nodes, kidneys, adrenals, testes, stomach, and caecum did not show any substance-related pathological changes. (Wiley et al. 1936 – quoted from BUA 1991, ACGIH 2001, Cavender & Sowinski 1994 and from IUCLID 2000).

67.2.1.2     Mice

Mice (20 animals) were exposed to ethylene glycol at concentrations of 350 to 400 mg/m³ 8 hours a day, 5 days per week for 16 weeks. Three mice died. The histological examination of brain, lung, heart, liver, pancreas, spleen, lymph nodes, kidneys, adrenals, testes, stomach, and caecum did not show any substance-related pathological changes. (Wiley et al. 1936 – quoted from BUA 1991, ACGIH 2001, Cavender & Sowinski 1994 and from IUCLID 2000).

67.2.1.3     Guinea pigs

Guinea pigs (15 male and female animals per group, no information about distribution between sexes) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 73 animals. (Coon et al. 1970).

In the continuous study, 3 animals died during exposure. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. Occasional foci of inflammatory cells were seen in kidneys from several animals, but this was not interpreted, by the authors, as being specific chemically induced changes.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed hepatic fatty changes in 2 of 8 animals and focal necrosis in the liver in 1 of 8 animals; focal necrosis of the liver was also seen in 1 of 3 control guinea pigs. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. The livers of 1 of 8 animals revealed areas of focal necrosis; this was not considered, by the authors, to be chemically induced. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.4     Rabbits

Male New Zealand albino rabbits (3 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 12 animals. (Coon et al. 1970).

In the continuous study, 1 rabbit died during exposure. All haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. One rabbit had hamartomatosis (a benign tumour-like nodule) in liver bile ducts, but this was not interpreted, by the authors, as being a specific chemically induced change. Moderate to severe eye irritation was observed and erythema, oedema, and discharge began after 3 days of exposure, the oedema being severe enough to result in virtual closure of the eyes. 

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, mild conjunctivitis was noted in one eye of each of two rabbits during the 4th and 5th weeks, which persisted to the end of the exposure; each of these rabbits also developed a small lesion over the irritated eye. These signs were, according to the authors, probably brought on by accidental trauma, which may have been aggravated by the exposure. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.5     Dogs

Male Beagle dogs (2 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 12 animals. (Coon et al. 1970).

In the continuous study, all haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 10 mg/m³, histopathological examination revealed mild congestion in the spleens of both exposed animals. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. None of the animals showed any signs of ocular or nasal irritation.

67.2.1.6     Monkeys

Male squirrel monkeys (2/3 animals per group) were exposed to ethylene glycol (vapour) continuously at a concentration of 12 ± 2 mg/m³ for 90 days (continuous study), or at concentrations of 10 ± 1 or 57 ± 14 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). The control group consisted of 8 animals. (Coon et al. 1970).

In the continuous study, all haematological data were within normal limits. Observations during necropsy revealed normal organs and tissues. Histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls.

In the repeated study, there were no deaths at both exposure levels and all haematological values were within normal limits. At 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals. The livers of 2 of 3 animals revealed areas of focal necrosis; this was not considered, by the authors, to be chemically induced. None of the animals showed any signs of ocular or nasal irritation.

Two chimpanzees were exposed by inhalation to air saturated with ethylene glycol (stated to be 256 mg/m³) for 28 days. A biopsy revealed that one animal had oxalate crystals in the kidney. Both animals experienced an unexplained rise in haemoglobin concentration and mean red cell volume, as well as a decreased ability to concentrate urine. (Felts 1969 (abstract) – quoted from LaKind et al. 1999, Cavender & Sowinski 1994).

When monkeys were exposed to an ethylene glycol aerosol (500 mg/m³) for up to 30 weeks, oxalate crystals were found in the kidneys (Harris 1969 (abstract) – quoted from LaKind et al. 1999).

67.2.2     Oral intake

67.2.2.1     Rats

Sprague-Dawley rats (10 males and females per group) received ethylene glycol in their drinking water for 10 or 90 days. In the 10-day study, the concentrations were 0, 0.5, 1.0, 2.0, or 4.0%. Based on a water consumption of 100 ml/kg b.w. per day, these concentrations correspond to 0, 554, 1108, 2216, or 4432 mg/kg b.w./day. In the 90-day study, females received the same concentrations in the drinking water whereas males received 0, 0.25 (227 mg/kg b.w./day), 0.5, 1.0, or 2%. (Robinson et al. 1990 – quoted from BUA 1991 and from IUCLID 2000).

In the 10-day study, high-dose (4%) male rats showed a strong reduction in body weight and in heart, liver, spleen and thymus weights. High-dose female rats had changes in blood parameters (decreased haemoglobin and haematocrit and markedly reduced numbers of erythrocytes and leucocytes) and decreased thymus weights. In male animals at the 2 and 4% dose levels, histopathological examination of the kidneys revealed a dose-dependent increase in the incidence and severity of kidney damage, which involved dilation, degeneration and necrosis of the renal tubules, acute inflammation and intratubular deposits of proteinaceous material and calcium oxalate crystals. In females, the effects on kidneys were limited to tubular dilation and intratubular deposition of proteinaceous material at the highest dose level. A NOAEL of 1% ethylene glycol in the drinking water (1108 mg/kg b.w./day) for 10 days of exposure can be considered.

In the 90-day study, 8 female and 2 male animals from the high-dose group (4% and 2%, respectively) died. Body weight gain was markedly reduced in high-dose (2%) males. In females, the leucocyte count was significantly reduced at 0.5, 2. and 4%. In male animals at the 1 and 2% dose levels and in female animals at the 2 and 4% dose levels, histopathological examination of the kidneys showed a dose-dependent increase in the incidence and severity of kidney damage, which involved dilation, degeneration and inflammation of the renal tubules, and renal pelvis. A NOAEL of 0.5% ethylene glycol in the drinking water (554 mg/kg b.w./day) for 90 days of exposure can be considered based on the renal effects in male animals.

In a 13-week study, ethylene glycol was administered in the feed to Fischer 344/N rats (10 males and 10 females) at dose levels of 0, 0.32, 0.63, 1.25, 2.5, or 5.0% (equivalent to 0, 160, 315, 625, 1250, or 2500 mg/kg b.w./day assuming than an adult rat consumes 50 g feed/kg b.w./day). Four high-dose male rats died. The body weight gain for male rats in the 2.5 and 5% dose groups was depressed by more than 10% when compared to the control group. The relative kidney weight was significantly increased in both male and female rats in the two highest dose groups (2.5 and 5%) and the relative thymus weight was significantly decreased in high-dose (5%) male rats. Serum urea nitrogen and serum creatinine levels were significantly elevated in male animals at the two highest dose levels (2.5 and 5%) and kidney lesions were observed in all male rats at these dose levels. Damaged kidneys contained calcium oxalate crystals mainly located within tubular lumens in the renal cortex, but were also occasionally found in tubules in the medulla. Crystals were also observed in the urinary bladder, the urethral lumen, and in the brain of some high-dose male rats (5%). Severe toxic nephrosis (distension and dilation of renal tubules, necrosis and regeneration of tubule epithelium, thickening of basement membranes, and fibrosis) was diagnosed in the 5% male dose group and moderate toxic nephrosis in the 2.5% male dose group. Toxic lesions in the kidneys of female rats, which were multifocal and tended to be subcapsular, were only observed in the high-dose group (5%); no crystals were observed. Based upon the renal effects observed in male rats, a NOAEL of 1.25% (equivalent to 625 mg/kg b.w./day) can be considered. According to the author, this dose level corresponds to 0.6 to 1.0 g/kg b.w./day. (Melnick 1984).

Wistar rats (15 animals of each sex per group) were administered dietary doses of 0, 0.05, 0.1, 0.25, or 1% (according to IUCLID corresponding to 0, 35/38, 71/85, 180/185, or 715/1128 mg/kg b.w. per day for males and females, respectively) for 16 weeks. In male rats at the two highest dose levels, increased levels of oxalic acid and oxalate crystals were found in the urine and histopathological examination revealed damage to the kidneys. In addition, in males at the highest dose level, increased kidney weight and impairment of renal function was observed. In female animals, the same effects were less severe and only occurred at the highest dose level. Based on the effects observed in male animals, a NOAEL of 71 mg/kg b.w. per day is considered. (Gaunt et al. 1974 – quoted from BUA 1991 and from IUCLID 2000).

Fischer 344 rats (130 males and females per group) were fed diets (0.1, 0.5, or 2.5%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. (DePass et al. 1986a).

High-dose (2.5%) male rats had a significant increase in mortality rate from the 9^th through the 16^th month of the study and the last high-dose male rat died after 474 days of the study. Other significant findings in high-dose male rats only included: increased water intake; decreased body weight gain; decreased red blood cell count, haematocrit, and haemoglobin concentration; increased neutrophilic leukocyte count; increased serum levels of creatinine and urea; increased urine volume; increased absolute and relative kidney weights after 6 and 12 months of treatment; and decreased absolute and relative liver weights after 12 months of treatment. In female rats of the highest dosage group, kidney weights were increased after 6 and 18 months of treatment, but not after 12 months. Calcium oxalate crystals were found in urine samples from all but one male of the high-dose animals after 12 months of treatment; at 18 and 24 months, all but one of the high-dose female rats had calcium oxalate crystals.

At the 6-month sacrifice, the incidence of renal lesions (tubular hyperplasia, tubular dilation, peritubular nephritis, and calcium oxalate crystalluria) was significantly increased in the high-dose males (2.5%) and calcium oxalate crystals were present in the urinary bladder in two high-dose male rats; these conditions were absent in the other male dose groups and in females.

At the 12-month sacrifice, all high-dose males (2.5%) had chronic nephritis (multiple severe histopathological changes including tubular dilation and proteinosis, glomerular shrinkage, tubular cell hyperplasia, and chronic interstitial nephritis) with calcium oxalate crystalluria and 50% had oxalate crystals in the urinary bladder; these findings were not present in males at the lower dose levels or in females.

By the time of the 18-month sacrifice, all of the high-dose males (2.5%) had died or were sacrificed moribund. In most of these animals, oxalate nephrosis was the primary cause of death. Kidneys from these animals had tubular obstruction by large crystals with secondary tubular dilation and degeneration. Calculi were sometimes found within the renal pelvic space, urethras, and urinary bladder, often with an associated hydronephrosis. Extrarenal lesions included cellular hyperplasia of the parathyroid glands and a significant increase in the incidence of splenic haemosiderosis.

Among the females sacrificed at 2 years, the incidences of haemosiderosis of the mesenteric lymph node (2.5%) and mild fatty metamorphosis of the liver (0.5 and 2.5%) were significantly increased. No biologically significant lesions were observed in the male rats sacrificed at 2 years.

Based on the effects observed in the kidneys, a NOAEL of 200 mg/kg b.w. per day is considered. For females, a NOAEL of 40 mg/kg b.w. per day can be considered based on the effects observed in the liver of high-dose females.

Sprague-Dawley rats (16 male and female animals per group) received ethylene glycol in their diet at concentrations of 0, 0.1, 0.2, 0.5, 1.0, or 4.0% (equivalent to 0, 50, 100, 250, 500 or 2000 mg/kg b.w./day assuming than an adult rat consumes 50 g feed/kg b.w./day). An increased mortality rate was observed in males of the two highest dose groups (1 and 4%) and in females of the highest dose group (4%). A significant decrease in growth was observed in male rats from week 16 at 4% and after week 70 at 1%, and in female rats after about one year at 4%. A significantly increased water consumption was noted in male rats at 1 and 4% and in female rats at 4%, and in these dose groups protein was found in the urine. The mean terminal kidney, lung and liver weights were lower (whether the reduction is significant is not stated in the publication) in male animals at 0.1% and above compared to controls. Calculi and crystal deposition in the kidneys were observed in male rats from 0.5% (only 1/16 animals at 0.5%) and in female rats at 4% and from 1%, respectively. Morphological changes in the kidneys also included degeneration of the tubular epithelium, manifested mainly as cytoplasmic vacuolisation; however, no details are provided at which dose levels these morphological changes were observed. According to the author, it appears probable that the NOEL is no higher than 0.2% (equivalent to 100 mg/kg b.w./day) ethylene glycol in the diet but may be less. (Blood 1965).

67.2.2.2     Mice

In a 13-week study, ethylene glycol was administered in the feed to B6C3F1 mice (10 males and 10 females) at dose levels of 0, 0.32, 0.63, 1.25, 2.5, or 5.0% (equivalent to 0, 480, 945, 1875, 3750, or 7500 mg/kg b.w./day assuming than an adult mouse consumes 150 g feed/kg b.w./day). There were no deaths, the relative weight gain data did not show any clear dose-related effects, and there were no differences in organ weights. Renal lesions diagnosed as mild toxic nephrosis (tubular dilation, cytoplasmic vacuolisation, and regenerative hyperplasia with piling up of nuclei) were observed in about half of the male high-dose mice (5%) and in one male mouse in the 2.5% dose group. There was no evidence of crystal formation in the affected tubules. A degenerative change (accumulation of an eosinophilic hyaline material in the cytoplasm of hepatocytes adjacent to or close to central veins) was present in the livers of all the male mice in the 2.5 and 5% dose groups. There were no adverse effects observed in female mice at any of the dose levels. Based upon the effects observed in kidneys and livers in male mice, a NOAEL of 1.25% (equivalent to 1875 mg/kg b.w./day) can be considered. (Melnick 1984).

B6C3F1 mice (60 animals of each sex per group) were fed diets containing ethylene glycol for 103 weeks. Male mice received 0, 6250, 12500, or 25000 ppm (equal to average daily levels of approximately 0, 1500, 3000, or 6000 mg/kg b.w./day) and female mice 0, 12500, 25000, or 50000 ppm (equal to average daily levels of approximately 0, 3000, 6000, or 12000 mg/kg b.w./day). There were no significant differences in survival between dosed and control groups. Mean body weights of exposed and control animals were similar and no treatment-related clinical findings or gross lesions were noted. Hepatocellular hyaline degeneration was seen in mid- and high-dose male and high-dose female mice. Pulmonary arterial medial hyperplasia was observed at an increased incidence in exposed females but not in exposed males. Incidence and severity of nephropathy were not affected by treatment in either sex. Small numbers of oxalate-like crystals, calculi, or both were noted in renal tubules, urethras, and/or urinary bladders in a few high-dose male mice. Based upon the effects observed in livers of female mice, a NOAEL of 12500 ppm (equal to approximately 3000 mg/kg b.w./day) can be considered. (NTP 1993).

CD-1 mice (80 males and females per group) were fed diets (0.1-0.05, 0.7-0.24, or 0.35-1.27%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. Renal tubular degeneration occurred slightly more frequently in high-dose females than in controls at the 18-month sacrifice, but was absent in the 15 high-dose females sacrificed at 24 month. A NOAEL of 1000 mg/kg b.w. per day can be considered. (DePass et al. 1986a).

67.2.2.3     Monkeys

Rhesus monkeys (2 males and 1 female) were given ethylene glycol in their diet at 0.2% (males) or 0.5% (female) for 3 years. No calculi or abnormal calcium deposits were demonstrated by X-ray examination and no other effects on organs and tissues were observed at the histopathological examination. (Blood et al. 1962).

67.2.3     Dermal contact

No data have been found.

67.3     Toxicity to reproduction

67.3.1     Inhalation

CD rats and CD-1 mice (25 animals per group) were exposed to a respirable ethylene glycol aerosol (mass median aerodynamic diameter (MMAD) 2.3 µm) on gestational days (GD) 6 to 15, 6 hours a day, by whole-body exposures (target concentrations: 0, 150, 1000, or 2500 mg/m³). Rats were sacrificed on gestation day 21, and mice on gestation day 18. (Tyl et al. 1995a).

The total concentrations of ethylene glycol (aerosol plus vapour) were 79, 89, or 84% of target concentrations, respectively (119 ± 13, 888 ± 149, or 2090 ± 244 mg/m³). The vapour phase was 82% of the total concentration for the 150 mg/m³ group; for the higher aerosol concentrations, the vapour phase was 19-20% of the total concentration.

All rat dams survived to scheduled termination. Food and water consumption, maternal body weights and weight gain, and maternal organ weights (other than liver) were unaffected by exposure. A significant increase in absolute and relative liver weight was observed at 2500 mg/m³. Gestational parameters (pre- and post-implantation loss, live foetuses/litter, sex ratio, and foetal body weight/litter) were unaffected by exposure. There was no significant increase in the incidence of any individual malformation, in the incidence of pooled external, visceral, or skeletal malformations, or in the incidence of total malformations by foetus or by litter. There were no significant increases in the incidence in any foetal external or visceral variations. There was some evidence of treatment-related reductions in ossification of the foetal skeleton, including an increase in the incidence of poorly ossified humerus (forelimb) and zygomatic arch (face) at the highest exposure level (2500 mg/m³) and an increase in the incidence of poorly ossified metatarsals and proximal phalanges of the hindlimb at 1000 mg/m³ but not at 2500 mg/m³. According to the authors, the NOAEL was 1000 mg/m³ for maternal and 150 mg/m³ for developmental toxicity.

All mouse dams survived to scheduled termination. One female at 2500 mg/m³ had a totally resorbed litter at termination; all other pregnant animals had one or more live foetuses at sacrifice. Clinical signs included only wet fur for all ethylene glycol exposed mice. Reduced body weight and body weight gain were observed at 1000 and 2500 mg/m³ both during and after the exposure period. Gravid uterine weight was also reduced at the two highest exposure levels so that body weight corrected for gravid uterine weight was unaffected by treatment. Liver and kidney weights were unaffected by treatment. The following gestational parameters were affected: the number of viable implantations per litter was reduced at 2500 mg/m³, the number of nonviable implantations per litter was increased at 1000 and 2500 mg/m³, the number of early resorptions was increased (not significantly) at 2500 mg/m³; the sex ratio was reduced at 1000 mg/m³ but not at 2500 mg/m³, and the foetal body weights per litter (male, female, and total) were reduced at 1000 and 2500 mg/m³. There was a significant increase in the incidence of a number of external, visceral, and skeletal malformations at 1000 and 2500 mg/m³, as well as in the incidence of pooled external, visceral, and skeletal malformations, and in the incidence of total malformations. Malformations were found in the head (exencephaly), face (cleft palate, foreshortened and abnormal face, and abnormal facial bones), and skeleton (vertebral fusions, and fused, forked, and missing ribs). The incidences of many foetal variations were also increased at the two highest dose levels, but only a few at the lowest dose level (150 mg/m³). According to the authors, the NOAEL was 150 mg/m³ for maternal and at or below 150 mg/m³ for developmental toxicity.

Ethylene glycol was teratogenic to mice by whole-body exposure to aerosol (1000 and 2500 mg/m³), see the study described above. According to the authors, the results were confounded by possible exposure from ingestion after grooming and/or from percutaneous absorption. Therefore, CD-1 mice (30 animals per group) were exposed to ethylene glycol aerosol (MMAD 2.6 ± 1.7 µm) on gestational days 6 to 15, 6 hours a day, by nose-only (target concentrations: 0, 500, 1000, or 2500 mg/m³) or whole-body exposures (target concentrations: 0 or 2100 mg/m³). Control environments were water aerosol (4200 mg/m³ for nose-only and 2700 mg/m³ for whole-body). On gestation day 18, the dams were sacrificed. (Tyl et al. 1995b).

In either exposure regimen, body weights, body weight gain (absolute or corrected for the weight of the gravid uterus), and liver weights (absolute or relative) were unaffected by treatment. Microscopic examination of maternal kidneys indicated no treatment-related incidence or severity of renal lesions. There were no treatment-related differences among groups in the number of corpora lutea per dam, the number of total or viable implantations per litter, or on foetal sex ratio. There was no significant increase in the incidence of any individual external or visceral malformation or variations, or of all external or visceral malformations or in total variations.

In the nose-only experiment, maternal clinical signs associated with the animals struggling while in restraint were observed. Maternal kidney weights were increased at concentrations of 1000 (absolute) and 2500 (absolute and relative) mg/m³ and there was a trend toward reduced gravid uterine weight at these concentrations as well. The percentage of live foetuses was reduced slightly (but not significantly) at the two highest concentrations. At the highest concentration (2500 mg/m³), the foetal body weights per litter were significantly reduced and the incidences of one skeletal malformation (fused ribs) and 18 skeletal variations were increased. According to the authors, exposure of CD-1 mice to a respirable ethylene glycol aerosol during organogenesis by nose-only inhalation resulted in minimal maternal toxicity at 1000 and 2500 mg/m³ and developmental toxicity at 2500 mg/m³; the NOAEL was 500 mg/m³ for maternal and 1000 mg/m³ for developmental toxicity.

In the whole-body dose group (2100 mg/m³), the gravid uterine weight, the percentage of live foetuses (due to an increase in late resorptions), and the foetal body weights per litter were significantly reduced. There was an increase in the incidence of a number of skeletal malformations, including fused thoracic arches, extra thoracic arches, fused lumbar arches and centra, fused ribs, and extra ribs between existing ribs, as well as in the incidence of pooled skeletal malformations and a total of 63 skeletal variations exhibited significantly increased incidences.

67.3.2     Oral intake

67.3.2.1     Rats

To assess the possible effects of ethylene glycol on reproductive performance, a three-generation reproduction study was performed in Fischer 344 rats. Ethylene glycol was administered in the diet at approximate doses of 40, 200, or 1000 mg/kg per day (weekly calculated doses ranged from 40-50, 200-300, or 1000-1300 mg/kg/day for males and from 40-60, 200-300, or 900-1200 for females). Two untreated diet control groups were included. At approximately 100 days of age, 10 males were mated to 20 females in each dose group. Necropsies and microscopic examinations of several organs and tissues were performed (5 animals of each sex from each dose level) on the F₂ parents and on the F₃ weanlings. There was no treatment-related effect on body weight gain or diet consumption, nor was there any mortality among parental rats. No treatment-related effect was observed for any of the reproductive indices (fertility index, gestation index, gestation survival index, survival indices, and days from first mating to litter) for all three generations, or on neonatal body weight at days 4, 14, or 21 postpartum. The histopathological examinations revealed no treatment-related findings in the F₂ parents and in the F₃ weanlings, including kidney damage. The NOAEL for reproductive toxicity was 1000 mg/kg b.w./day. (DePass et al. 1986b).

Fischer 344 rats were fed ethylene glycol in their diet from gestation day 6 to 15 so that the animals received doses of 0, 40, 200, or 1000 mg/kg b.w./day. Animals were sacrificed on gestation day 21 and the foetuses were examined. The only effects found were a non-significant increase in pre-implantation losses at the highest dose level and a delay in ossification of the foetal skeleton. The incidence of malformations was not significantly different to the negative controls. No maternal effects were observed at any of the dose levels; however, only body weight of the dams was examined. (Maronpot et al. 1983 – quoted from BUA 1991 and from IUCLID 2000).

In order to determine a NOAEL for developmental toxicity of ethylene glycol administered orally, CD rats received 0, 150, 500, 1000, or 2500 mg/kg b.w. per day by gavage on gestation days 6 to 15. At the highest dose level, water consumption was increased during treatment and body weights were reduced throughout gestation; liver and kidney weights were increased at sacrifice (gestation day 21). Relative liver weights were also increased at 1000 mg/kg/day. Effects observed in foetuses at 2500 mg/kg/day included hydrocephaly; gastroschisis; umbilical hernia; fused, duplicated, or missing arches, centra, and ribs; poor ossification in thoracic and lumbar regions; and reduced body weights. At 1000 mg/kg/day, reduced body weights; duplicated or missing ribs, centra, and arches; and poor ossification were observed. According to the authors, the NOAEL for developmental toxicity was 500 mg/kg b.w./day. (Neeper-Bradley et al. 1995 – abstract quoted from TOXLINE 1995-1998).

Timed-pregnant CD rats (at least 20 animals per group) were dosed by gavage with ethylene glycol in distilled water on gestational days 6 through 15 at doses of 0, 1250, 2500, or 5000 mg/kg b.w. No maternal deaths or distinctive clinical signs were noted, except for piloerection which was seen in all treated groups but not in controls. Maternal body weight gain during treatment was significantly reduced in all dose groups. Gravid uterine weight was reduced at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. Absolute liver weight was significantly decreased at the high dose and relative kidney weight was increased in the mid- and high-dose groups. Dose-related increases in post-implantation loss per litter were observed with the high dose significantly above controls. The number of live foetuses per litter and foetal body weight per litter was significantly reduced at the mid and high doses. The percentage of malformed live foetuses per litter and/or the percentage of litters with malformed foetuses were significantly elevated in all dose groups and more than 95% of litters were affected at the high dose. A wide variety of malformations (external, visceral and skeletal) were observed with the most common being craniofacial and neural tube closure defects and axial skeletal dysplasia. (Price et al. 1985).

Price et al. (1988 – quoted from BUA 1991 and from IUCLID 2000) also have administered ethylene glycol by gavage to CD rats from gestation day 6 to 20 at doses of 0, 250, 1250, or 2250 mg/kg b.w./day. The offspring were reared by untreated dams and examined in respect to postnatal growth and survival rate, bodily development, the onset of sexual maturity, locomotive activity, and performance in a complex test of learning ability. At the two highest dose levels, there was a significant increase in the duration of gestation; microscopic examination revealed an increase in kidney damage. At the highest dose level, maternal body weight gain was reduced. In offspring from dams exposed at the highest dose level, foetal mortality was increased, there was a reduction in live litter size and in the weight of the neonates, and there was a significant increase in the number of malformations (particularly in the form of hydrocephaly and abnormalities of the axial skeleton). Prenatal exposure to ethylene glycol had no adverse effect on postnatal learning behaviour.

Ethylene glycol was administered to pregnant Wistar rats from gestation day 6 to 15 orally by a stomach tube at dose levels of 253, 638, 858, 1073, or 1595 mg/kg b.w. The foetal body weight and crown-rump length were significantly reduced from 858 mg/kg and 1.8 to 43.6% of foetuses among these groups presented gastroschisis, exencephaly, meningoencephalocele, harelip, and rib malformation; malformation frequencies showed a dose-response relationship. (Longzhan et al. 1989).

67.3.2.2     Mice

Examination of testicular weight in mice, which were administered ethylene glycol by gavage at doses of 0, 500, 1000, 2000, or 4000 mg/kg b.w. per day, 5 days a week for 5 weeks, gave no indication of any testicular damage (Nagano et al. 1984 – quoted from BUA 1991).

In a continuous breeding study, CD-1 mice (20 animals of each sex in the dose groups; 40 animals of each sex in the control group) were given ethylene glycol in their drinking water at concentrations of 0, 0.25, 0.5, or 1% for 14 weeks. As a rough estimate, the dose (on a mg/kg body weight basis) was calculated using the average daily water consumption multiplied by the concentration of the chemical in that dose group and divided by the body weight. Between days 98 to 105, the average doses were 0, 410, 840, and 1640 mg/kg b.w., respectively. (Lamb et al. 1985).

No treatment-related effects were observed on body weight or water consumption, or in clinical signs of toxicity. At the highest dose level (1%), significant decreases in the number of litters per fertile pair, the mean number of live pups per litter, and the mean live pup weight were observed as compared to control F₀ mice. In the F₁ generation, the number of live pups per litter and the live pup weight were lower in the dosed group (1%), but differences were not significant. Facial anomalies were noted in a number of offspring of high-dose mice (1%) and an examination for skeletal defects demonstrated a pattern including reduction in the size of bones in the skull, fused ribs, and abnormally shaped sternebrae and vertebrae; examination by light microscopy of bones from treated mice did not reveal histological alterations. At least six pups from three different litters had cleft lip when observed grossly at birth. The NOAEL for reproductive effects was 0.5% (corresponding to an average dose of 840 mg/kg b.w./day).

In order to determine a NOAEL for developmental toxicity of ethylene glycol administered orally, CD-1 mice received 0, 50, 150, 500, or 1500 mg/kg b.w. per day by gavage on gestation days 6 to 15. There were no apparent treatment-related effects in dams. Effects observed in foetuses at 1500 mg/kg/day included reduced body weights; fused ribs and arches; poor ossification in thoracic and lumbar centra; and increased occurrence of an extra 14^th rib. At 500 mg/kg/day, slight reductions in foetal body weight and increased incidences of extra ribs were observed. According to the authors, the NOAEL for developmental toxicity was 150 mg/kg b.w./day. (Neeper-Bradley et al. 1995 – abstract quoted from TOXLINE 1995-1998).

Timed-pregnant CD-1 mice (at least 20 animals per group) were dosed by gavage with ethylene glycol in distilled water on gestational days 6 through 15 at doses of 0, 750, 1500, or 3000 mg/kg b.w. No maternal deaths or distinctive clinical signs were noted, except for piloerection which was seen in all treated groups but not in controls. Maternal body weight gain during treatment was significantly reduced in mid- and high-dose groups. Gravid uterine weight was reduced at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. Absolute liver weight was significantly decreased at the mid and high dose. Dose-related increases in postimplantation loss per litter were observed. The number of live foetuses per litter was significantly reduced at the high dose and foetal body weight per litter was significantly reduced at the mid and high doses. The percentage of malformed live foetuses per litter and/or the percentage of litters with malformed foetuses were significantly elevated in all dose groups and more than 95% of litters were affected at the high dose. A wide variety of malformations (external, visceral and skeletal) were observed with the most common being craniofacial and neural tube closure defects and axial skeletal dysplasia. (Price et al. 1985).

67.3.2.3     Rabbits

New Zealand White (NZW) rabbits (23-24 inseminated animals per group) were administered ethylene glycol by gavage on gestational day 6 through 19 at doses of 0, 100, 500, 1000, or 2000 mg/kg/day. Dams were sacrificed at gestation day 30.

Profound maternal toxicity (42% mortality, three early deliveries, and one spontaneous abortion) was observed at the highest dose (2000 mg/kg b.w./day). At necropsy, there were no significant effects on gravid uterine weight, and liver or kidney weights. Kidney damage observed at 2000 mg/kg b.w./day was limited to the cortical renal tubules and included intraluminal oxalate crystals, epithelial necrosis and tubular dilatation and renal tubular degeneration. There were no effects on pre- or postimplantation loss, the number of foetuses per litter, foetal body weight per litter, or sex ratio (percent male foetuses per litter), and no evidence of teratogenicity based on evaluation of external, visceral including craniofacial, skeletal or total malformations, or variations at any dose level. The NOAEL for maternal toxicity was 1000 mg/kg b.w./day and the NOAEL for developmental toxicity was 2000 mg/kg b.w./day. (NTP 1991, Tyl et al. 1993 – abstract quoted from Toxline 1990-1994).

67.3.3     Dermal contact

CD-1 mice (30 animals per group) were exposed to ethylene glycol on gestation days 6 to 15, 6 hours per day by occluded cutaneous application at 0, 12.5, 50, or 100% ethylene glycol (0.1 ml/animal, equivalent to approximately 0, 404, 1677, or 3549 mg/kg b.w./day). There were no treatment-related maternal effects, and no differences in pre- or postimplantation loss or in foetal body weights/litter, and no increased incidences of any foetal malformations. Two skeletal variations were observed at the highest exposure level. According to the authors, the NOAEL for maternal and developmental toxicity was the highest exposure level (approximately 3549 mg/kg b.w./day). (Tyl et al. 1995 – abstract quoted from TOXLINE 1995-1998).

67.4     Mutagenic and genotoxic effects

67.4.1     In vitro studies

Ethylene glycol was negative when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 at concentrations from 1 to 10000 µg/plate with and without metabolic activation (Various studies quoted in IUCLID 2000, BUA 1991, and ATSDR 1997).

A negative result has been reported for ethylene glycol in the SOS chromotest in Escherichia coli PQ37 when tested at concentrations of up to 60000 µg/plate with and without metabolic activation (von der Hude et al. 1988).

When tested in the DNA damage and repair assay in Escherichia coli (WP2, WP2uvrA, WP67, CM611, WP100, W3100polA+, p3478polA-) at concentrations up to 60000 µg/plate with and without metabolic activation, negative results were obtained (McCarroll et al. 1991 – quoted from IUCLID 2000, BUA 1991 and from ATSDR 1997).

Ethylene glycol was negative when tested in the gene conversion assay in Saccharomyces cerevisiae with and without metabolic activation. According to IUCLID, genetic changes induced by 11% ethylene glycol appeared to be due to elevated osmotic pressure; no further details are given. (BIBRA 1988 – quoted from IUCLID 2000).

A negative result has also been obtained for aneuploidy induction in the fungus Neurospora crassa when tested with and without metabolic activation (Griffiths 1979, 1981 – quoted from ATSDR 1997).

A negative result has been reported for gene mutation in the HGPRT assay in Chinese Hamster Ovary cells with and without metabolic activation (Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991). In another HGPRT assay in Chinese Hamster Ovary cells, there was no dose-related increase in the incidence of mutagenic events either in the presence or absence of metabolic activations; according to IUCLID, the result of the assay was ambiguous (Union Carbide Corporation 1981 – quoted from IUCLID 2000).

Ethylene glycol has been reported to yield a negative result when tested for point mutations in the mouse lymphoma assay in L5178Y with and without metabolic activation (Brown et al. 1980 – quoted from IUCLID 2000 and from BUA 1991, McGregor et al. 1991 – quoted from IUCLID 2000 and from NTP 1993).

Negative results were obtained for sister chromatid exchanges when ethylene glycol was tested in Chinese Hamster Ovary cells with and without metabolic activation (Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991, Galloway et al. 1985,1987 – quoted from NTP 1993) or without metabolic activation at concentrations for 2 to 20% (v/v) (Union Carbide Corporation 1981 – quoted from IUCLID 2000).

A negative result was also obtained for chromosome aberrations when ethylene glycol was tested in Chinese Hamster Ovary cells with and without metabolic activation (Galloway et al. 1985,1987 – quoted from NTP 1993, Ballantyne 1985, Slesinski et al. 1986 – both quoted from IUCLID 2000 and from BUA 1991). When tested in another assay at concentrations from 10 to 100 mg/ml, ethylene glycol did not produce an increase in the incidence of chromosome aberrations; according to IUCLID, the result of the assay was ambiguous (Union Carbide Corporation 1985 – quoted from IUCLID 2000).

Negative results were also obtained for unscheduled DNA synthesis when ethylene glycol was tested in rat hepatocytes with and without metabolic activation (Slesinski et al. 1986, Union Carbide Corporation 1981 – quoted from IUCLID 2000 and from BUA 1991).

67.4.2     In vivo studies

Ethylene glycol has been tested for mutagenic effects in the micronucleus assay in Swiss mice by oral administration of 2.5, 3.125, 6.25, or 12.5 ml/kg b.w. (corresponding to 2.8, 3.5, 7.0, or 13.9 g/kg b.w.; 4, 4, 4, or 2 animals in the dose groups, respectively) and by intraperitoneal injection of 1.25, 2.5, or 6.25 ml/kg b.w. (corresponding to 1.4, 2,8, or 7.0 g/kg b.w.; 5, 5, or 1 animal in the dose groups, respectively). A control group of 19 animals was included. Animals in the high-dose oral group (13.9 g/kg b.w.) showed toxic effects. At all, but the lowest oral dose level (2.8 g/kg b.w.), there was an increase in the numbers of micronuclei in polychromatic erythrocytes when compared to the control. According to IUCLID, a weak positive but not dose dependent result was obtained. (Conan et al. 1979).

When tested in mice for chromosome aberrations following an intraperitoneal injection of 2.5 ml/kg b.w., a negative result was obtained (Conan et al. 1979 – quoted from BUA 1991).

In a combined 3-generation reproduction and dominant lethal study, ethylene glycol was administered to Fischer 344 rats (20 females and 10 males) in their diet at approximate dose levels of 40, 200, or 1000 mg/kg b.w. per day. Two control groups received the same diet without ethylene glycol. Males from each dosage group of the F₂ generation, which had received an ethylene glycol containing diet for 155 days, were bred with 15 untreated females at weekly intervals for 3 weeks. On day 12 of gestation, the females were sacrificed, and uteri and ovaries were examined for the numbers of living and dead foetuses. No significant changes were observed in any of the test groups. (DePass et al. 1986b).

67.5     Carcinogenic effects

67.5.1.1     Rats

Fischer 344 rats (130 males and females per group) were fed diets (0.1, 0.5, or 2.5%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. The only tumour type for which there was a significant difference was fibroadenoma of the mammary gland in females at the lowest dose level (0.1%). According to the authors, this finding was most probably unrelated to ethylene glycol treatment because of the absence of an effect at the two higher dose levels. Non-neoplastic findings are described in 4.2.2.1. (DePass et al. 1986a).

In Sprague-Dawley rats (16 male and female animals per group), which received ethylene glycol in their diet at concentrations of 0, 0.1, 0.2, 0.5, 1.0, or 4.0% (equivalent to 0, 50, 100, 250, 500 or 2000 mg/kg b.w./day) for 2 years, mammary tumours developed in many female rats towards the end of the experiment. According to the author, the distribution of the tumours in both males and females was such that it was impossible to correlate incidence with treatment. Few tumours were identified in organs within the body and none was associated with the occurrence of calculi. (Blood 1965).

67.5.1.2     Mice

In B6C3F1 mice (60 animals of each sex per group), which were fed diets containing ethylene glycol for 103 weeks (male mice: approximately 0, 1500, 3000, or 6000 mg/kg b.w./day; female mice: approximately 0, 3000, 6000, or 12000 mg/kg b.w./day), no treatment-related neoplasms were observed at the 15-month interim evaluations or at the end of the 2-year studies. Non-neoplastic findings are described in 4.2.2.2. (NTP 1993).

CD-1 mice (80 males and females per group) were fed diets (0.1-0.05, 0.7-0.24, or 0.35-1.27%) yielding approximate dosages of 40, 200, or 1000 mg/kg b.w. per day of ethylene glycol for 24 months. Two untreated control groups were included. The only tumour type for which there was any evidence of a possible increased incidence was lymphosarcoma in females. The time-adjusted incidence was significantly increased according to one of three trend tests. Comparisons among groups for differences in tumour proportions indicated that the observed results could have occurred by chance alone. Non-neoplastic findings are described in 4.2.2.2. (DePass et al. 1986).

68     Regulations

68.1     Ambient air

Denmark (C-value):        -

68.2     Drinking water

Denmark:                       -

68.3     Soil

Denmark:                       -

68.4     Occupational Exposure Limits

Denmark:                       10 ppm (26 mg/m³), notation H (At 2002).

                                      Atomised 10 mg/m³ (At 2002).

ACGIH:                         39 ppm (100 mg/m³) (ACGIH 2001)

68.5     Classification

Ethylene glycol is classified for acute toxic effects (Xn;R22 – harmful if swallowed) (MM 2002).

68.6     IARC

-

68.7     US-EPA

Oral reference dose (RfD): 2 mg/kg b.w. per day. The oral RfD is based on the NOAEL of 200 mg/kg b.w. per day for renal toxicity in a 2 year feeding study in rats (DePass et al. 1986 – quoted in IRIS 2002) and the application of a uncertainty factor of 100 (10 for interspecies extrapolation and 10 for differences in individual human sensitivity) (IRIS 2002).

69     Summary

69.1     Description

Ethylene glycol (EG) is a clear, colourless, slightly viscous, hygroscopic liquid with a sweet taste. It is miscible with water and has a low vapour pressure (0.06 mmHg at 20 °C).

69.2     Toxicokinetics

EG is rapidly absorbed and distributed following inhalation, oral and dermal administration. In one inhalation study in rats, 75-80% of inhaled EG (vapour or aerosol) was distributed immediately after exposure. Total recovery of oral doses in rats and mice is approximately 90-100%, indicating substantial absorption. After dermal application, approximately 30% of a dose was absorbed through rat skin, whereas mice absorbed 85-100% of the administered dose.

The metabolism of EG occurs in the liver and kidney. The initial step is a conversion of the parent compound to glycolaldehyde, which is further oxidised to glycolic acid. Glycolic acid is then converted to glyoxylic acid, which is converted either to carbon dioxide or to oxalic acid. Generally, metabolism begins immediately after administration of EG, and excretion of most of the parent compound and metabolites is complete 12 to 48 hours after dosing. The major excretory end products are carbon dioxide in exhaled air, and glycolate and unchanged EG in the urine.

69.3     Human toxicity

Twenty male volunteers exposed for 30 days, 20-22 hours a day, to EG atmospheres (aerosol, diameters of droplets 1-5 µm) containing mean concentrations of 17 to 49 mg/m³ did not experience any serious signs of toxicity, but there were complaints of irritation of the throat. The irritation became common when the volunteers re-entered the chamber in which the concentration of EG (during the absence of volunteers) was raised to about 140 mg/m³ and concentrations greater than about 200 mg/m³ were intolerable due to strong irritation of the upper respiratory tract. No significant alterations of the haematological, clinically chemical, or clinically pathological parameters studied, including the concentrations of urea nitrogen and creatinine in the blood of the exposed volunteers were observed.

Several deaths due to accidental or intentional ingestion of EG have been reported; the minimal lethal oral dose for humans has been estimated to be about 1.6 g/kg b.w. (adults). The clinical signs which follow acute poisoning after ingestion of EG can be divided into three (possibly four) stages: 1) effects on the central nervous system, which occur 30 minutes to 12 hours after ingestion; 2) effects on the cardiopulmonary system occurring 12 to 72 hours after ingestion, this stage may also be characterised by severe metabolic acidosis; 3) effects on the kidneys, which occur 24 to 72 hours after ingestion, this stage is characterised by profound metabolic acidosis; and 4) degenerative effects on the central nervous system occurring 6 or more days after ingestion, symptoms which are uncommon.

EG has not shown a particularly irritating potential to eyes or skin and was not shown to have a strong sensitising potential although some case reports are available. Prolonged dermal exposure can result in skin maceration.

No data on toxicity to reproduction, mutagenic and genotoxic effects, or carcinogenic effects of EG in humans have been found.

69.4     Animal toxicity

69.4.1     Single dose toxicity

An LC₅₀-value (one hour) of 10.9 g/m³ has been reported in rats. In a study of rats, all animals survived an 8-hour exposure to a saturated atmosphere (ca. 200 mg/m³ (calculated)).

The reported oral LD₅₀-values ranged from >2.0 to 11.3 g/kg b.w. in rats, from 5.89 to 15.4 g/kg b.w. in mice, from 7.0 to 9.3 g/kg b.w. in rabbits, from 4.0 to 8.2 g/kg b.w. in guinea pigs, from 4 to 8.2 g/kg b.w. in dogs, and of 1.67 and 4.7 g/kg b.w. in cats. A minimal lethal dose of 3.8 g/kg b.w. has been reported for rats, of 1 g/kg b.w. for cats, and of 6.7/7.3 g/kg b.w. for dogs.

Dermal LD₅₀-values of 9.53 and 10.6 g/kg b.w. have been reported for the rabbit.

EG did not show irritating properties when applied to the skin of rabbits.

Moderate to severe eye irritation has been observed in rats and rabbits exposed continuously for 90 days to EG (vapour) at a concentration of 12 mg/m³; guinea pigs, dogs, and monkeys exposed similarly showed no effects on the eyes. Following exposure to a concentration of 57 mg/m³ of EG (vapour, 8 hours a day, 5 days per week for 6 weeks), rats, guinea pigs, rabbits, dogs, and monkeys did not show any signs of ocular irritation.

EG (10, 20, or 50 % solution in water) caused slight oedema and erythema under occlusive conditions in the eyes of rabbits whereas instillation of neat EG produced moderate to severe oedema and erythema. Irritation consisting of chemosis, swelling, and conjunctival redness has also been observed in rabbit eyes following instillation of 4 and 40% EG in balanced salt solutions. The lowest non-irritating concentration of EG has been reported to be 20% when applied as 0.1 ml solution 5 times a day for 21 consecutive days. In other studies, neat EG has been reported to be only slightly irritating one hour after instillation of 0.1 ml of the fluid or to show an extremely low potential for eye irritation.

No data on sensitisation in experimental animals have been found.

69.4.2     Repeated dose toxicity

Rats, guinea pigs, rabbits, dogs and monkeys were exposed to EG (vapour) either continuously at a concentration of 12 mg/m³ for 90 days (continuous study), or at concentrations of 10 or 57 mg/m³ for 8 hours a day, 5 days per week for 6 weeks (repeated study). In the continuous study, one rat, one rabbit and 3 guinea pigs died during exposure as well as four control rats; moderate to severe eye irritation was observed in rats and rabbits; and histopathological examination showed inflammatory changes in the lungs of exposed animals and to a lesser degree in controls. In the repeated study, at 10 mg/m³, histopathological examination revealed mild congestion in the spleens of both dogs, hepatic fatty changes in 2/8 guinea pigs and in 1/8 rats, and focal necrosis in the liver of 1/8 guinea pigs and of 1/8 rats; focal necrosis of the liver was also seen in 1 of 3 control guinea pigs; at 57 mg/m³, histopathological examinations revealed non-specific inflammatory changes in the lungs and occasionally the hearts of exposed animals.

In a 90-day drinking water study, a dose-dependent increase in the incidence and severity of kidney damage (dilation, degeneration, and inflammation of the renal tubules, and renal pelvis) were observed in male rats at concentrations from 1% EG in the drinking water and in female rats from 2%; the NOAEL for renal effects (in males) in this study was 0.5% EG in the drinking water (corresponding to about 550 mg/kg b.w./day).

In a 13-week feeding study in rats, kidney lesions were observed at dose levels from 2.5% in male rats (toxic nephrosis) and at 5% in female rats; the relative kidney weight was significantly increased in both male and female rats from 2.5% and serum urea nitrogen and serum creatinine levels were significantly elevated in male animals from 2.5%. The NOAEL for renal effects (in males) in this study was 1.25% (equivalent to 625 mg/kg b.w./day, or according to the author: 600-1000 mg/kg b.w./day). In another dietary study in rats of similar duration (16 weeks), damage to the kidneys were observed from 0.25% in male rats and at 1% in female rats; the NOAEL for renal effects (in males) in this study was 0.1% (corresponding to about 70 mg/kg b.w./day).

In a 2-year feeding study in rats, kidney lesions were observed at a dose level of 2.5% in male rats and all male rats at this dose level had died after 16 months of exposure due to oxalate nephrosis; kidney lesions were not observed in female rats at dose levels up to 2.5%. Kidney weights were increased in both male and female rats at 2.5%. The NOAEL for renal effects (in males) in this study was 0.5% (corresponding to 200 mg/kg b.w./day). Mild fatty metamorphosis of the liver was observed in female rats at 2.5% (corresponding to 1000 mg/kg b.w./day); a NOAEL of 0.5% (corresponding to 200 mg/kg b.w./day) can be considered for liver effects in female rats. In another 2-year dietary study in rats, kidney damage was observed from 0.5% in male rats (crystal deposition in the kidney, degeneration of the tubular epithelium in 1/11 animals) and in female rats at 4%; the NOAEL for renal effects (in males) in this study was 0.2% (equivalent to 100 mg/kg b.w./day).

In mice, mild toxic nephrosis (only one animal) and a degenerative change in the livers were observed in male mice from a dietary level of 2.5% EG for 13 weeks; no effects were seen in female mice. The NOAEL in this study was 1.25% (equivalent to 1875 mg/kg b.w./day).

In a 2-year feeding study in mice, hepatocellular hyaline degeneration was seen in female mice at dietary levels from 2.5%. Incidence and severity of nephropathy were not affected in either sex at dietary levels of up to 2.5% in males and of up to 5% in females. The NOAEL in this study was 1.25% (equal to 3000 mg/kg b.w./day). In another 2-year feeding study in mice, no adverse effects on the kidneys were observed at dietary levels up to 1000 mg/kg b.w./day (the highest dose level in the study).

69.4.3     Toxicity to reproduction

When rats were exposed to a respirable EG aerosol by whole-body exposures (6 hours a day, gestational days 6 to 15), there was some evidence of treatment-related reductions in ossification of the foetal skeleton at 2500 mg/m³ and an increase in the incidence of poorly ossified metatarsals and proximal phalanges of the hindlimb at 1000 mg/m³; the only maternal effect observed was a significant increase in liver weight (absolute and relative) at 2500 mg/m³. The NOAEL was 1000 mg/m³ for maternal and 150 mg/m³ for developmental toxicity.

In mice exposed similarly as the rats, several gestational parameters were affected from 1000 mg/m³ and there was a significant increase in the incidence of a number of external, visceral, and skeletal malformations. The incidences of many foetal variations were also increased from 1000 mg/m³, but only a few at the lowest dose level (150 mg/m³). Reduced maternal body weight and body weight gain and reduced gravid uterine weight were observed from 1000 mg/m³. The NOAEL was 150 mg/m³ for maternal and at or below 150 mg/m³ for developmental toxicity.

In a subsequent nose-only study (aerosol, 6 hours a day, gestational days 6 to 15) in mice, foetal body weights per litter were significantly reduced and the incidences of one skeletal malformation (fused ribs) and 18 skeletal variations were increased at 2500 mg/m³; maternal kidney weights were increased from 1000 mg/m³. The NOAEL was 500 mg/m³ for maternal and 1000 mg/m³ for developmental toxicity.

In a three-generation dietary reproduction study in rats, no treatment-related effects were observed in the F₂ parents and in the F₃ weanlings, including kidney damage; the NOAEL for reproductive toxicity was 1000 mg/kg b.w./day (the highest dose level in the study). In a continuous breeding study in mice, EG was administered in the drinking water for 14 weeks; the NOAEL for reproductive effects was 0.5% (corresponding to an average dose of 840 mg/kg b.w./day) with foetotoxic effects, including malformations, being observed at the higher concentration of 1%.

In Fischer 344 rats, the NOAEL for developmental toxicity as well as for maternal toxicity (only body weights of the dams were examined) was 1000 mg/kg b.w./day (the highest dose level in the study) when EG was administered in the diet from gestation day 6 to 15. When EG was administered by gavage (from gestation day 6 to 15) to CD rats, the NOAEL for developmental toxicity was 500 mg/kg b.w./day with effects (reduced body weights; duplicated or missing ribs, centra, and arches; and poor ossification) being observed at 1000 mg/kg b.w./day; maternal effects (increased relative liver weight) was observed as well at this dose level. Similarly, another gavage study in CD rats showed that administration of EG (from 1250 mg/kg b.w./day, the lowest dose level in the study) during organogenesis produced severe dose-related developmental toxicity, including malformations, at dose levels where no serious maternal effects (reduced maternal body weight gain at 1250 mg/kg b.w./day) were observed. However, in a third gavage study, where EG was administered to CD rats from gestation day 6 to 20, no toxicity were observed in offspring at dose levels up to 1250 mg/kg b.w./day; maternal effects at this dose level included kidney damage. In Wistar rats, a NOAEL of 638 mg/kg b.w./day for developmental toxicity (foetotoxicity as well as malformations) was observed following administration of EG by a stomach tube from gestation day 6 to 15; no information was given with regard to maternal effects.

When EG was administered by gavage (from gestation day 6 to 15) to CD-1 mice, the NOAEL for developmental toxicity was 150 mg/kg b.w./day with effects (slight reductions in foetal body weight and increased incidences of extra ribs) being observed at 500 mg/kg b.w./day; no maternal effects were observed at any dose level (up to 1500 mg/kg b.w./day). Another gavage study in CD-1 mice showed that administration of EG (from 750 mg/kg b.w./day, the lowest dose level in the study) during organogenesis produced severe dose-related developmental toxicity, including malformations, a dose level where no maternal effects were observed.

In New Zealand White (NZW) rabbits, the NOAEL for maternal toxicity was 1000 mg/kg b.w./day and the NOAEL for developmental toxicity was 2000 mg/kg b.w./day (the highest dose level in the study), when EG was administered by gavage on gestational day 6 through 19.

Following occluded cutaneous application of EG to CD-1 mice on gestation days 6 to 15 (6 hours per day),  the NOAEL for maternal and developmental toxicity was the highest exposure level (approximately 3550 mg/kg b.w./day).

69.4.4     Mutagenic and genotoxic effects

EG has shown negative results in the following in vitro test systems: in the Ames test (several tests in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537; TA1538), in the SOS chromotest in Escherichia coli PQ37, in the DNA damage and repair assay in Escherichia coli (WP2, WP2uvrA, WP67, CM611, WP100, W3100polA+, p3478polA-), for gene conversion in Saccharomyces cerevisiae, for aneuploidy induction in the fungus Neurospora crassa, in a cytogenetic assay in Chinese Hamster Ovary (CHO) cells, for gene mutation in the HGPRT assay in CHO cells, in the mouse lymphoma assay, for sister chromatid exchanges and chromosome aberrations in CHO cells, and for unscheduled DNA synthesis in rat hepatocytes. In one HGPRT assay and in one test for chromosome aberration, both in CHO cells, the result of the assay was reported as being ambiguous. Most of the assays were performed both with and without metabolic activation.

Negative results have also been reported in in vivo studies: in the dominant lethal assay following oral administration in the feed to Fischer 344 rats and for chromosome aberrations in mice following intraperitoneal injection. In a micronucleus assay in mice, an increase in the numbers of micronuclei was observed following oral administration of very high doses (2.8 to 13.9 g/kg b.w.), except at the lowest dose level, and following intraperitoneal injection (doses of 1.4 to 7.0 g/kg b.w.).

69.4.5     Carcinogenic effects

No evidence of a carcinogenic effect of EG was observed in Fischer 344 rats administered diets yielding dosages of up to approximately 1000 mg/kg b.w./day for 24 months, in Sprague-Dawley rats receiving dietary concentrations of up to 4% (equivalent to 2000 mg/kg b.w./day) for 2 years, in B6C3F1 mice fed diets containing EG for 103 weeks (male mice: up to 2.5% equivalent to approximately 6000 mg/kg b.w./day; female mice: up to 5% equivalent to approximately 12000 mg/kg b.w./day), or in CD-1 mice administered diets yielding dosages of up to approximately 1000 mg/kg b.w./day for 24 months.

70     Evaluation

Ethylene glycol (EG) is rapidly and almost completely absorbed (inhalation, rats: 75-80% of inhaled EG (vapour or aerosol); oral, rats and mice: 90-100%; dermal, rats: 30%; dermal, mice: 85-100%), distributed, metabolised and cleared (almost completely 12 to 48 hours after dosing) following exposure. It should be noted that dermal uptake generally is greater in rodents than in humans because the skin of rodents is thinner than that of humans.

EG is metabolised by oxidation via glycol­aldehyde and glycolic acid to glyoxylic acid, which is converted either to carbon dioxide or to oxalic acid. The kinetic study published by Pottenger et al. (2001) indicated that the metabolic conversion of EG to glycolic acid was approaching saturation at EG blood levels obtained following oral administration (gavage) of 2500 mg/kg b.w., and that glycolic acid was being formed at a maximum rate between 1000 and 2500 mg/kg b.w.

The major excretory end products are carbon dioxide in exhaled air, and glycolate and unchanged EG in the urine. The study published by Pottenger et al. (2001) showed that urinary elimination demonstrated dose-dependency, with the high dose groups (2500 mg/kg b.w.) eliminating almost 70% of the administered dose in urine, compared with about 16% in the low dose groups (10 mg/kg b.w.); the shift in urinary elimination was mainly due to increased urinary glycolic acid and EG, and not to increased elimination of oxalic acid. The study also showed that oxalate was a very minor metabolite in both blood and urine at all dose levels (up to 2500 mg/kg b.w.).

There are numerous case reports in the literature of poisoning in humans due to accidental or intentional ingestion of EG; the minimal lethal oral dose for humans has been estimated to be about 1600 mg/kg b.w. for adults. The clinical symptoms of acute EG poisoning in humans can be divided into three (possibly four) fairly distinct stages (effects on the CNS, cardiopulmonary effects, renal effects, and delayed neurological effects); toxicity in stage 2 and 3 is characterised by severe metabolic acidosis. The severity of these stages and the advance from one stage to the next depends greatly on the amount of EG absorbed.

The acute toxicity of EG in experimental animals closely mirrors the acute effects seen in humans; one exception is the fourth clinical stage as no studies have been found that reported this stage in animals. EG is of low acute toxicity in experimental animals, except the cat, with reported oral LD₅₀-values ranging from >2000 to 15400 mg/kg b.w.; a minimal lethal dose of 3800 mg/kg b.w. has been reported for rats, of around 7000 mg/kg b.w. for dogs and, of 1000 mg/kg b.w. for cats. Comparing the lethal oral dose in humans to the minimum lethal dose in experimental animals, EG appears to be two to five times more acutely toxic to humans and cats, on a body weight basis, than to rats and dogs. EG is classified for acute toxic effects following oral administration according to the EU classification criteria.

The very limited data on acute inhalation and dermal toxicity in experimental animals also indicate a low acute toxicity by these routes with a reported LC₅₀-value (one hour) of 10.9 g/m³ in rats and dermal LD₅₀-values of around 10600 mg/kg b.w. for the rabbit.

EG has not shown a particularly irritating potential to eyes or skin in humans and did not show irritating properties when applied to the skin of rabbits. Prolonged dermal exposure to humans can result in skin maceration.

The data on eye irritation in experimental animals are conflicting but overall, the data indicate an eye irritating potential following instillation of either neat EG or solutions of EG to rabbit eyes. Moderate to severe eye irritation has been observed in rats and rabbits exposed continuously for 90 days to EG (vapour, 12 mg/m³) but not following exposure to a concentration of 57 mg/m³ (vapour, 8 hours a day, 5 days per week for 6 weeks); guinea pigs, dogs, and monkeys exposed similarly showed no effects on the eyes.

Male volunteers complained of irritation of the throat following exposure to 17 to 49 mg/m³ (aerosol, 20-22 hours a day for 30 days); the irritation became common at about 140 mg/m³ when the volunteers re-entered the exposure chamber in which the concentration of EG (during the absence of volunteers) was raised and concentrations greater than about 200 mg/m³ were intolerable due to strong irritation of the upper respiratory tract.

EG is not considered to have a sensitising potential in humans although some case reports are available. No data on sensitisation in experimental animals have been found.

Male volunteers (exposed for 30 days, 20-22 hours a day, aerosol, mean concentrations of 17 to 49 mg/m³) did not experience any serious signs of toxicity and no indications of renal toxicity (alterations in urea nitrogen and creatinine in the blood) were observed. Similarly, no indications of renal toxicity were observed in rats, guinea pigs, rabbits, dogs and monkeys exposed to EG (vapour) either continuously (12 mg/m³ for 90 days), or repeatedly (10 or 57 mg/m³ for 8 hours a day, 5 days per week for 6 weeks). Inflammatory changes were observed in the lungs of exposed animals and to a lesser degree in controls as well as some effects in the liver in a few animals; most of the observed effects were interpreted, by the authors, as being unrelated to the exposure to EG, however, these interpretations cannot be evaluated from the data provided in the publication (Coon et al. 1970).

Repeated oral administration of EG to rats results primarily in toxic effects in the kidneys. Male rats are far more sensitive to the renal effects of EG than are female rats as renal effects occur in male rats at dose levels from about 200-250 mg/kg b.w./day (16-week and 2-year feeding studies) but in female rats at dose levels above 1000 mg/kg b.w./day. Mice appear to be relatively resistant to EG induced kidney damage when compared to rat as incidence and severity of nephropathy were not affected in either sex at dietary levels (2-year feeding study) of up to 6000 mg/kg b.w./day in males and of up to 12000 mg/kg b.w./day in females.

The incidence and severity of the renal effects appear to depend on the dose level as well as on the exposure duration. A NOAEL for renal effects (in male rats) of about 550 mg/kg b.w./day can be considered from a 90-day drinking water study (Robinson et al. 1990) and of about 625 mg/kg b.w./day from a 13-week feeding study (Melnick 1984) whereas 2-year feeding studies have revealed NOAELs of 100 (Blood 1965) and 200 mg/kg b.w./day (DePass et al. 1986a), respectively. This difference in the NOAELs could be a result of different sensitivity in the rat strains used in the two studies (Blood: Sprague-Dawley; DePass et al.: Fischer 344/N), but no data are available to further elucidate this aspect. However, the validity of the study by DePass et al. is considered to be better than that of the study by Blood, e.g., 130 animals of each sex per group compared to 16 animals of each sex per group; more detailed descriptions of results including incidence and severity of renal effects in the various dose groups. The lowest NOAEL (about 70 mg/kg b.w./day) for damage to the kidneys in male Wistar rats has been observed in a 16-week dietary study (Gaunt et al. 1974 – quoted from BUA 1991 and IUCLID 2000); however, BUA and IUCLID do not give any details about the kidney damage (type, incidence, severity) and the study report is not public available. Overall, a NOAEL for renal effects in male rats, the most sensitive species, of 200 mg/kg b.w./day is considered taken into account the reliability of the various studies as discussed above. According to NTP (1993), the study by DePass et al. (1986a) was considered adequate to evaluate the chronic toxicity of EG in F344 rats; therefore, NTP only has conducted a 2-year study in mice.

According to DePass et al. (1986), the greater susceptibility of male rats to EG induced renal toxicity may be the result of more efficient conversion of EG to toxic metabolites including oxalate in male rats, as well as to the more rapid progression of spontaneous nephropathy in the male. High-dose females had significant amounts of urinary oxalate crystals at 12, 18, and 24 months, so conversion of EG to oxalate clearly occurred in females also. The critical factor responsible for the more severe nephrotoxicity in male rats was probably the greater incidence and severity of spontaneous nephropathy in the male.

No data on toxicity to reproduction in humans have been found.

Dietary exposure of male and female F344 rats to EG at dose levels up to 1000 mg/kg b.w./day (the highest dose level in the study) for three generations produced no effects on fertility, fecundity, or reproductive performance. When EG was administered to CD-1 mice in the drinking water for 14 weeks (continuous breeding study), reduced fertility and fecundity, and foetotoxic effects, including malformations were observed at about 1640 mg/kg b.w./day; the NOAEL was about 840 mg/kg b.w./day.

Administration of EG via the gastrointestinal route (gavage) at high concentrations has resulted in developmental toxicity, including teratogenicity in rats and mice. Developmental toxicity was observed in CD rats in two gavage studies at dose levels from about 1000 mg/kg b.w./day (gavage) and in Wistar rats from about 860 mg/kg b.w./day whereas one gavage study did not show any developmental toxicity in CD rats at 1250 mg/kg b.w./day. When EG was administered to F344 rats in the diet, no developmental effects were observed at dose levels up to 1000 mg/kg b.w./day (the highest dose level in the study). No serious maternal effects were noted at the dose levels resulting in developmental toxicity. Mice appear to be far more sensitive to the developmental effect exerted by EG with severe developmental toxicity, including malformations, being observed in a gavage study at dose levels from 750 mg/kg b.w./day (the lowest dose level in the study), a dose level where no maternal effects were observed. In another gavage study, lower dose levels were administered and a NOAEL for developmental toxicity of 150 mg/kg b.w./day can be considered from this study. One gavage study in rabbits indicates that this species is fairly resistant to the developmental effects exerted by EG as no developmental effects were observed at dose levels up to 2000 mg/kg b.w./day (the highest dose level in the study).

Following dermal application of EG to CD-1 mice, no developmental effects were observed at dose levels up to about 3550 mg/kg b.w./day (the highest dose level in the study).

Developmental toxicity, including teratogenicity, has been observed in CD-1 mice following whole-body exposures to EG respirable aerosol at concentrations from 1000 mg/m³ (6 hours a day); CD rats exposed similarly exhibited developmental toxicity, but no teratogenicity at the same exposure levels. This is consistent with the results observed in the oral studies discussed above, in which mice appeared to be far more sensitive to the developmental effect exerted by EG than rats. The NOAEL for developmental effects in the whole-body study was 150 mg/m³ for rats and at or below 150 mg/m³ for mice with NOAELs for maternal effects of 1000 and 150 mg/m³, respectively (no concentrations between 150 and 1000 mg/m³). Analysis of EG on the fur of rats and mice during and after the exposure period (2500 mg/m³) showed significant amounts of EG on the fur, which, according to the authors (Tyl et al. 1995a,b), alone could have produced the effects seen in mice if it were ingested by grooming and/or percutaneously absorbed. Therefore, a nose-only study was performed in CD-1 mice in order to evaluate the toxicity of EG aerosol from inhalation exposure alone (500, 1000, and 2500 mg/m³). In this study, the NOAEL for maternal and developmental effects, including teratogenicity, was 500 and 1000 mg/m³, respectively. Assuming (for pregnant mice) an inhalation rate of 25 ml/min (corresponding to 0.036 m³/day), a body weight of 0.035 kg, and 100% absorption of EG by inhalation, the NOAELs correspond to about 500 and 1000 mg/kg b.w./day, respectively.

Most of the mutagenicity and genotoxicity tests available indicate that EG is not a mutagenic or genotoxic substance although some positive results have been reported. In the micronucleus assay in mice, the increased numbers of micronuclei was observed following administration (oral, intraperitoneal injection) of very high doses (2.8 to 13.9 g/kg b.w.) and thus, the result is not considered as being reliable. Overall, EG is considered not to be a mutagenic or genotoxic substance.

No evidence of a carcinogenic effect of EG was observed at dietary concentrations of up to approximately to 2000 mg/kg b.w./day for 2 years in rats or of up to approximately 12000 mg/kg b.w./day for 2 years in mice.

No data on mutagenic and genotoxic effects, or carcinogenic effects of EG in humans have been found.

The toxicity of EG is primarily a result of the effects of its metabolites although the effects on the central nervous system observed shortly after acute ingestion is partly attributed to unmetabolised EG.

The metabolic acidosis is usually attributed to the acidic metabolites of EG and recent studies of cases of human EG poisoning (discussed in Jacobsen & McMartin 1997) have demonstrated that the major determinant of the metabolic acidosis is the degree of glycolic acid accumulation as glycolate accumulation correlates with the increase in anion gap or decrease in arterial bicarbonate concentrations observed in poisoned humans, as well as in animals.

The mechanism of the renal toxicity is not yet known. Calcium oxalate precipitation within the renal tubules has long been accepted as an important pathogenic factor for the development of renal toxicity; however, there is no evidence directly linking oxalate precipitation with development of renal tubular necrosis and renal damage can occur at exposure levels where no or few oxalate crystals are detected. The renal toxicity has also been suggested to occur from a metabolite-induced cytotoxicity, such as from glycolaldehyde or glyoxylate, which are both highly toxic in vitro, or via the metabolic acidosis resulting from accumulation of glycolic acid. As a result of the decrease in the pH in body fluids, an elevated anion gap develops and the serum osmolal gap across cells increases, resulting in renal oedema that compromises intrarenal blood flow and promotes renal failure. These suggestions are in concordance with the data published by Pottenger et al. (2001) showing that oxalate was a very minor metabolite in rats in both blood and urine at oral (gavage) dose levels up to 2500 mg/kg b.w. and the data discussed in Jacobsen & McMartin (1997) showing that glycolate accumulation correlates with the increase in anion gap or decrease in arterial bicarbonate concentrations observed in poisoned humans, as well as in animals.

Although the mechanism(s) behind the developmental effects is not yet known, a link between maternal metabolic acidosis and developmental toxicity has been suggested with glycolic acid being the predominant toxic metabolite.

70.1.1     Conclusion

The critical effects following exposure to EG are the effects in the kidneys, which are observed in both humans and experimental animals; the developmental effects observed in experimental animals; and the irritative effects observed in humans and experimental animals following inhalation of EG.

In female rats, effects on the liver (mild fatty metamorphosis) has been observed with a NOAEL of about 200 mg/kg b.w./day; however, the liver effects observed are not considered to be as serious as the renal lesions observed in male rats although a NOAEL of about 200 mg/kg b.w./day for renal effects in male rats has been considered as well.

The mechanism(s) behind the nephrotoxic and developmental effects are not known but are probably due to the metabolic acidosis resulting from an accumulation of the EG metabolite glycolic acid. Glycolic acid is a major metabolite in both humans and experimental animals whereas oxalate appears to be a minor metabolite.

Based on the results reported in the available oral studies, male rats are far more sensitive to the renal effects than are female rats and mice (both sexes). No data are available in order to evaluate the sensitivity of humans to the renal effects as no long term studies in humans are available. Following acute ingestion of EG, the same type of renal effects are observed in humans as in experimental animals. Therefore, humans are considered to be as sensitive as male rats to the nephrotoxic effects of EG; a NOAEL of 200 mg/kg b.w./day has been considered for renal effects in male rats from a 2-year dietary study as discussed above. The exposure levels in the available inhalation studies (volunteers: 17-49 mg/m³ for 30 days; experimental animals: 12 mg/m³ continuously for 90 days or 10-57 mg/m³ for 8 hours a day, 5 days per week for 6 weeks) are considered to be far too low to result in any renal effects.

Mice appear to be far more sensitive to the developmental toxicity exerted by EG than are rats and rabbits. No data on reproductive toxicity in humans are available. Therefore, humans are considered to be as sensitive as mice to the developmental effects of EG. In a nose-only inhalation study, the NOAEC for developmental effects, including teratogenicity, in mice was 1000 mg/m³ while the NOAEC was at or below 150 mg/m³ in a whole-body inhalation study (no concentrations between 150 and 1000 mg/m³). Assuming (for pregnant mice) an inhalation rate of 25 ml/day (corresponding to 0.036 m³/day), a body weight of 0.035 kg, and 100% absorption of EG by inhalation, these NOAECs correspond to about 1000 and 150 mg/kg b.w./day, respectively. A NOAEL for developmental toxicity in mice following gavage of 150 mg/kg b.w./day can be considered as discussed above.

Male volunteers complained of irritation of the throat following exposure to mean concentrations of 17 to 49 mg/m³ (aerosol, 20-22 hours a day for 30 days). Moderate to severe eye irritation has been observed in rats and rabbits exposed continuously for 90 days to EG (vapour, 12 mg/m³) but not following exposure to a concentration of 57 mg/m³ (vapour, 8 hours a day, 5 days per week for 6 weeks); furthermore, the data indicate an eye irritating potential following instillation of either neat EG or solutions of EG to rabbit eyes. Based on these data, a LOAEC for irritative effects of 17 mg/m³ is considered. Assuming (for adults) an inhalation rate of 20 m³/day, a body weight of 70 kg, and 100% absorption of EG by inhalation, this LOAEC corresponds to about 5 mg/kg b.w./day, which is far below the NOAELs considered for renal (200 mg/kg b.w./day) and developmental toxicity (150 mg/kg b.w./day).

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Appendix 10: Propylene glycol (PG) 
72     General description

72.1     Identity

Molecular formula:          C₃H₈O₂

Structural formula:           CH₃-CHOH-CH₂OH

Molecular weight:            76

CAS-no.:                        57-55-6

Synonyms:                      Propanediol

                                      Monopropylene glycol

                                      Propane-1,2-diol

                                      alpha-Propylene glycol

                                      Sirlene

                                      1,2-Propanediol

                                      1,2-Dihydroxypropane

                                      Methylethylene glycol

                                      Trimethyl glycol

                                      1,2-Propylene glycol

72.2     Physico / chemical properties

Description:                    Propylene glycol (PG) is a colourless viscous liquid. It is soluble in water and hygroscopic.

Melting point:                  -59 °C

Boiling point:                   189 °C

Density:                          1.036 g/ml (at 20°C)

Vapour pressure:             0.07 mmHg (9.3 Pa) (at 20°C)

Concentration of

saturated vapours:           291 mg/m³             (calculated)

Conversion factor:           1 ppm = 3.16 mg/m³ (at 20°C and 760 mmHg)

                                      1 mg/m³ = 0.316 ppm     

Solubility:                        Water:  ≥ 100 g/l (at 21°C). Also soluble in alcohol.

Partition coefficient

Log K_OW :                      -0.92

References:                    ATSDR (1997), ChemFinder (2002), LaKind et al. (1999).

73     Toxicokinetics

73.1     Inhalation

No data were found on toxicokinetics of PG via the inhalation route.

73.2     Oral intake

Data indicated that absorption from the gastrointestinal tract is fairly rapid. The maximum plasma concentration of PG in humans was reached within 1 hour after oral exposure (ATSDR 1997).

In dogs, gavage doses of 2, 8 and 12 ml PG/kg water (corresponding to 1036, 4144 and 6216 mg PG/kg b.w.) lead to maximum blood concentrations within 30 minutes for the low dose, and after 2-4 hours for the mid- and high doses (Lehman et al. 1937 – quoted from Mortensen 1993).

In humans, the maximum plasma concentration of PG was reached within 1 hour after oral exposure. No details on the dose were given. (Yu et al. 1985 – quoted from ATSDR, 1997).

The major route of metabolism for PG is by liver alcohol dehydrogenase to lactaldehyde, then to lactic acid and pyruvic acid. An alternative route of metabolism is via phosphorylated glycol. The two metabolites, lactic and pyruvic acids, enter the energy-generating process (i.e. tricarboxylic acid cycle and/or gluconeogenic pathway). Pyruvic acid can also be metabolised further to carbon dioxide and water. (Mortensen 1993, LaKind 1999).

Metabolism of PG in humans was non-linear, based on a saturable clearance. Elimination half-life following oral exposure was about 3.8 hours. The total body clearance in humans was about 100 ml/kg b.w./hour (corresponding to 103.6 g/kg b.w./hour) (Yu et al. 1985 - quoted from ATSDR 1997 and Mortensen 1993).

In another investigation, 20-25% of orally administered PG was eliminated via the urine in 10 hours. (Hanzlik et al. 1939 – quoted from Mortensen 1993).

Dose-dependent elimination was seen in rats, with saturation of the pathways at doses above 5880 mg/kg b.w. and a maximum elimination rate of 0.63 g PG/kg b.w./hour (Morshed et al. 1988 - quoted from ATSDR 1997). 

In cats, PG has been shown to be metabolised to d- and l-lactate. In rats, approximately 33% of an oral dose PG was excreted as the unchanged substance in urine, while dogs excreted 45% unchanged. (Van Winkle 1941, Lehman & Newman 1937 - quoted from Mortensen 1993). 

73.3     Dermal contact

Absorption through damaged skin was studied in 45 patients with second and third degree burns over more than 20% of their total body surface. The sulfadiazine preprations applied dermally over a period of 3-7 contained PG, which was detected in the serum of 24 patients at average levels of 0.08 mg/ml with a range of 0-1.3 mg/ml. PG was detected in the urine of 40 patients at average levels of 1.3 mg/ml, with a range of 0-23.0 mg/ml. (Kulick et al. 1985 – quoted from ATSDR 1997).

73.4     Mode of action

No data were found.

74     Human toxicity

74.1     Single dose toxicity

Acute effects include primarily CNS depression and lactic acidosis form extremely high doses.

74.1.1     Inhalation

Ninety-three asthma-patients showed no adverse clinical effects from 15 minutes inhalation through a mask of an aerosol consisting of 0.005% isoproterenol-hydrochloride in a  40 % PG isotonic saline solution (Cohen & Crandall 1964).

74.1.2     Oral intake

A 15-month infant was given a vitamin C preparation containing 22.5 ml PG (equivalent to 23.8 g) as vehicle. The child experienced hypoglycaemia, irregular heart rate, sinus arythmia, tachypnea, tachycardia and perspiration. The effects disappeared upon cessation of treatment. (Martin & Finberg 1970 - quoted from LaKind et al. 1999).

No effects on the basal metabolic rate were demonstrated in a study of 3 human subjects administered 50 ml (51800 mg) PG (Hanzlik et al. 1939 - quoted from LaKind et al. 1999).

74.1.3     Dermal contact

An severely burnt 8-month infant (large surface of the skin with second and third-degree burns was treated topically over 70 hours with silver sulfadiazine containing 76.7 mg PG/g. The PG dose was calculated to 630 g/kg b.w. The treatment resulted in a peak serum PG level of 1059 mg/dl and caused acute metabolic acidosis and cardio-respiratory arrest, which may be due to the high serum PG levels. (Fligner et al. 1985 - quoted from LaKind et al. 1999).

74.1.4     Irritation and sensitisation

Cases of contact dermatitis have been reported from therapeutic and cosmetic products containing PG as a vehicle. Numerous patch test studies have been designed to determine the incidence and nature of PG skin reactions. Most of the positive reactions were found to be primary irritation, but several investigators were unable to conclude whether reactions to PG exposure were dermal irritation or true allergy. 

In a Draize test, 204 subjects were treated with 12 % and 89 subjects wit 60% PG in petrolatum over a 3-5 week period. No allergic response was seen at challenge with 72 hour contact to 12 % PG. (Marzulli & Maibach 1974 – quoted from Mortensen 1993).

In a modified Draize sensitisation study, 13 of 203 subjects showed a significant response to PG, by none of these subjects reacted to provocative use testing with 100 % PG. The authors concluded that PG was at lest a minimal irritant. (Trancik & Maibach 1982 - quoted from LaKind et al. 1999).

Frosch & Kligman (1977 - quoted from LaKind et al. 1999) classified 100% PG as a moderate skin irritant.

In another test, undiluted PG was applied to 1556 subjects for 20-24 hours. Skin reactions were seen in 12.5% of subjects, 70% of which were considered irritative and 30 % allergic, depending on onset time and area involved. Twelve out of 42 subjects with skin reaction also showed reaction were then tested with 10% PG in water. (Hannuksela et al. 1975 - quoted from Mortensen 1993).

Andersen (1980) pointed at the difficulty to evaluate a positive reaction and its clinical relevance, as false positive reactions occur commonly, and impurities may play a role. The incidence of PG allergy in the population was considered to be very low in relation to the extended use in consumer products.

74.2     Repeated dose toxicity

74.2.1     Inhalation

Exposure of children and adults to up to 94 mg PG/m³ (mean of 69 mg PG/m³) over weeks to months did not result in any adverse effects. (Harris & Stokes Jr, 1943 - quoted from A&H 1983).

No other data were found.

74.2.2     Oral intake

A child was given 2-4 ml of a vitamin D preparation containing 98% PG, twice a day for up to 13 months. The treatment corresponds to an exposure of 114-228 mg PG/kg b.w. day. After the 13 months, the child began to experience repeated seizures followed by a period of unconsciousness within 2 to 4 hours after the second daily dose. The symptoms disappeared when the treatment ceased. (Arulanantham & Genel 1978 - quoted from LaKind et al. 1999).

74.2.3     Dermal contact

No data were found.

74.3     Toxicity to reproduction

No data were found.

74.4     Mutagenic and genotoxic effects

No data were found.

74.5     Carcinogenic effects

No data were found.

75     Animal toxicity

75.1     Single dose toxicity

75.1.1     Inhalation

Rabbits were exposed to of 10% PG aerosol for 20 or 120 minutes. No deaths were reported. In the tracheal epithelium, the goblet cells were degenerated at both observation times, whereas ciliated cells were slightly affected at 20 min., and seriously at 120 minutes. No details of the aerosol particle size or other exposure conditions were given. (Konrádová et al. 1978 - quoted from ATSDR and LaKind et al. 1999).

Dogs were exposed for 15 minutes to a 10 or a 20% PG aerosol and haemolysis or haemodynamic effects were investigated. No effects on the blood were seen. No details of the aerosol particle size or other exposure conditions were given. (Konrádová et al. 1978 - quoted from ATSDR and LaKind et al. 1999).

75.1.2     Oral intake

Reported LD₅₀-values in rats for PG range from 21 to 33.5 g/kg b.w., and minimum lethal doses of 19.8 and 20.9 g/kg were reported for the substance. (IUCLID 2000, LaKind et al. 1999).

Rats were administered single doses of 10 ml of 10, 20, 50 or 100 % PG solutions/kg (equivalent to 1.04, 2.08, 5.2 and 10.4 g PG/kg b.w.). No deaths were reported. At the two highest doses, PG produced decreased respiration and marked CNS depression, including ataxia, ptosis (hanging eye lid), decrease of spontaneous motor activity and of body/limb tone, and of respiration. (Singh et al. 1982 - quoted from LaKind 1999).

Rats administered a single dose of 23.5 g/kg PG developed acute haemorrhagic enteritis and extensive adrenocortical haemorrhage. Widespread lymphocyte depletion was also evident. (Clark et al. 1979 - quoted from LaKind 1999).

Single oral doses of 730 or 2940 mg PG/kg b.w. were administered to adult female albino Wistar rats to examine haematological effects. PG was found to decrease packed cell volume for up to 2 days. Red blood cell count was reversibly decreased. (Saini et al. 1996 - quoted from LaKind et al. 1999).

LD₅₀-values of 22, 23.9, 24.8 and 31.9 g/kg b.w. were reported in mice for PG (LaKind et al. 1999 and IUCLID 2000). The study of Singh et al. reported above also included mice, in which similar effects as for the rats were described. (Singh et al. 1982 - quoted from LaKind 1999).

Single doses of 23310 mg PG/kg b.w. administered to mice by stomach tube caused slight microscopic changes in the kidney with nuclear pyknosis and vacuolar degeneration of the cytoplasm. A few cortical tubules contained protein debris. (Laug et al. 1939 - quoted from LaKind et al. 1999).

In rabbits, the LD₅₀-values for PG were reported to range from 18 to 19.3 g/kg b.w. (IUCLID 2000). Dosing by stomach tube with 13.73 to 21 g/kg b.w. PG resulted in increased respiratory rate, loss of equilibrium, depression, analgesia and coma. Deaths occurred in 18 to 36 hours. (Braun & Cartland 1936 - quoted from LaKind et al. 1999).

LD₅₀-values in guinea pigs of 18.35, 18.9 and 19.6 g/kg b.w. were reported (LaKind et al. 1999).

An LD₅₀-value in dogs of 22 g/kg b.w. has been cited (IUCLID 2000).

75.1.3     Dermal contact

A LD₅₀-value in rabbits of 20800 mg/kg b.w. is reported without any details (Raw Material Data Handbook 1974 – quoted from IUCLID 2000).

75.1.4     Irritation and sensitisation

Several dermal irritation studies with PG in rabbits were conducted. Very few details were reported. The results ranged from none to mild irritation. (IUCLID 2000).

PG was reported to be not irritating to mildly irritating to rabbits eyes in a number of eye irritation tests in rabbits reported with very few details (IUCLID 2000).

No studies on sensitisation were found.

75.2     Repeated dose toxicity

75.2.1     Inhalation

Nineteen Sprague-Dawley rats/sex/dose were exposed nose-only to an aerosol containing 0, 160, 1000 or 2200 mg PG/m³ (0, 51, 321 or 707 ppm), 6 hours/day, 5 days/week over 90 days. Treatment related nose bleeding occurred from week 2 through the study. The authors attributed the effect to dehydration of the tissues. At the two highest dose levels, the number, size and mucous content of the goblet cells in the nasal epithelium were significantly increased. Slight dose related body weight reduction was reported females. Significant but not dose-related changes in haematological parameters were seen. No toxicological effects were seen on liver, kidney, spleen or lung. (Suber et al. 1989).

Twenty-nine monkeys (Macacus Rhesus) were continuously exposed over 13 months to levels of 32-112 ppm PG (not further specified. equivalent to 101-354 mg/m³). Thirteen animals died or were killed because of illness due to infection. No effects were seen on body weights, respiratory system, gastrointestinal tract, liver, kidney or spleen. The haemoglobin count was elevated at 112 ppm compared to controls. However, as many of the monkeys suffered from parasite infection and lung mites, the authors concluded that the haematological effects were not related to PG. (Robertson et al. 1947 - quoted from ATSDR 1997).

75.2.2     Oral intake

Inbred albino rats were administrated 2.45 % or 4.9 % PG (approximately 1225 or 2450 mg/kg b.w./day). The duration of exposure was not given in the reference. No effects of PG were reported on growth rate, food and water consumption, survival, gross and microscopic lesions in lung, heart, liver, spleen, kidney, adrenal or testis. (Morris et al. 1942 – quoted from LaKind et al. 1999).

Six male Wistar rats/group were dosed with 0 or 2942 mg PG/kg/day in the water for 10, 20 or 30 days. No death was observed. Body weights were reduced 41% at 10 days, but were then increased at 20 and 30 days. Enzyme activity in the gastrointestinal tract was increased, but no effects on the jejunal surface were seen.  (Morshed et al. 1991 – quoted from ATSDR 1997).

Rats were given drinking water containing 1, 2, 5, 10, 25 or 50 % PG v/v (corresponding to approximately 1320, 2640, 6600, 13200 or 26400 mg PG/kg b.w./day) for 140 days (20 weeks). Animals of the two highest exposure groups died within 10 weeks. No adverse effects were seen up to 10%  PG (13200 mg). (Clayton & Clayton 1982 – quoted from LaKind et al. 1999 and IUCLID 2000).

In a two-year feeding study in CD-1 rats, levels of 0, 6250, 12500, 25000 or 50000 ppm PG (corresponding to approximately 0, 200, 400, 900 or 1700 mg/kg b.w./day in males and 300, 500, 1000 or 2100 mg/kg b.w./day in females) were used. No significant compound-related effects were observed in any treated group when compared to controls with respect to behaviour, body weights, organ weights, haematology, or microscopic examination of the heart, lung, liver, kidney and adrenal. (Gaunt et al. 1972 - quoted from ATSDR 1997, LaKind et al. 1999 and IUCLID 2000).

Rats administered up to 1834 mg/kg b.w./day for 2 years showed very slight liver damage, but no renal pathology (Clayton & Clayton 1982 – quoted from LaKind et al. 1999).

Cats fed 1200 ppm PG in the diet for 2 weeks showed increased haptoglobin concentration (Weiss et al. 1992 – quoted from ATSDR 1997).

Cats were fed 1600 or 8000 mg PG/kg b.w./day for 3 or 5 weeks. At the high dose, the animals developed decreased activity, mental depression and slight to moderate ataxia. The high dose cats also had polyuria and polydipsia, and showed decreased erythrocyte counts Heinz body formation and hypercellularity of the bone marrow, while the level of D-lactate was 44 fold higher than controls. No effects were reported at the low dose. (Christopher et al. 1989 and 1990 – quoted from LaKind et al. 1999 and ATSDR 1997).

In a 12-week study, kittens administered 5 or 10 % PG in the diet (corresponding approximately to 1100 and 2400 mg/kg b.w./day, respectively) showed increased Heinz body formation. (Hickman et al. 1990 – quoted from LaKind et al. 1999).

Cats administered 0, 80, 443, 675, 1763 or 4239 ppm PG/kg feed for 13 weeks showed increased Heinz-body formation from 443 ppm. This effect was dose related as the haemosiderin content in the liver and the spleen from 673 ppm PG. (DOW 1979 – quoted from IUCLID 2000).

Male and female cats were treated in their diet with 0, 6, or 12 % PG (corresponding to 0, 2100 or 3560 mg/kg b.w./day) for 13 weeks. Haematological parameters were examined with 2 weeks interval in the treatment period. Haemoglobin was significantly decreased in the low, but not the high dose group. Erythrocyte count was significantly decreased in both treated groups, but not in controls. Reticulocyte aggregate numbers were significantly increased in the 12 % group and Heinz bodies significantly increased in both treated groups. (Bauer et al. 1992 – quoted from LaKind et al. 1999).

Cats exposed in the diet to 2400 mg PG /kg b.w./day for 17 weeks showed Heinz body formation (Weiss et al. 1990 - quoted from ATSDR 1997).

Dogs were exposed to PG in the drinking water at levels of 5% PG twice a day for females and 600 ml of 10 % PG for males daily over 9 months. No functional deficits in the kidney or liver were reported and no pathological changes were found in these organs. (VanWinkle & Neuman 1941– quoted from LaKind et al. 1999).

Groups of 5 male and 5 female dogs were given 2000 or 5000 mg/kg b.w./day for 2 years. An additional “control” group were given 63500 mg dextrose/kg/day. At 2000 mg/kg b.w./day, no effects were seen on body weights or on any organs. At 5000 mg/kg b.w./day slight decrease in erythrocyte count, haemoglobin and haematocrit, which was reversible, was noted, but no effects on the bone marrow could be shown. Increased urinary output and decreased water intake were noted. The relevance of these findings was not discussed in any of the references. (Weil et al. 1971 – quoted from ATSDR 1997, LaKind et al. 1999 and IUCLID 2000).

75.2.3     Dermal contact

No effects on the kidney were seen from PG doses of 0.16 to 5.0 ml/kg b.w. applied to the shaved abdominal skin or rabbits for 30 days (Hanzlik et al. 1947 - quoted from LaKind et al. 1999).

75.3     Toxicity to reproduction

75.3.1     Inhalation

75.3.1.1     Fertility

White rats exposed continuously to a concentration of 55-112 ppm PG for 18 months showed no adverse effects on the ability to produce live young, or on survival of the offspring (Roberson et al. 1947).

No data were found on developmental effects of PG after inhalation.

75.3.2     Oral intake

75.3.2.1     Fertility

No effects on reproductive parameters were reported from administration to CD-1 mice of 5 % PG in the drinking water (equivalent to 10100 mg/kg b.w./day over 14 weeks in a continuous breeding study. The offspring were examined for reproductive parameters, but did not show any effect of PG either. (Gulati et al. - quoted from LaKind et al. 1999).

A multigeneration, continuous breeding feeding study in Swiss albino mice was performed, using concentrations of 1, 2.5 or 5% in the drinking water (corresponding to 1820, 4800 or 10100 mg/kg b.w./day) over 98 days from day 1 before mating in the parent generation (F₀). No significant effects were seen on the fertility of this generation. No effects were noted in the offspring (F₁) or the following generation (F₂) for mating or fertility indices, mean number of liver pups per litter, proportion of pups born live, or gender of pups born live. (Morrissey 1989 - quoted from LaKind et al. 1999).

No significant differences were seen on fertility rate, numbers of resorptions, average litter size or birth weight in CD1-mice treated by gavage with 10000 mg PG/kg b.w./day on gestation days 8-12. (Kavlok et al. 1987 - quoted from LaKind et al. 1999).

75.3.2.2     Developmental toxicity

Testing for developmental effects in several mammalian species using a 10-day exposure identified no effects at the highest dose tested, e.g. respectively 1600 mg/kg b.w./day for Wistar rats and CD-1 mice; 1550 mg/kg b.w./day for golden hamsters and 1230 mg/kg b.w./day for Dutch-belted rabbits. No differences in numbers of soft tissue changes or skeletal abnormalities between treated groups and control groups, and no effects on maternal or fetal survival were noted. (Food and Drug Research Laboratories Report No 1573j-1574j 1973 – quoted from LaKind et al. 1999).

In a developmental screening test, mice were administered 10000 ppm PG via gavage on days 8 to 12 of gestation. No significant differences were reported for maternal deaths, numerous reproductive endpoints, postnatal pup weight gain or abnormalities in the pups. No further details were found. (Kavlok et al. 1987 – quoted from LaKind et al. 1999).

75.3.3     Dermal exposure

No data were found.

75.4     Mutagenic and genotoxic effects

75.4.1     In vitro studies

PG was negative in an Ames test conducted without metabolic activation in Salmonella typhimurium strains TA98, TA 100, TA 1535 and TA 1537. (Pfieffer & Dunkelberg 1980 – quoted from IUCLID 2000).

PG was also negative in another Ames test in strains TA 92, TA 94, TA 98, TA 100, TA 1535 and TA 1537 conducted with metabolic activation. (Ishidate et al. 1984 – quoted from IUCLID 2000).

A third Ames test was conducted in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 at concentrations of 20-5000 mg PG/plate with and without metabolic activation. The result of this study was also negative. (BASF 1981 – quoted from IUCLID).

A chromosome aberration assay was conducted twice in human lymphocytes, using concentrations of 476, 1910 and 3810 mg PG/l, according to OECD guideline 473. PG did not cause a statistically significant increase in the proportion of metaphases containing chromosome aberrations. (EC GmbH 1990 – quoted from IUCLID 2000).

A chromosome aberration test conducted in human embryonic lung cells (WI-38) with 0.001, 0.01 and 0.1 mg PG/ml was negative (Litton Bionetics Inc. 1974 - quoted from IUCLID).

In another chromosome aberration test, Chinese hamster lung fibroblast (CHL) cells were exposed to 32 mg PG/ml without metabolic activation. The test was positive. However, when the concentration was doubled and metabolic activation added, no increase in chromosome aberrations was found (Ishidate et al. 1984, 1988 - quoted from Mortensen 1993).

A DNA damage and repair assay conducted with PG in Chinese hamster V79-cells with and without activation was negative (Swenberg et al. 1976 - quoted from IUCLID).

A cell transformation assay conducted with PG in Syrian hamster embryo cells was negative (Mutat Res 1983 - quoted from IUCLID).

75.4.2     In vivo studies

No chromosome aberrations in the bone marrow were seen in a cytogenetic assay in rats following administration by gavage of a single dose or 5 doses of 30, 2500 and 5000 mg PG /kg b.w. The same dosing regime was used in a dominant lethal assay which was negative. (Litton Bionetics 1974 – quoted from IUCLID 2000).

A dominant lethal assay in mice treated intraperitoneally with a single dose of 10 mg PG/kg b.w was negative (Kennedy Jr. et al. 1975 – quoted from IUCLID 2000 and A&H 1983).

75.5     Carcinogenic effects

75.5.1     Inhalation

No data were found.

75.5.2     Oral intake

No treatment-related increase in neoplasms was seen in a two-year study in rats treated with up to 2500 mg/kg b.w./day in the diet. Other effects seen in this study are reported under section 4.2.2. (Gaunt et al. 1972 – quoted from ATSDR 1997).

75.5.3     Dermal contact

No increase in tumours was observed after twice weekly application of PG to the skin of Swiss mice for 120 weeks, at doses up to 2 mg. No further details were given. (Stenbak & Shubik 1974 - quoted from ATSDR 1997).

76     Regulations

76.1     Ambient air

-

76.2     Drinking water

-

76.3     Soil

-

76.4     Occupational Exposure Limits

-

76.5     ADI

EU:                                25 mg/kg b.w. (SCF 1993)

76.6     EU-Classification

The substance is not adopted on Annex I to directive 65/548/EEC (MM 2002)

76.7     IARC

   -

76.8     US-EPA

   -

77     Summary

77.1     Description

Propylene Glycol (PG) is a colourless viscous liquid. PG is soluble in water and hygroscopic and has a low vapour pressure.

77.2     Toxicokinetics

PG has been shown to be rapidly absorbed form the gastrointestinal tract and following dermal absorption through damaged skin. It is metabolised to lactic acid and pyruvic acid, which enter the energy production, or for pyruvic acid is further metabolised to CO₂ and water. 20- 45% PG is found unchanged in urine.

77.3     Human toxicity

77.3.1     Single dose toxicity

No reports of deaths caused by PG were found. Acute effects included CNS depression and acidosis was reported from exposure to very high doses.

77.3.2     Irritation and sensitisation

Numerous patch tests have been performed to examine the incidence and nature of PG skin reactions. However, the results were difficult to interpret. PG showed weak irritative and none to slight allergenic response.

77.3.3     Repeated dose toxicity

No effects were seen from repeated exposure to up to 94 mg PG/m³ over several weeks. Ingestion by a child of 114-228 mg/kg b.w./day for 13 months resulted in CNS-depression with seizures and unconsciousness.

No further data regarding effects in humans were found.

77.4     Animal toxicity

77.4.1     Single dose toxicity

Rabbits exposed to aerosol concentrations of 10% PG showed degeneration of the goblet cells in the trachea, and the ciliated cells were affected.

Reported oral LD₅₀-values for rats and mice range from approximately 20 to 30 g/kg b.w., while the LD₅₀-values for rabbits and guinea pigs ranged from 18 to 20 g/kg b.w.; an LD₅₀-valus of 22 g PG/kg b.w. in dog was reported. Signs of toxicity include CNS-depression and respiratory depression and reversible haematological effects. Slight hyperaemia or the gastrointestinal tract and one case of haemorrhagic enteritis in rats have also been reported.

A dermal LD₅₀-value in the rabbit was reported to be 20.8 g/kg b.w.

77.4.2     Irritation and Sensitisation

Various reports describe PG as not irritating to mildly irritating to the skin and eyes of rabbits. No sensitisation studies in animals were found.

77.4.3     Repeated dose toxicity

Nose bleeding was reported from inhalation exposure of rats to aerosols at  concentrations of 160, 1000 and 2200 mg PG/m³ over 90 days.

Repeated inhalation exposure of rats to aerosols with 1000 or 2200 mg PG/m³ over 90 days caused multiplication and enlargement of the goblet cells and their mucous content in the nasal epithelium.

Rats exposed by inhalation to 2200 mg PG/m³ as an aerosol over 90 days showed an effect (not dose-related) on haematological parameters.

Dogs treated via the diet with 5000 mg PG/kg b.w/day over 2 years and cats treated for shorter periods of time (2, 3, 5, 13 or 17 weeks) with doses of 1100 to 8000 mg PG/kg b.w./day showed haematological changes with decreased erythrocyte count and Heinz body formation.

No effects were seen in rats and in dogs treated with 2000 mg/kg b.w./day for 2 years. An ADI for PG from food of 25 mg/kg b.w/day has been defined on basis of this NOAEL.

77.4.4     Toxicity to reproduction

PG did not cause fertility effects in rats by inhalation or oral administration. No developmental effects in rats, mice, hamsters or rabbits treated by the oral route. No dermal data were available.

77.4.5     Mutagenic and genotoxic effects

PG was tested in in vitro bacterial assays and in mammalian cells for chromosome aberrations, DNA-damage and repair and cell transformation. All tests but one, a chromosome aberration assay in Chinese hamster lung cells conducted without metabolic activation, were negative.

An in vivo chromosome aberration test in rats and two dominant lethal in vivo tests, in rats and mice, were negative.

77.4.6     Carcinogenic effects

No carcinogenic effect was reported from PG in a 2-year oral study in rats or a 120-week dermal study in mice.

78     Evaluation

Data on human toxicity of propylene glycol (PG) are scarce. The toxicological data on experimental animals indicate that PG has a low order of toxicity.

PG has a very low acute oral toxicity in several species with LD₅₀-values of 18-33.5 g/kg b.w.. At high oral doses, central nervous system depression occurs as demonstrated in dogs treated with 8000 mg/ kg b.w.

No effects on the respiratory tract have been reported from human exposure to PG used as a vehicle for medical purposes. No systemic effects in rabbits and dogs were reported from short-time inhalation exposure of 10% PG. However, no details on exposure conditions or particle size of the aerosol were given in the reference. Local effects in the respiratory tract involving goblet cells have been reported in rats and rabbits.  Haemorrhage from the nose and hyperaemia of the intestines is probably related to the highly hygroscopic character of PG and the effects are probably due to dehydration of the tissues.

PG is a mild irritant to rabbit skin. No data on sensitisation to PG in animals were found. Contact dermatitis in humans from PG has been reported. The majority of these reactions are of irritative nature. However, a few may be of allergic nature.  On basis of the low number of persons sensitised to PG in comparison with widespread exposure due to extensive use of the substance, the sensitising potential of PG is considered to be low.

Effects on the erythrocytes with decreased erythrocyte counts and formation of Heinz bodies was described at high doses in dogs treated orally with 5000 mg PG/kg b.w./day over 2 years, and more consistently and at lower doses in cats exposed to PG by the oral route for 2-17 weeks to doses as low as 1100 mg/kg b.w./day. Cats thus appear to be more sensitive to PG, which could be explained by a different metabolism in cats possibly yielding a higher level of the parent compound in the blood. No haematological signs have been reported in humans from exposure to PG. Therefore, the effect is considered of lesser relevance to humans.

Short-term studies in rats and dogs showed no adverse effects at levels of around 10 % of the diet (equivalent to approximately 13200 mg/kg b.w. in rats). The NOAEL from long-term feeding studies in rats and dogs was 2000 mg/kg b.w./day. PG is considered of low toxicity following repeated exposure.

No effects on reproduction were reported from fertility and developmental studies in mice, rats, hamsters and rabbits.

Several negative in vitro assays for different mutagenicity end-points are available. One in vitro chromosome aberration test showed a positive result without metabolic activation. However, 3 in vivo tests were negative. On basis of all the evidence available, PG is evaluated not to be a mutagenic substance.

No carcinogenic effect has been reported.

Based on the available data, the critical effects of PG are considered to be the effects on the skin and its dehydrating potential to mucous membranes.

79     References

A&H (1983). Propylenglykol. Arbete och Hälsa 27, 1-38. Nordiska expertgruppen för gränsvärdesdokumentation. Arbetarskyddsverket.

Andersen KE (1980). Hudreaktioner fremkaldt af propylenglykol. Ugeskr Læg 142, 2475-2478.

ATSDR (1997). Toxicological profile for ethylene glycol and propylene glycol. US Department of Health & Human Services. Public Health Service, Agency for Toxic Substances and Disease Registry.

Cohen BM and Crandall C (1964). Physiologic benefits of “Thermo Fog” as a bronchodilator vehicle: Acute ventilation responses of 93 patients. Am J Med Sci 247, 57-61.

ChemFinder (2002). Propylene glycol. Http://www.chemfinder.com.

IUCLID (2000). Propane- 1,2-diol. In: International Uniform Chemical Information Database. Existing Chemicals 2000. ECB, JRC, Ispra, Italy.

LaKind JS, McKenna EA, Hubner RP and Tardiff RG (1999). A review of the comparative mammalian toxicity of ethylene glycol and propylene glycol. Crit Rev Toxicol 29, 331-369.

MM (2002). The Statutory Order from the Ministry of the Environment no. 439 of June 3, 2002, on the List of Chemical Substances.

Mortensen B (1993). Propylene Glycol. In: Health effects of selected chemicals 2. Nordic Council of Ministers. Nord 1993: 29, 181-208.

SCF (1993). Opinion on propylene glycol. In: Reports of the Scientific Committee for Food. EU Commission.

Suber RL, Deskin R, Nikiforov I, Fouillet X and Coggins CRE (1989). Subchronic nose-only inhalation study of propylene glycol in Sprague-Dawley rats. Fd Chem Toxic, 27, 573-583.

Appendix 11: 2-Butoxyethanol (EGBE) 
80     General description

80.1     Identity

Molecular formula:          C₆H₁₄O₂

Structural formula:           CH₃-CH₂-CH₂-CH₂-O-CH₂-CH₂-OH

Molecular weight:            118.2

CAS-no.:                        111-76-2

Synonyms:                      2-Butoxyethanol

                                      n-Butoxyethanol

                                      Butyl Cellosolve

                                      Butylglycol

                                             Glycol butyl ether

                                      Monobutyl ethylene glycol ether

80.2     Physical / chemical properties

Description:                    Colourless liquid with a faint, mild ethereal odour.

Melting point:                  -70, -75 °C

Boiling point:                   171 °C

Density:                          0.9019 g/ml (at 20°C)

Vapour pressure:             0.76 mmHg (101.3 Pa) (at 20°C)

                                      0.88 mmHg (117.3 Pa) (at 25°C)

Concentration of

saturated vapours:           4900 mg/m³ (at 20°C and 760 mmHg) (calculated).

Conversion factor:           1 ppm = 4.91 mg/m³        (at 20°C and 760 mmHg).

                                      1 mg/m³ = 0.204 ppm

Solubility:                        Water: Miscible.

LogP_{octanol/water}:                0.81

References:                    EPA (1999), ATSDR (1998).

81     Toxicokinetics

81.1     Absorption, distribution, excretion

81.1.1     Inhalation

When seven male volunteers were exposed to EGBE (20 ppm (98 mg/m³)) for 2 hours during light physical exercise (bicycle ergometer), the respiratory uptake of EGBE averaged 1.2 mg/minute (57% of the inspired amount). The concentration of EGBE in blood reached a plateau level of 0.9 mg/l within 1-2 hours and was no longer detectable 2-4 hours after the end of exposure. The apparent values of elimination half-time, mean residence time, total blood clearance, and steady-state volume of distribution were 40 minutes, 42 minutes, 1.2 l/minute, and 54 l, respectively, for EGBE. The half-life for EGBE in urine was 1.36 hours. The amount of EGBE excreted in urine was less than 0.03% of the total uptake, while that of 2-butoxyacetic acid (2-BAA) ranged from 15-55%. Relative to the uptake of EGBE, the excretion averaged 41% on an equimolar basis. (Johanson et al. 1986 – quoted from ATSDR 1998, ECETOC 1995b).

In a similar study in five healthy male volunteers, the minimum and maximum observed concentrations of 2-BAA in blood ranged from 2.4 to 6.7 mg/l; the blood level peaked after 2-4 hours. (Johanson & Johanson 1991 – quoted from ATSDR 1998).

Healthy male volunteers (four individuals) were exposed to 50 ppm (245 mg/m³) EGBE vapour for 2 hours by mouth (through a respiratory valve). The concentration of EGBE increased during the first hour and appeared to approach steady state at about 0.35 mg/l at the second hour. The respiratory uptake rate was 1.3 mg/minute. (Johanson & Boman 1991 – quoted from ATSDR 1998).

When rats (three to five animals per group) were exposed (nose-only) to 4.3, 49, or 438 ppm (21, 240, or 2150 mg/m³) [¹⁴C]EGBE for 6 hours, the respiratory uptake was 531, 541, and 423 mikrogram/ppm at 4.3, 49, and 439 ppm, respectively. A less-than-proportional amount of EGBE was, according to ATSDR, inhaled at the high dose because of decreased minute volume. 2-BAA was the major metabolite of EGBE in plasma. Ratios of ethylene glycol to 2-BAA in plasma were higher than those in urine. The majority of the inhaled [¹⁴C]EGBE was eliminated in the urine, with free 2-BAA being the major urinary metabolite, accompanied by lesser amounts of ethylene glycol and EGBE glucuronide. A small proportion (5-8%) of the retained EGBE was exhales as ¹⁴C-carbon dioxide. Faeces accounted for 1-2% of the absorbed dose. At 4.3 ppm, about 60% of the urinary radioactivity was excreted during the 6-hour exposure, whereas at 438 ppm, only 10% was excreted during the exposure period. The production of carbon dioxide most closely followed the production of ethylene glycol and may represent the metabolism of this metabolite. (Sabourin et al. 1992 – quoted from ATSDR 1998).

In another study in rats, eight male Sprague-Dawley rats were exposed to 20 or 100 ppm (98 or 490 mg/m³) EGBE 24 hours per day for 1, 2, 3, 4, 6, 8, 10, or 12 days. The concentrations of EGBE and 2-BAA in blood, muscle, liver, and testis increased rapidly during the first 1-3 days of exposure and continued to increase, but more slowly, during the remaining days. The average peak concentration of EGBE in the blood was 1.8 mg/l in the 20-ppm group and 8.5 mg/l in the 100-ppm group. Respiratory uptake averaged 37 mg/day at 20 ppm and 186 mg/day at 100 ppm. The urinary excretion rate for 2-BAA in the 20-ppm group averaged 24 mg/day, whereas the 100-ppm group excreted 122 mg/day; the observed urinary excretion rate corresponded to 64% of the calculated respiratory uptake and renal clearance of 0.53 l/hour for both groups. (Johanson 1994 – quoted from ATSDR 1998).

81.1.2     Oral intake

Case reports of intentional poisonings with EGBE indicate that EGBE is absorbed in humans following oral exposure.

When male F-344 rats were given a single oral dose of [¹⁴C]EGBE at 125 or 500 mg/kg, detectable exhalation of radioactivity began 1-2 hours after treatment indicating rapid absorption and distribution. Approximately 18 or 10% of the respective doses was exhaled as ¹⁴C-carbon dioxide in 48 hours. Cumulative urinary excretion of radioactivity amounted to approximately 32 or 18% of the respective doses at 8 hours, to approximately 64 or 30% at 24 hours, and to approximately 70 or 40% at 48 hours. Faecal excretion of radioactivity amounted to 2-3% of the doses in 48 hours. Thus, by adding the 48-hour excretion of radioactivity in the expired air and urine, at least 88% of the 125 mg/kg dose and at least 50% of the 500 mg/kg dose was absorbed. According to ATSDR, the fact that the percentage of radioactivity excreted was less at the higher dose than at the lower dose may indicate saturation of a metabolic pathway rather than lower absorption of the high dose.

EGBE was distributed to all tissues, with the highest levels (determined 48 hours after dosing) detected in the forestomach. The increase in the tissue concentration was not proportional to the increase in dose. While the ratio of the administered doses was 1:4, the ration of the tissue concentration in most tissues was greater than 1:10. According to ATSDR, this may be related to saturation of EGBE metabolism at the high dose.

The major route of elimination was the urine (40-70%), followed by exhalation (11-18%). A small portion was excreted in the bile (8% at the 500-mg/kg dose) 8 hours after dosing, and a small amount was detected in the faeces (2-3%).

(Ghanayem et al. 1987 – quoted from ATSDR 1998).

In male F-344 rats given single gavage doses of 8.6 or 126 mg/kg [¹⁴C]EGBE, 59% of the low dose of radioactivity and 38-70% of the high dose of radioactivity was excreted in the urine during the first 24 hours. Excretion of ¹⁴C-carbon dioxide in the expired air amounted to 7.2 or 8%, respectively, in 24 hours. Thus, at least 66% of the low dose and 46-78% of the high dose was absorbed. (Corley et al. 1994 – quoted from ATSDR 1998).

Male F-344 rats were administered [¹⁴C]EGBE in the drinking water at concentrations of 290, 860, or 2590 ppm (corresponding to 28, 47, or 141 mg/kg b.w./day according to ATSDR) for 24 hours. The majority of the radioactivity was excreted in urine (50-60% as 2-BAA, 10% as ethylene glycol) or exhaled as ¹⁴C-carbon dioxide (8-10% of the dose). Less than 5% of the dose was exhaled as unmetabolised EGBE. Ethylene glycol was excreted in urine, representing approximately 10% of the dose of EGBE. Elimination in the faeces was a minor pathway (1.6-2.8% of the dose). Unmetabolised EGBE and its glucuronide conjugate were also identified in the urine. The maximum excretion occurred during the first 12-24 hours after the start of exposure. (Medinsky et al. 1990 – quoted from ATSDR 1998).

No quantitative or qualitative alterations of metabolism or disposition were observed in rats by repeated exposure to EGBE as compared to treatment with a single dose. Elimination of ¹⁴C-carbon dioxide and EGBE-derived radioactivity in the urine of rats receiving multiple doses of EGBE was essentially the same as for rats receiving a single dose. The ratios of free 2-BAA, the EGBE-glucuronide and the EGBE-sulphate conjugates, and parent EGBE excreted in the urine of rates treated for 4 or 8 days were relatively similar to those from rats treated with a single dose. (Ghanayem et al. 1992 – quoted from ATSDR 1998).

81.1.3     Dermal contact

Dermal absorption of EGBE was evaluated in workers using window-cleaning agents containing EGBE in concentrations ranging from 0.9 to 21.2% in volume (Vincent et al. 1993 – quoted in ATSDR 1998). Positive correlations between exposure levels of EGBE and 2-BAA in the urine indicate dermal (and probably some pulmonary) absorption of EGBE by these workers.

Experimental evidence exists that dermal absorption of EGBE vapours may contribute to the overall absorption of EGBE during vapour exposure (Johanson & Boman 1991 – quoted in ATSDR 1998, EPA 1999, ECETOC 1995b). Healthy male volunteers were exposed to 50 ppm (245 mg/m³) EGBE vapour for 2-hour, followed by 1 hour of no exposure, followed by 2 hours of body-only exposure (exposed in a chamber while breathing fresh air via respirator) to 50 ppm. Capillary blood samples (finger prick, assuming that the finger prick blood samples represented mixed arterial blood) were collected at regular intervals and analysed for EGBE. The average concentration in blood and the calculated rate of uptake of EGBE was about 3-4 times higher during dermal exposure than during inhalation exposure, suggesting that about 75% of the total uptake during normal vapour exposure could be accounted for by dermal absorption.

The results obtained in the above study have been questioned by Corley et al. (1994 – quoted in EPA 1999). They reanalysed the data assuming that the finger prick blood samples represented venous blood draining the skin prior to mixing systemically. These simulations resulted in a dermal absorption contributing no more than 22% of the total uptake of EGBE in a whole-body exposure.

Corley et al. (1997 – quoted in ATSDR 1998, EPA 1999) observed a much lower dermal absorption (<1500 times) of EGBE vapour when blood samples were taken from an unexposed arm compared to blood samples taken from the exposed arm (finger prick sample).

About two-thirds of the 2-BAA excreted in the urine was in the form of the N-butoxyacetyl glutamine conjugate. No free EGBE, free og conjugated ethylene glycol ether, or glycolic acid were detected in the urine. 2-BAA was eliminated in the urine primarily during the first 12-hour collection period.

Dermal uptake has been estimated under worst-case resting conditions (respiration rates at their lowest, no clothing worn) and under more realistic conditions (35% of the body surface area exposed). Under the worst-case conditions, 15-27% of the total uptake of EGBE vapour would be through the skin, while dermal uptake would be 4.5-8.5% of the total if only 25% of the body surface was exposed. Under simulated exercise, uptake was estimated as 4.6-8.7% and 1.2-2.3% of the total assuming 100 or 25% of the body surface area was exposed, respectively. (Corley et al. 1997 – quoted from ATSDR 1998).

Percutaneous absorption of liquid EGBE has been investigated in male volunteers keeping two or four fingers immersed in neat EGBE for 2 hours. The presence of EGBE in blood and 2-BAA in urine confirmed that EGBE enters the systemic circulation. The excretion rate of 2-BAA increased during the first hours after exposure, reached a maximum at about 5 hours, and then declined, with an average half-life of 3.1 hours. Seventeen percent of the absorbed dose was excreted as 2-BAA in 24 hours. (Johanson et al. 1988).

EGBE is also absorbed through animal skin. Percutaneous absorption in rats appears to be 20-30% of the applied dose (Bartnik et al. 1987, Sabourin et al. 1992, 1993 – both quoted in ATSDR 1998).

Only a small amount of the applied dose (0.3-2%) was still present at the application site 72 hours following dosing. The majority of the absorbed dose was excreted in the urine (82-83%); 3-5% was found as carbon dioxide, 3-6% in the faeces, and 3-13% in the carcass. Free 2-BAA was the main metabolite (66-70%), followed by the EGBE-glucuronide (13-15%9, and ethylene glycol (4-6%). (Sabourin et al. 1992, 1993 – quoted in ATSDR 1998).

81.2     Elimination

According to ATSDR, there is no available evidence to suggest that the route of administration has any substantial effect on the subsequent metabolism of EGBE in humans, but data from animals indicate that the route of exposure may make some differences in the relative amounts of metabolites produced.

The proposed metabolic pathway in rats and humans for EGBE is shown in Figure 2.2. EGBE is oxidised in the liver by alcohol dehydrogenase to butoxyacetaldehyde (BAL), which is further oxidised to 2-BAA by acetaldehyde dehydrogenase. In humans, 2-BAA can be conjugated with glycine or glutamine to form N-butoxyacetyl glycine or N-butoxyacetyl glutamine, respectively, or it can be further metabolised (humans, rats) to carbon dioxide. EGBE can also undergo O-dealkylation (by a cytochrome P450 dealkylase CYP2E1) to form ethylene glycol, and butyraldehyde and butanol. In rats, EGBE can also be conjugated with sulphate to form butoxyethanol sulphate, or with glucuronic acid to form the glucuronide. (EPA 1999, ATSDR 1998).

The two main oxidative pathway of EGBE metabolism in rats are alcohol dehydrogenase and O-dealkylation by CYP2E1. EGBE may also form conjugates with glucuronide and sulphate to some extent. Primarily because 2-BAA is excreted in the urine of both rats and humans following exposure to EGBE, it has been suggested that the former pathway would be applicable to both rats and humans. However, the other three proposed metabolic pathways of EGBE may be applicable only to rats as the metabolites of these pathways (ethylene glycol, EGBE-glucuronide, and EGBE-sulphate) have been observed only in the urine of rats and not in the urine of humans. In addition, approximately two-thirds of the 2-BAA formed by humans is conjugated with glutamine and, to a lesser extent, glycine; these 2-BAA conjugation pathways have not been detected in the rat. (Medinsky et al. 1990, Corley et al. 1997, Bartnik et al. 1987, Ghanayem et al. 1987 – quoted from EPA 1999).

81.3     Mode of action

The most characteristic toxic effect of EGBE in experimental animals is on the haematological system and includes elevated osmotic fragility of erythrocytes, haemolysis, decreased haematocrit (Hct), haemoglobinuria, haemoglobinaemia, and haemolytic anaemia. Compensatory erythropoiesis occurring in response to haemolysis results in an increase in reticulocytes, increased mean cell volume, increased or normal mean cell haemoglobin, and decreased mean cell haemoglobin concentrations. Most of the other toxic effects of EGBE observed in animal studies may be secondary to the haemolytic effects. (ATSDR 1998, NTP 2000).

Figure 2.2. Proposed metabolic pathway for EGBE in rats and humans. From EPA (1999 – adapted from Medinsky et al. 1990 and Corley et al. 1997).

The EGBE-induced haemolysis is a direct effect on the erythrocyte membrane causing osmotic fragility and intravascular lysis. Significant species differences have been reported with rats appearing to be particularly sensitive to EGBE induced haemolysis, whereas humans appear to be more resistant to this effect. (ECETOC 1995a, ATSDR 1998, NTP 2000).

The mechanism(s) of action is not fully understood, but the metabolite of EGBE, 2-butoxyacetic acid (2-BAA), shows a significantly greater haemolytic effect in vitro than EGBE (ECETOC 1995a, EPA 1999, ATSDR 1998). Furthermore, studies in rats showed that pyrazole and cyanamide as metabolic inhibitors of alcohol and aldehyde dehydrogenases, respectively, protected or significantly reduced the EGBE-induced haematotoxicity (EPA 1999).

Spermatogenesis is adversely affected by the lower ethylene glycol ethers (EGME, EGEE) and their acetates with EGME being the most potent glycol ether inducing testicular toxicity. There is no evidence however, that higher homologues, as EGBE, are testicular toxicants. (ECETOC 1995a).

The developmental effects of ethylene glycol ethers are mediated by alkoxy acetic acid metabolites. However, ethylene glycol ethers with alkyl chains of more than 2 carbon atoms, as EGBE, do not appear to be selectively toxic to the foetus. For EGBE, foetotoxic effects have been observed only in the presence of maternal toxicity. In an in vitro study, the alkoxy acetic acids methoxyacetic acid (MAA) (from ethylene glycol monomethyl ether (EGME)) and ethoxyacetic acid (EEA) (from ethylene glycol monomethyl ether (EGEE)) showed similar developmental effects, whereas 2-BAA (from EGBE) was only slightly active. (ECETOC 1995a).

82     Human toxicity

Haematological effects have been identified as the critical end-point in toxicological studies on EGBE. Humans appear to be less sensitive to the haematological effects of EGBE than are rats, mice, rabbits, and guinea pigs. (EPA 1999).

82.1     Single dose toxicity

82.1.1     Inhalation

Three controlled studies have been conducted by Carpenter et al. (1956 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

In the first study, two men were exposed for 4 hours to an EGBE concentration of 113 ppm (555 mg/m³). Effects observed included nasal and ocular irritation, a metallic taste in the mouth, and belching. Erythrocyte osmotic fragility was not affected.

In a second study, two men and one woman were exposed to 195 pm (957 mg/m³) for two 4-hour periods, separated by a 30-minute recess. There was no change in the blood pressure, erythrocyte fragility, or pulse rate. Irritation of the nose and throat followed by ocular irritation and disturbed taste was noted.

In the third study, two men and two women were exposed for 8 hours to an EGBE concentration of 100 ppm (490 mg/m³). No changes in blood pressure, erythrocyte fragility, or pulse rate were observed. Irritation of the nose and throat followed by ocular irritation and a disturbing metallic taste were noted.

When seven male volunteers were exposed to EGBE (20 ppm (98 mg/m³)) for 2 hours during light physical exercise (bicycle ergometer), none of the subjects complained of or showed any signs of adverse effects (pulmonary ventilation, respiratory frequency, heart rate, electrocardiograms) (Johanson et al. 1986 – quoted from ECETOC 1995b, ATSDR 1998, NTP 2000).

82.1.2     Oral intake

In twenty-four children (aged 7 months to 9 years) observed subsequent to oral ingestion of at least 5 ml of glass window cleaner containing EGBE (0.5-9.9%), no symptoms of EGBE poisoning and no haemolysis were observed in any of the children (Dean & Krenzelok 1991 – quoted from EPA 1999, ATSDR 1998).

Poisoning cases by ingestion of household products containing EGBE have been reported (Rambourg-Schepens et al. 1988, Gijsenbergh et al. 1989, Bauer et al. 1992, Gualtieri et al. 1995  – quoted from EPA 1999, ECETOC 1995b, ATSDR 1998, NTP 2000). The intakes of EGBE were 30-60 ml, 25-30 g (corresponding to 400-500 mg/kg b.w.), ca. 46 ml, and 79-105 ml (corresponding to 1130-1500 mg/kg b.w.) in the four case reports, respectively. The symptoms observed in various studies included cardiovascular effects (increased heart rate, decreased blood pressure), haematological effects (decreased haemoglobin concentration and haematocrit, and thrombocytopenia), metabolic acidosis, haematuria, and haemoglobinuria.

82.1.3     Dermal contact

No data have been located.

82.1.4     In vitro studies

Blood from male and female young volunteers was unaffected by 4-hour incubations with 2-butoxyacetic acid (2-BAA, the toxic metabolite of EGBE) at concentrations up to 4.0 mM (470 mg/l) (60-235 mg/l produced total rat erythrocyte haemolysis). There was a slight increase in haemolysis of human erythrocytes incubated with 8 mM (945 mg/l) of 2-BAA with female erythrocytes showing a slightly greater sensitivity than male erythrocytes. (Ghanayem 1989, Ghanayem 1989 et al. 1989 – quoted from EPA 1999, ECETOC 1995b, ATSDR 1998).

Using a sensitive assay for erythrocyte deformability and haemolysis, human erythrocytes were unaffected following exposure to 2-BAA at concentrations up to 2mM (235 mg/l) for up to 4 hours (rat erythrocytes were haemolysed and showed decreased deformability). The possibility that certain human subpopulations, including the aged and those predisposed to haemolytic disorders, might be at an increased risk from exposure to EGBE has been investigated using blood from the elderly (mean age 71.9 years; range 64-79 years, five men and four women), from patients with sickle cell disease (seven patients), and from patients with spherocytosis (three individuals). Erythrocytes from these potentially sensitive groups were unaffected by incubations with 2-BAA at concentrations up to 2.0 mM (235 mg/l) for up to 4 hours. (Udden 1994, Udden & Patton 1994 – quoted from EPA 1999, NTP 2000; Udden 1992 – quoted from ECETOC 1995b).

The deformability of human erythrocytes incubated at 2-BAA concentrations of 7.5-10 mM (885-1180 mg/l) displayed a slight but significant decrease that was accompanied by slight increases in osmotic fragility and MCV. These effects were judged pre-haemolytic and corresponded to similar changes reported in rat erythrocytes, but at approximately 15-fold lower concentrations (0.5 mM) in rat erythrocytes than in human erythrocytes. (Udden 1995 – quoted from EPA 1999).

82.2     Irritation

Irritation of nose and throat, and eyes have been reported following exposure to airborne EGBE at concentrations from 490-957 mg/m³ for 4-8 hours; irritation was not reported following exposure to EGBE (98 mg/m³) for 2 hours. See 3.1.1 for further details.

82.3     Sensitisation

Volunteers (214 men and women) were given 0.2 ml 10% EGBE in patches applied to the intra-scapular area of the back. The dosed area was occluded. Testing included dosing, removal of the patch within 24 hours, evaluating the exposed area at 48 hours, and application of an identical patch to the same location. The subjects received nine such applications over a 6-week period. By the end of the 6 weeks, 25% of the subjects showed slight or definite erythema. Following application to a naïve area at 6 weeks, slight erythema was observed in seven subjects at 48 hours and 12 at 72 hours; one subject had definite erythema at 72 hours. No other effects were noted. It was concluded that there were no dermal effects of 10% EGBE under the conditions of the study. (Greenspan et al. 1995 – quoted from ATSDR 1998, NTP 2000).

82.4     Repeated dose toxicity

In a cross-section study, 31 male workers (22-45 years old, employed for 1-6 years) exposed to low levels of EGBE were monitored by Haufroid et al. (1997 – quoted from EPA 1999, ATSDR 1998). The average airborne concentration of EGBE was 2.9 mg/m³ (20 workers were exposed to an average concentration of 3.7 mg/m³ and 11 workers to 2.3 mg/m³). Red blood cell (RBC) count, haemoglobin (Hgb), haematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), haptoglobin (Hp), reticulocyte numeration (Ret), and osmotic resistance (OR, a measure of osmotic fragility) as well as hepatic and renal creatinine and urinary retinal binding protein parameters was investigated. Single determinations of 2-BAA (toxic metabolite of EGBE) in post-shift urine samples were used to assess exposure to EGBE. A significant correlation was observed between post-shift free urinary 2-BAA concentrations and EGBE in air. No difference between exposed and control workers was observed for RBC counts, Hgb, MCV, MCH, Hp, Ret, and OR. Statistically significant changes were observed for Hct (3.3% decrease) and MCHC (2.1% increase). No significant differences were observed in hepatic and renal biomarkers. According to ATSDR (1998), the differences in Hct and MCHC may be consistent with haemolysis and may be early indicators of potential adverse effects, but because the changes were in the range of normal clinical values, the concentration in the air of EGBE (2.9 mg/m³) was considered as a NOAEC.

82.5     Toxicity to reproduction

No data have been located regarding toxicity to reproduction in humans following exposure to EGBE.

82.6     Mutagenic and genotoxic effects

No increases in the frequencies of micronuclei or sister chromatid exchanges were observed in peripheral blood lymphocytes of varnish production workers exposed to both EGBE and to 2-ethoxyethanol (EGEE) (Söhnlein et al. 1993 – quoted from ATSDR 1998, NTP 2000).

82.7     Carcinogenic effects

No data have been located regarding carcinogenic effects in humans following exposure to EGBE.

83     Animal toxicity

Numerous studies have examined the effects of EGBE in experimental animals. Haematological effects have been identified as the critical end-point in the toxicological studies following both acute and repeated exposures. In addition to the haematological effects, effects in the liver, spleen, and kidney have also been observed following exposure to EGBE; the available data indicate that these effects are secondary to the haematological effects. Below, the focus has therefore been put on the most relevant studies for an evaluation of the haematological effects primarily based on the citations of the relevant studies in EPA (1999), ATSDR (1998), and ECETOC (1995b).

83.1     Single dose toxicity

83.1.1     Inhalation

For rats, an LC₅₀-value (4 hours exposure, whole-body) of 2385 mg/m³ has been reported for males and of 2210 for females; haemoglobinuria and effects in kidneys were observed. In another study, the LC₅₀-value (4 hours exposure) was reported to be about 2455 mg/m³. (ECETOC 1995b, ATSDR 1998, NTP 2000).

An LC₅₀-value (7 hours exposure) of 3440 mg/m³ has been reported for mice. One of six guinea pigs died after 4 hours of exposure to saturated vapour (ca. 4900 mg/m³); no haemoglobinuria was observed. (ECETOC 1995b, ATSDR 1998, NTP 2000).

Haematological effects of EGBE have been observed in animals after acute inhalation exposure. Female rats exposed to 432 ppm (2120 mg/m³) for 2-8 hours exhibited haemolysis at 2 hours, haemoglobinuria at 3 hours, and hemin crystalluria at 4 hours during exposure. Elevation of erythrocyte fragility was seen in rats exposed to 62 ppm (305 mg/m³) for 4 hours. Haemoglobinuria has been observed in rats exposed to 203 ppm (995 mg/m³) for 7 hours and in mice exposed to 200 ppm (980 mg/m³) for 7 hours. One male monkey and one female monkey exposed to 200 ppm (980 mg/m³) EGBE for 7 hours did not show any alteration of erythrocyte fragility or evidence of haemoglobinuria. (Carpenter et al. 1956 – quoted from ATSDR 1998).

83.1.2     Oral intake

LD₅₀-values ranging from 530 to 3000 mg/kg have been reported for rats; the LD₅₀-value was age and sex dependent, with females and older animals being more susceptible to toxicity. Signs of toxicity included congested or haemorrhagic lungs, mottled livers, congested kidneys, and haemoglobinuria (ECETOC 1995b, ATSDR 1998, NTP 2000).

LD₅₀-values of 1230 and 1519 mg/kg have been reported for mice, from 320 to 3100 mg/kg in rabbits, and from 950 to 1415 mg/kg in guinea pigs (ECETOC 1995b, ATSDR 1998, NTP 2000).

Administration of a 2000 mg/kg oral dose of EGBE to guinea pigs caused complete mortality of females and 60% mortality of males. A 20% mortality was observed following administration of 1000 mg/kg. Clinical signs and gross necropsy indicated toxicity was due to irritation of the stomach. There was no evidence of haemolytic toxicity. (Shepard 1994 – quoted from EPA 1999).

Haemolysis and haemoglobinuria are common observations in animals treated orally with EGBE for acute duration (ATSDR 1998).

In rats, haemoglobinuria was observed following a single dose (gavage) of 3000 mg/kg in male rats and 1500 mg/kg in female rats (Carpenter et al. 1956 – quoted from ATSDR 1998).

Male rats receiving a single dose of 500 mg/kg by gavage exhibited haemolysis of erythrocytes accompanied by a drastic increase in the concentration of free haemoglobin in the plasma and haemoglobinuria was observed in all rats. These effects were also observed following a single dose of 125 mg/kg, although to a lesser degree. (Ghanayem et al. 1987 – quoted from ATSDR 1998).

In male rats given 250 mg/kg by gavage, haemolysis was noted, characterised by an increase in mean cell volume and haematocrit, followed by a decline in haemoglobin concentration and RBC count. MCH and MCHC initially increased and then declined. Guinea pigs dosed once with 250 mg/kg showed no adverse haematological effects. (Ghanayem & Sullivan 1993 – quoted from ATSDR 1998).

The effect of age on toxicity of EGBE has been assessed by comparing effects in treated young male rats (4-5 weeks old) and adult male rats (9-13 weeks, 5-6 months, or 16 months) given single doses (gavage in water) of 0, 32, 63, 125, 250, or 500 mg/kg). Evaluations included haematology (red blood cell (RBC) counts and white blood cell (WBC) counts), organ weights, and histology (liver, spleen, bladder, kidney, and testes). Haematological effects were found to be dose- and age-dependent, with older rats being more sensitive than younger rats. Significant decreases in RBC counts, Hct and Hgb occurred in both adult and young rats, with the younger rats exhibiting significantly less pronounced responses. Histopathological changes in the liver, consisting of focal coagulative necrosis of hepatocytes were observed in adult rats at the two highest dose levels; no effects were seen in the liver of young rats. Phagocytised haemoglobin was found in the hepatic parenchymal cells and Kupffer cells, which is consistent with the role of these cells in haemoglobin degradation. Histopathological changes in the kidney, consisting of haemoglobin casts in the proximal tubules, were observed in mature rats from 125 mg/kg b.w./day; no effects were seen in young rats. (Ghanayem et al. 1987 – quoted from EPA 1999, ATSDR 1998).

Based upon the findings in this study, a LOAEL of 32 mg/kg for haematological effects in the aged rats has been used for establishing an acute oral ’Minimal Risk Level MRL’ of 0.4 mg/kg (ATSDR 1998).

83.1.3     Dermal contact

LD₅₀-values ranging from 406 to 1804 mg/kg have been reported for rabbits, from 1200 to 4800 mg/kg (1 week occluded exposure) for guinea pigs, of 255 or 271 mg/kg for guinea pigs on intact or abraded skin, respectively, and of 2273 mg/kg for rats (ECETOC 1995b, ATSDR 1998).

Dermal exposure of animals has also resulted in haemolysis of red blood cells and haemoglobinuria (ATSDR 1998).

Haemolytic effects were observed in 2/3 rats within 6 hours following a single application of 260 mg/kg EGBE placed on the dorsal shaved skin and covered with a glass capsule, and haemoglobinuria in 1/3 rats. A dose of 500 mg/kg increased MCV, decreased RBC count and haemoglobin level, and haemoglobinuria; no effects were observed at 200 mg/kg (Bartnik et al. 1987 – quoted from EPA 1999, ATSDR 1998).

83.1.4     In vitro studies

Ghanayem (1989 – quoted from EPA 1999, ECETOC 1995b) has studied the metabolic and cellular basis of EGBE-induced haemolysis of rat erythrocytes in vitro. EGBE is not metabolised when incubated with blood from rats and caused no haemolysis or metabolic alterations at concentrations up to 10 mM (1180 mg/l). A concentration of 20 mM (2360 mg/l) EGBE was required to produce significant haemolysis of rat blood. Incubation of rat blood with the metabolites of EGBE 2-butoxyacetaldehyde (2-BAL) and 2-butoxyacetic acid (2-BAA) caused a time- and concentration-dependent increase in cell swelling (increased Hct) followed by haemolysis. This response was more pronounced for 2-BAA, with nearly complete haemolysis observed following a 4-hour incubations at 2.0 mM (235 mg/l); BAL produced only slight haemolysis under the same conditions. The addition of aldehyde dehydrogenase and its cofactors to rat blood followed by BAL produced a potentiation of the haemolytic effects. Addition of cyanamide, an aldehyde dehydrogenase inhibitor, significantly decreased the effects either with or without added aldehyde dehydrogenase.

83.2     Irritation

83.2.1     Skin irritation

Dermal irritation from EGBE has been studied in rabbits using both the Draize protocol (24-hour occluded exposure) and the EU protocol (4-hour occluded exposure. For both protocols, 0.5 ml of undiluted EGBE was placed on the skin. EGBE was considered a severely irritant by the Draize protocol and an irritant by the EU protocol. (Zissu 1995 – quoted from ATSDR 1998).

EGBE has been reported to be slightly irritating to rabbit skin (4 hour, non occluded exposure) and moderately irritating in percutaneous toxicity studies over 24 hours (Tyler 1984 – quoted from ECETOC 1995b).

Several other studies cited in ATSDR (1998) have also reported EGBE to be a skin irritant in rabbits.

Female rabbits exposed to EGBE (³72 mg/kg) for 8 hours developed cutaneous lesions accompanied by necrosis of epidermis and dermis on the 4^th day after exposure; skin lesions healed within a 2-week period (Duprat & Gradiski 1979 – quoted from ATSDR 1998).

Severe skin irritation has been noted in guinea pigs after application of EGBE (dose not specified) (Eastman Kodak 1988 – quoted in ATSDR 1998).

83.2.2     Eye irritation

In several studies cited in ATSDR (1998), EGBE has been reported to be severely irritating when instilled (undiluted) in the eyes of rabbits; moderate to extensive conjunctivitis, moderate corneal damage, and/or slight iritis were observed.

According to ECETOC (1995b), EGBE is severely irritating to the rabbit eye.

Moderate corneal injury was observed in rabbits in which 0.5 ml of a 15% dilution of EGBE was placed in the conjunctival sac; no effects were observed with a dilution of 5% (Union Carbide 1980 – quoted from ATSDR 1998).

In a Draize test performed in rabbits, scores for different concentrations (given in parenthesis as percent) tested at 24 hours post-instillation (0.1 ml in polyethylene glycol) were 66 (100%) indicating severe irritation, 49 (70%) and 39 (30%) indicating moderate irritation, and 2 (20%) and 1 (10%) indicating mild irritation (the maximal obtainable score in the system is not stated in the reviews). In an assessment that measured corneal thickness, the highest concentrations was classified as severely irritating, the 70% concentration was moderately irritating, and the lower concentrations were mildly irritating. (Kennah et al. 1989 – quoted from EPA 1999, ATSDR 1998).

In another test for ocular irritation in rabbits (no further details given), mean erythema scores and percent corneal thickening indicated that the substance should be classified as an eye irritant (Jacobs & Marten 1989 – quoted from EPA 1999).

83.2.3     Respiratory tract irritation

Male mice exposed to 153-1666 ppm (750-8200 mg/m³) EGBE for 10-15 minutes exhibited a 20% decreased in respiratory rate at the lowest concentration and a 40% decrease at the highest concentration; the decrease occurred during the first few seconds of exposure and is an indicator of respiratory irritation (Kane et al. 1980 – quoted from EPA 1999).

83.3     Sensitisation

EGBE has been tested for dermal sensitisation in guinea pigs using the maximized Magnusson and Kligman test. The animals were given an injection of EGBE with Freund’s adjuvant behind the shoulders at the beginning of the 1^st week of the study. On the 8^th day, the animals received a 48-hour topical application of EGBE (doses not stated). On the 24^th day of the study, 0.5 ml of a 1% EGBE solution was applied for 48 hours to the left sheared flank using an occlusive patch. The intensity of the erythema and oedema was scored, and histopathological examinations were completed 24 hours after the removal of the patch. EGBE did not result in dermal sensitisation under the conditions of this study. (Zissu 1995 – quoted from ATSDR 1998).

EGBE was not sensitising in a guinea pig maximisation test (0.5% injection induction, 25% application induction, 10% application challenge) (Unilever 1989 – quoted from ECETOC 1999b).

83.4     Repeated dose toxicity

83.4.1     Inhalation

83.4.1.1     Rats

Wistar rats (23 animals/group) were exposed by inhalation to 0, 135, or 320 ppm EGBE (0, 663, or 1570 mg/m³) 7 hours/day, 5 days/week for 5 weeks. The highest dose level resulted in an increased percentage of circulating immature granulocytes, a decrease in Hgb concentration and RBC count, and an increase in RTC count. These haematological changes were, according to the authors, not severe and reversed 3 weeks after discontinuing exposures. No effect on the WBC count was observed. (Werner et al. 1943 – quoted from EPA 1999, NTP 2000).

Rats (15 animals/sex) were exposed to EGBE vapours at concentrations of 54, 107, 203, 314, or 432 ppm (265, 525, 997, 1540, or 2120 mg/m³) 7 hours/day, 5 days/week for 6 weeks. Erythrocyte osmotic fragility was observed immediately after a single 7-hour exposure to 107 ppm or higher; fragility in females exceeded that for males. In almost all animals, these high fragility values returned to normal after the overnight exposure free period. (Carpenter et al. 1956 – quoted from EPA 1999, NTP 2000).

In a 90-day study, F344 rats (16 rats/sex) were exposed to EGBE vapours for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, or 77 ppm (0, 25, 123, or 378 mg/m³). High-dose males exhibited slight (5%) but statistically significant decreases in red blood cell (RBC) counts and haemoglobin (Hgb) levels that were accompanied by increases in mean corpuscular haemoglobin (MCH). At the end of the study, these effects had either decreased or returned to the range of the control values. The NOAEC was determined to be 25 ppm (123 mg/m³). (Dodd et al. 1983 – quoted from EPA 1999; ATSDR 1998, NTP 2000).

In a 14-week NTP study (NTP 2000), F344 rats (10 animals/sex/group) were exposed via inhalation to concentrations of 0, 31, 62.5, 125, 250, or 500 ppm of EGBE (0, 152, 307, 614, 1228, or 2455 mg/m³) 6 hours/day, 5 days/week.

One female rat in the 250 ppm group was killed moribund during week 8; four females in the 500 ppm group were killed moribund during week 1 and one during week 5. Final mean body weights of females exposed to 500 ppm were significantly less than those of the controls. Clinical findings included abnormal breathing, pallor, red urine stains, nasal and eye discharge, lethargy, and increased salivation and/or lachrymation.

The haematological evaluation showed an exposure concentration-related anaemia, evidenced by decreases in Hct, Hgb, and RBC counts, occurred from 125 ppm in males and in all exposed groups of females. Females appeared to be more slightly sensitive than males as evidenced by approximately 25-35% decreases in Hct and Hgb in females compared to approximately 20-25% decreases in males, and a 44% decreased in RBC counts for 500 ppm females compared to a 34% decreased for 500 ppm males. The anaemia was characterised as microcytic, normochromic, and responsive.

Liver and kidney weights were significantly increased in females from 125 ppm and in males from 250 ppm and at 500 ppm, respectively; thymus weights of 500 ppm females were significantly decreased. The histopathological lesions noted in rats at terminal sacrifice were similar between males and females and were consistent with haemolytic anaemia and haemoglobinuria and included haematopoietic cell proliferation of the spleen, primarily erythroid (in females from 62.5 ppm; in males from 250 ppm); bone marrow hyperplasia (in females from 62.5 ppm; in males from 250 ppm); pigmentation (haemosiderin) of the hepatic Kupffer cells (in females from 62.5 ppm; in males from 125 ppm); and deposition of pigment (haemosiderin) in the renal cortical tubules (in females from 125 ppm; in males from 250 ppm). In addition, minimal forestomach inflammation and epithelial hyperplasia were noted in male rats from 250 ppm and epithelial hyperplasia in one female at 250 and at 500 ppm. The 31 ppm exposure level (152 mg/m³) was a LOAEC for haematological changes in female rats and 62.5 ppm (307 mg/m³) a NOAEC in male rats.

NTP (2000) has also performed a 2-year inhalation study in F344 rats (50 animals/sex/group) exposed to concentrations of 0, 31, 62.5, or 125 ppm of EGBE (0, 152, 307, or 614 mg/m³) 6 hours/day, 5 days/week. For haematology and bone marrow analysis, additional groups of rats (27/sex/group) were exposed similarly to 0, 62.5 or 125 ppm of EGBE (0, 307, or 614 mg/m³) for evaluation at 3, 6, and 12 months and nine male and nine female rats were exposed to 31 ppm (152 mg/m³) for evaluation at 3 (haematology only) and 6 months.

No effect on survival was observed. Mean body weight of 125 ppm female rats were generally less than those of controls. Non-neoplastic effects included hyaline degeneration of the olfactory epithelium (males: 13/48, 21/49, 23/49, 40/50; females: 13/50, 18/48, 28/50, 40/49); Kupffer cell pigmentation in the livers (males: 23/50, 30/50, 34/50, 42/50; females: 15/50, 19/50, 36/50, 47/50), and splenic fibrosis in males (11/50, 14/50, 19/50, 20/50). The severity of the nasal lesion was not affected by exposure and is considered to be the most common age-related change in the nasal passages of rats; overall, the lesion was considered to be incidental and likely not related to EGBE exposure. The Kupffer cell pigmentation resulted from haemosiderin accumulation, a recognised secondary effect of the haemolytic activity of EGBE. Neoplastic findings are described in 4.7.

The most consistent exposure-related effect on the haematopoietic system was an exposure concentration-related mild macrocytic, normochromic, and responsive anaemia, evidenced by decreases in Hct, Hgb, and RBC counts, with females being more affected than males. Significant decreases in Hct values, Hgb concentration and RBC counts occurred at 3 and 6 months in females from 31 ppm and in males at 125 ppm, and at 12 months in males and females from 62.5 ppm. Evidence of macrocytosis was demonstrated by increases in MCV (at 3 months from 31 ppm in both males and females) accompanied by elevation in the MCHV. Increases were also observed in reticulocyte and nucleated erythrocyte counts (both sexes), which is consistetnt with an erythropoietic response to the anaemia. Increases in bone marrow cellularity occurred in 125 ppm females at all time points.

The LOAEC for haematological changes was 31 ppm (152 mg/m³).

83.4.1.2     Mice

Mice (10 males per group) were exposed to EGBE vapours at concentrations of 100, 200, or 400 ppm (490, 980, or 1965 mg/m³) 7 hours/day for 30, 60, or 90 days. An increase in erythrocyte osmotic fragility occurred at all concentrations and was consistent throughout the exposures. In all animals, erythrocyte osmotic fragility was normal after a 17-hour rest period. (Carpenter et al. 1956 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

In a 14-week NTP study (NTP 2000), B6C3F1 mice (10 animals/sex/group) were exposed via inhalation to concentrations of 0, 31, 62.5, 125, 250, or 500 ppm of EGBE (0, 152, 307, 614, 1228, or 2455 mg/m³) 6 hours/day, 5 days/week.

An exposure concentration-related anaemia, evidenced by decreases in Htc, Hgb, and RBC counts, occurred in males from 125 ppm and in all exposed groups of females; the anaemia was slightly more pronounced in females than in males. The anaemia was characterised as normocytic, normochromic, and responsive; nocrmocytic and normochromic erythrocytes were demonstrated by the lack of change in MCV and MCHC, and responsiveness by increased reticulocyte counts. Platelet counts were increased from 250 ppm in females and at 500 ppm in males.

Histopathological effects observed in mice surviving to the end of the study consisted of exposure-related increases in the incidences of haematopoietic cell proliferation and haemosiderin pigmentation of the spleen (in females from 250 ppm; in males from 125 ppm); haemosiderin pigmentation in hepatic Kupffer cells (in females from 250 ppm; in males at 500 ppm); and renal tubular haemosiderin pigmentation (in males and females at 250 ppm); and epithelial hyperplasia and inflammation (in females from 125 ppm; in males at 500 ppm).

The NOAEC for haematological changes was 62.5 ppm (307 mg/m³) in males and the LOAEC was 31 ppm (152 mg/m³).

NTP (2000) has also performed a 2-year inhalation study in B6C3F1 mice (50 animals/sex/group) exposed to concentrations of 0, 62.5, 125, or 250 ppm of EGBE (0, 307, 614, or 1228 mg/m³) 6 hours/day, 5 days/week. For haematology and bone marrow analyses, additional groups of mice (30/sex/group) were exposed similarly and evaluated at 3, 6, and 12 months.

Survival was significantly decreased in male mice at 125 and 250 ppm (54 and 53%, respectively). Mean body weights were generally less in exposed animals than for controls, with females experiencing greater and earlier reductions. Non-neoplastic effects included forestomach ulcers and epithelial hyperplasia (in males at 125 ppm; in females in all exposed groups); haematopoietic cell proliferation in the spleen (in males from 125 ppm; in females at 250 ppm); haemosiderin pigmentation in the spleen (in males in all exposed groups; in females from 125 ppm); Kupffer cell pigmentation (haemosiderin) in the livers (in males from 125 ppm; in females in all exposed groups); hyaline degeneration of the olfactory epithelium (in females in all exposed groups); and bone marrow hyperplasia (in males from 125 ppm). Neoplastic findings are described in 4.7.

An exposure-related anaemia, evidenced by decreases in Htc, Hgb, and RBC counts, occurred in males from 125 ppm and in all exposed groups of females; the anaemia was slightly more pronounced in females than in males. The anaemia was characterised as minimal normocytic, normochromic, and regenerative. The LOAEC for haematological changes was 62.5 ppm (307 mg/m³).

83.4.1.3     Dogs

Dogs (2 animals/group) were exposed by inhalation to 0, or 415 ppm EGBE (0 or 2040 mg/m³) 7 hours/day, 5 days/week for 12 weeks. Decreased Hgb concentration and RBC count, and increased hypochromia, polychromatophilia, and microcytosis were observed. These haematological changes were, according to the authors, not severe and reversed 5 weeks after the end of exposure. (Werner et al. 1943 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

83.4.1.4     Monkeys

One rhesus monkey exposed to 210 ppm (1030 mg/m³) EGBE for up to 30 days exhibited elevated osmotic fragility after the fourth exposure and reduced RBC count and haemoglobin values after 30 exposures. At the end of the study, RBC counts and haemoglobin concentration were reduced to levels half that at the start of the study. A male and a female monkey exposed to 100 ppm (490 mg/m³) for 90 days had increased erythrocyte fragility and decreased numbers of erythrocytes; the female was more severely affected. (Carpenter et al. 1956 – quoted from NTP 2000, ATSDR 1998).

83.4.2     Oral intake

83.4.2.1     Rats

Rats (24 male F344 rats per group) were gavaged with EGBE in water at doses of 0, 500, or 1000 mg/kg for 4 days. Six rats per dose were examined at 1, 4, 8, and 22 days after the last dose. Haematology evaluations showed marked dose-related effects on circulating RBCs and WBCs. Most of the RBC changes subsequently returned to normal. Body weight gain was reduced throughout the post-treatment period at the highest dose level. Changes in relative organ weights (increased liver and spleen weights at both dose levels; increased kidney and reduced thymes weights at the highest dose level) were evident on post-treatment day 1. These changes returned to normal by post-treatment day 22 except for liver and spleen weights at the highest dose level, which remained increased about 5 and 20%, respectively. (Grant et al. 1985 – quoted from EPA 1999, NTP 2000).

When rats were administered EGBE via gavage for a 12-day period at dose levels of 0 or 125 mg/kg, significant haemolysis occurred, which became more pronounced up to the third day of dosing; gradual recovery was observed up to day 12. Relative spleen weights increased up to the sixth day of dosing and declined thereafter. (Ghanayem et al. 1992 – quoted from EPA 1999).

Ten adult male rats were given 222, 443, or 885 mg/kg b.w./day undiluted EGBE by gavage 5 days/week for 6 weeks. Haematology parameters were determined following the last treatment. Dose-related changes were observed in the RBC counts (decreased RBC counts and Hgb concentration, and increased MCH and haemoglobinuria) of all treatment groups. At the two higher dose-levels, decreased MCHC was observed as well. Increased relative liver weight was observed at all dose levels; histological examination revealed haemosiderin deposition from 443 mg/kg b.w./day and hepatocytomegaly at 885 mg/kg b.w./day. Focal haemosiderin accumulation was observed in the proximal tubules at the two higher dose levels. Splenic congestion was seen at all doses and enlarged dark spleens were observed in 3/9 mid-dose rats and in 4/8 high-rats. The LOAEL was determined to be 222 mg/kg b.w./day, the lowest dose tested. (Krasavage 1986 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

In a 13-week NTP study (NTP 1993 – quoted from EPA 1999, NTP 2000), F344 rats (10 animals/sex/group) received EGBE in the drinking water at doses of 0, 69/82, 129/151, 281/304, 367/363, or 452/470 mg/kg b.w./day in male/female rats, respectively. Male rats evaluated at 13 weeks showed significantly reduced RBC counts from 281 mg/kg b.w./day, and reduced Hgb concentration and reduced platelets from 367 mg/kg b.w./day. Female rats showed significantly reduced RBC counts and Hgb concentration from 82 mg/kg b.w./day, and increased RTCs and reduced platelets from 304 mg/kg b.w./day. Histopathological examination of the livers revealed hepatocellular alterations at all dose levels in both sexes; centrilobular hepatocellular degeneration from 281 and 304 mg/kg b.w./day in males and females, respectively; and brown to green granular pigment staining strongly positive for iron in Kupffer cell cytoplasm in males at 452 mg/kg b.w./day and in females from 151 mg/kg b.w./day. These lesions, particularly in the females, displayed both increased dose-related incidence and increased dose-related severity. Bone marrow hyperplasia was found from 281 and 363 mg/kg b.w./day in males and females, respectively; haematopoietic cell proliferation and congestion in the spleen from 367 and 363 mg/kg b.w./day in males and females, respectively; and increased haemosiderin pigmentation in the spleen was found from 129 and 151 mg/kg b.w./day in males and females, respectively. No histopathological changes were observed in the kidneys, urinary bladders, thymus, lymph nodes, testes, or epididymides. The lowest dose level tested (69/82 mg/kg b.w./day in males/females, respectively) was considered a LOAEL.

In a study designed to investigate effects in the immune system, rats (6 animals/group) were exposed to EGBE in the drinking water at doses of 0, 180/204, or 506/444 mg/kg b.w./day in males and females, respectively, for 21 days. All rats were injected subcutaneously with heat aggregated aqueous keyhole limpet haemocyanin (KLH) antigen on days 7 and 13 following the start of dosing. No dose-related changes in organ weights (spleen, thymus, liver, kidney, and testis) or histology (thymus, liver, kidney, and testis) were observed. Natural killer cell cytotoxic response was significantly enhanced in the low-dose group, but not in the high-dose group (a decrease in this parameter is an indication of compromised non-specific immune system integrity; an increased is not considered an adverse effect). No significant alterations were noted in other immune parameters (serum anti-KLH IgG antibody levels, delayed-type hypersensitivity reaction, interleukin 2 and interferon production, and spleen cell counts). (Exon et al. 1991 – quoted from EPA 1999, ATSDR 1999).

83.4.2.2     Mice

EGBE was administered by gavage to male mice at doses of 0, 357, 714, or 1430 mg/kg b.w./day 5 days/week for 5 weeks. All of the high-dose animals died before study termination; no mortality was observed in the lower dose groups. Haematology was evaluated at the end of the study. Mean RBC counts were significantly lower than the control value in the 357 and 714 mg/kg b.w./day groups; WBC counts were not affected. No differences in testes weight or histology was found. The LOAEL was 357 mg/kg b.w./day, the lowest dose tested. (Nagano et al. 1979 – quoted from EPA 1999).

In a 13-week NTP study (NTP 1993 – quoted from EPA 1999, NTP 2000), B6C3F1 mice (10 animals/sex/group) received EGBE in the drinking water at doses of 0, 118/185, 223/370, 553/676, 676/861, or 694/1306 mg/kg b.w./day in male/female mice, respectively. Mean final body weight and body weight gain were slightly reduced at the three highest dose levels. Haematological evaluations were not performed. No histopathological effects were observed in the liver, kidneys, urinary bladders, spleen, thymus, lymph nodes, bone marrow, testes, or epididymides.

83.4.3     Dermal contact

Repeated application of EGBE either neat or as a dilute (aqueous) and occluded to rabbits at dose levels of 18, 90, 180, or 360 mg/kg 6 hours/day, nine consecutive applications, produced haemoglobinuria in high-dose males and in females at the two highest dose levels. Recovery was noted following a 14-day observation period. (Tyler 1984 – quoted from EPA 1999).

In a 13-week study, occluded dermal administration of EGBE to rabbits (6 hours/day, 5 days/week) at exposure levels of 10, 50, or 150 mg/kg produced no observable haematological effects (Tyler 1984 – quoted from EPA 1999, ATSDR 1998).

In guinea pigs, dermal administration of EGBE at 2000 mg/kg produced no clinical signs of toxicity or treatment-related signs of organ toxicity (Shepard 1994 – quoted from EPA 1999).

83.5     Toxicity to reproduction

Due to the known testicular and developmental toxicity of two lower ethylene glycol ethers (EGME, EGEE), the reproductive and developmental toxicity of EGBE has been studied in a variety of well-conducted oral, inhalation and dermal studies using rats, mice and rabbits.

EGME seems to be the most potent glycol ether inducing testicular toxicity, but there is no evidence however, that higher homologues, as EGBE, are testicular toxicants. The developmental effects of ethylene glycol ethers are mediated by alkoxy acetic acid metabolites. However, ethylene glycol ethers with alkyl chains of more than 2 carbon atoms, as EGBE, do not appear to be selectively toxic to the foetus. For EGBE, foetotoxic effects have been observed only in the presence of maternal toxicity. In an in vitro study, the alkoxy acetic acids methoxyacetic acid (MAA) (from ethylene glycol monomethyl ether (EGME)) and ethoxyacetic acid (EEA) (from ethylene glycol monomethyl ether (EGEE)) showed similar developmental effects, whereas 2-BAA (from EGBE) was only slightly active. (ECETOC 1995a).

83.5.1     Inhalation

83.5.1.1     Rats

In a 90-day study, F344 rats (16 rats/sex) were exposed to EGBE vapours for 6 hours/day, 5 days/week at concentrations of 0, 5, 25, or 77 ppm (0, 25, 123, or 378 mg/m³). No changes in testicular weight or in the pathology of the epididymides and testes of male rats were observed at any exposure level; reproductive organs of female rats were not examined histologically. Other findings are described in 4.4.1.1. (Dodd et al. 1983 – quoted from EPA 1999; ATSDR 1998).

In a 14-week NTP study (NTP 2000), no effects were noted in reproductive organs of F344 rats (10 animals/sex/group) exposed via inhalation to concentrations of 0, 31, 62.5, 125, 250, or 500 ppm of EGBE (0, 152, 307, 614, 1228, or 2455 mg/m³) 6 hours/day, 5 days/week. Other findings are described in 4.4.1.1.

NTP (2000) has also performed a 2-year inhalation study in F344 rats (50 animals/sex/group) exposed to concentrations of 0, 31, 62.5, or 125 ppm of EGBE (0, 152, 307, or 614 mg/m³) 6 hours/day, 5 days/week. No effects were noted in the reproductive organs. Other findings are described in 4.4.1.1.

Rats (15/group) were exposed to 0, 150, or 200 ppm (0, 737, or 980 mg/m³) EGBE via inhalation for 6 hours/day during gestation days 7-15. High-dose rats showed some evidence of haematuria on the first day of exposure, but no effects were noted thereafter. No adverse effects were seen in the offspring. (Nelson et al. 1984 – quoted from EPA 1999, ECETOC 1995b, NTP 2000).

Pregnant F344 rats (36/group) were exposed to 0, 25, 50, 100, or 200 ppm (0, 123, 246, 491, or 982 mg/m³) EGBE via inhalation for 6 hours/day on gestational days 6-15. A 50% decrease in viable implants and in live foetuses per litter, and an eight-fold increase in non-viable implants were observed at 200 ppm, but not at the lower concentrations. Foetotoxicity was observed in the form of retarded skeletal ossification of vertebral arches or centra, sternebrae, or phalanges at 100 and 200 ppm. There were no significant increases in external, visceral, skeletal, or total malformations in the foetuses due to treatment. Maternal toxicity was also evident at 100 and 200 ppm as an increased incidence of haematuria, reduced RBC count, decreased weight gain, and reduced food consumption. The NOAEL for maternal and developmental toxicity was 50 ppm (246 mg/m³). (Tyl et al. 1984 – quoted from EPA 1999, ATSDR 1998, ECETOC 1995b, NTP 2000).

83.5.1.2     Mice

In a 14-week NTP study (NTP 2000), no effects were noted in reproductive organs of B6C3F1 mice (10 animals/sex/group) exposed via inhalation to concentrations of 0, 31, 62.5, 125, 250, or 500 ppm of EGBE (0, 152, 307, 614, 1228, or 2455 mg/m³) 6 hours/day, 5 days/week except for testicular degeneration in 2/4 mice from the 500 ppm group that died or were killed moribund. Other findings are described in 4.4.1.2.

NTP (2000) has also performed a 2-year inhalation study in B6C3F1 mice (50 animals/sex/group) exposed to concentrations of 0, 62.5, 125, or 250 ppm of EGBE (0, 307, 614, or 1228 mg/m³) 6 hours/day, 5 days/week. No effects were noted in the reproductive organs. Other findings are described in 4.4.1.2.

83.5.1.3     Rabbits

Pregnant rabbits (24/group) were exposed to 0, 25, 50, 100, or 200 ppm (0, 123, 246, 491, or 982 mg/m³) EGBE via inhalation for 6 hours/day on gestational days 6-18. Decreases in total implants and implant viability were observed at 200 ppm. Foetal skeletal ossification of sternebrae and rudimentary rib was delayed at 200 ppm. Maternal toxicity was also evident at 200 ppm as an increased incidence of clinical signs, reduced gravid uterine weight, and decreased weight gain; no effect on haematological parameters was observed. The NOAEL for maternal and developmental effects was 100 ppm (491 mg/m³). (Tyl et al. 1984 – quoted from EPA 1999, ATSDR 1998, ECETOC 1995b, NTP 2000).

83.5.2     Oral intake

83.5.2.1     Rats

Male rats were given single gavage doses of 0, 174, 434, or 868 mg/kg b.w. of 2-butoxyacetic acid (BAA, a metabolite of EGBE). Occasional significant decreases were observed in the weight of the prostate and seminal vessels, but the decreases were not time or dose related. No treatment-related lesions were noted following histological examination of the testes, epididymides, and prostate. (Foster et al. 1987 – quoted from EPA 1999, NTP 2000).

In male F344 rats (24/group) gavaged with EGBE in water at doses of 0, 500, or 1000 mg/kg for 4 days, no testicular effects were observed. Other findings are described in 4.4.2.1. (Grant et al. 1985 – quoted from EPA 1999, ATSDR 1998).

When male and female F344 rats were exposed to EGBE in the drinking water for 2 weeks at doses of 0, 73/77, 108/102, 174/152, 242/203, or 346/265 mg/kg b.w./day in males and females, respectively, there were no effects on testicular weight and no histopathological lesions in the testes or epididymides (NTP 1993 – quoted from EPA 1999, ATSDR 1998).

In male rats (6 animals/group) exposed to EGBE in the drinking water at doses of 0, 180, or 506 mg/kg b.w./day for 21 days, no effects were observed regarding testis weight, testicular histopathology, and fertile parameters. Other findings are described in 4.4.2.1. (Exon et al. 1991 – quoted from EPA 1999, ATSDR 1998).

Ten adult male rats were given 222, 443, or 885 mg/kg b.w./day undiluted EGBE by gavage 5 days/week for 6 weeks. No effects were found on testicular weight and in histopathological lesions in the testes, seminal vesicles, epididymides, or prostate at any exposure level. Other findings are described in 4.4.2.1. (Krasavage 1986 – quoted from EPA 1999, ATSDR 1998).

In a 13-week NTP study (NTP 1993 – quoted from EPA 1999, ATSDR 1998), no effects on testicular weight and no histopathological lesions in the testes, epididymides, or seminal vesicles were found in F344 rats (10 animals/group) administered EGBE in the drinking water at doses of up to 452 mg/kg b.w./day. Spermatozoa concentrations were significantly decreased compared to controls from 281 mg/kg b.w./day but not in a dose-related manner; there were no significant effects on spermatid heads, spermatid counts, or percent mobile spermatozoa. No histopathological lesions were found in the mammary glands, ovaries, or uterus at doses of up to 470 mg/kg b.w./day. Other findings are described in 4.4.2.1.

Pregnant F344 rats (28-35 per group) were administered EGBE by gavage in distilled water at doses of 0, 30, 100, or 200 mg/kg b.w./day on gestation days 9-11, or doses of 0, 30, 100, or 300 mg/kg b.w./day on gestation days 11-13. Maternal effects in either dosing sequence included marked reductions in body weight and/or weight gain; increases in kidney and spleen weights; severe haematotoxicity as evidenced by a decrease in HCT, Hgb, and RBC counts and an increase in RCTs at doses greater than or equal to 100 mg/kg b.w./day. These effects were dose related. No indications of developmental toxicity were observed at the two lower dose levels. Viability of embryos was reduced at 200 but not 300 mg/kg b.w./day. A decreased platelet count was noted in the foetuses at 300 mg/kg b.w./day. No foetal malformations, including cardiovascular malformations, were noted at any dose. The NOAEL for maternal toxicity was 30 mg/kg b.w./day and for developmental toxicity 100 mg/kg b.w./day. (Sleet et al. 1989 – quoted from EPA 1999, ATSDR 1998).

83.5.2.2     Mice

When male and female B6C3F1 mice were exposed to EGBE in the drinking water for 2 weeks at doses of 0, 93/150, 148/237, 210/406, 370/673, or 627/1364 mg/kg b.w./day in males and females, respectively, there were no treatment-related gross lesions in any of the reproductive organs, histopathological examinations were not performed (NTP 1993 – quoted from EPA 1999).

When EGBE was administered by gavage to male mice at doses of 0, 357, 714, or 1430 mg/kg b.w./day 5 days/week for 5 weeks, no changes in testes weight or histology were found. Other findings are described in 4.4.2.2. (Nagano et al. 1979 – quoted from EPA 1999, ATSDR 1998).

In a 13-week NTP study (NTP 1993 – quoted from EPA 1999, ATSDR 1998), no effect was observed in testis weight, no histopathological changes were observed in the seminal vesicle, testes, and epididymides, and no consistent treatment-related effects on sperm parameters in B6C3F1 mice (10 animals/group) administered EGBE in the drinking water at doses of up to 694 mg/kg b.w./day. No histopathological lesions were found in the mammary glands, ovaries, or uterus at doses of up to 1306 mg/kg b.w./day. Other findings are described in 4.4.2.2.

A continuous breeding study has been performed in Swiss CD-1 mice exposed to EGBE in drinking water at doses of 0, 700, 1300, or 2000 mg/kg b.w./day for 7 days pre-mating and 98 days as breeding pairs (Heindel et al. 1990 – quoted from EPA 1999, ATSDR 1998, ECETOC 1995b, NTP 2000). In the high-dose and mid-dose groups, 13/30 and 6/20 females, respectively, died during the study. Toxic effects in adults at these dose levels included decreased body weight gain, increased kidney and liver weights, and dose-related decreases in water consumption. No significant effects on the weights of testes, seminal vesicles, or prostate, and no effects on sperm parameters were observed. Decreased pup weight and a decrease in the number of litters produced per pair and in the size of each litter were observed in the mid- and high-dose groups. A significant reduction (5%) of live pup weight was also observed in the low-dose group, but no adverse effect on fertility. At the completion of the continuous breeding phase, a crossover mating trial was performed to determine which sex was more affected by treatment. F₀ males and females from the 1300 mg/kg b.w./day group were mated with male and female control animals. The exposed mice had significantly lower body weights and increased relative kidney weights, but reproductive organ weights, sperm motility and morphology, and oestrous cycle length and frequency did not differ from controls. The proportion of successful copulation was the same in all groups, and no developmental effects were observed in the offspring of any group. However, the number of fertile females was significantly reduced in the groups where treated females were mated with control males. No adverse effect on fertility was noted when offspring of the low-dose groups were mated; there were insufficient numbers of offspring to assess the two highest dose groups. The NOAEL for maternal and reproductive effects is 700 mg/kg b.w./day, which is a LOAEL for developmental effects as a slight decrease in pup weight was observed at this dose.

CD-1 mice received 0, 350, 650, 1000, 1500, or 2000 mg/kg b.w./day EGBE via gavage on days 8-14 of gestation. Maternal toxicity included mortality of 3/6 animals in the 1000 mg/kg b.w./group and 6/6 in the 2000 mg/kg b.w./day group. Treatment-related clinical observations were lethargy, abnormal breathing, and green or red vaginal discharge. An increased number of resorptions and a reduced number of viable foetuses were observed from 1000 mg/kg b.w./day. The LOAEL for maternal effects was 350 mg/kg b.w./day and the NOAEL for prenatal effects was 650 mg/kg b.w./day. In a post-natal study, reproductive effects were evaluated in CD-1 mice administered EGBE via gavage at 0, 650, or 1000 mg/kg b.w./day on days 8-14 of gestation. Maternal body weight was lowered at the high dose. No adverse reproductive or developmental effects were observed. (Wier et al. 1987 – quoted from EPA 1999, ATSDR 1998, ECETOC 1995b, NTP 2000).

83.5.3     Dermal contact

Females rats were given EGBE by dermal application to the shaved interscapular skin during days 6-15 of gestation, four time per day at 1800 or 5400 mg/kg b.w./day. In the high-dose group, 10/11 rats died between days 3 and 7 of treatment. Signs associated with treatment included red-stained urine, ataxia, inactivity, rough coats, and necrosis of the tail tip. At the lower dose, body weight was slightly reduced; there was no evidence of embryo- or foetotoxicity, nor were any gross malformations or variations observed. (Hardin et al. 1984 – quoted from EPA 1999, NTP 2000).

83.6     Mutagenic and genotoxic effects

83.6.1     In vitro studies

NTP has reported negative responses for mutagenicity when EGBE was tested in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 at concentrations up to 10 mg/plate with or without metabolic activation (Zeiger et al. 1992 – quoted from EPA 1999, ECETOC 1995b, NTP 2000).

EGBE was negative for mutation in Salmonella typhimurium strains TA98, TA100, and TA102 both with and without rat S9 activation at concentration up to 9 mg/plate; a weak mutagenic response was seen in strain TA97a at 4.5 mg/plate with (S9 mix) and without metabolic activation (Hoflack et al. 1995 – quoted from EPA 1999, ATSDR 1998, ECETOC 1995b, NTP 2000).

In order to confirm this result, testing was conducted in Salmonella typhimurium strains TA97a and TA100, as well as Escherichia coli WP2uvrA. EGBE was negative in these strains when evaluated at 0.5, 1.0, 2.5, 5.0, 8.5, and 10 mg/plate in the presence and absence of Arochlor-induced rat liver S9 mix. (Gollapudi et al. 1996 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

No mutagenic activity was observed when EGBE was investigated using bacteriophage T4D with Escherichia coli B, CR63, and K12 when tested at concentrations from 20 to 111 µl/ml, but EGBE had a severe toxic effect on phage yield (Kvelland 1988 – quoted from EPA 1999, ECETOC 1995b).

When tested for 5 hours at up to toxic doses, EGBE or its metabolite 2-butoxyacetaldehyde (BAL) were not found to be mutagenic in an in vitro gene mutation assay using Chinese hamster ovary (CHO) cells. High toxicity was reported at 4.5 mg/ml. (Chiewchanwit & Au 1995 – quoted from EPA 1999, ATSDR 1998, NTP 2000).

Both EGBE and BAL weakly induced gene mutations in Chinese hamster V79 cells at high concentrations (from 7.5 mg/l) (Elias et al. 1996 – quoted from EPA 1999).

EGBE did not induce sister chromatid exchanges (SCEs) or chromosomal aberrations in CHO cells with or without liver S9 mix; EGBE induced cell cycle delay (an indication of cytotoxicity) (NTP 1993 – quoted from EPA 1999, NTP 2000).

EGBE was reported not to induce SCEs in Chinese hamster ovary cells exposed to concentrations up to 0.25% (2.5 mg/ml) for 2 hours in the presence of metabolising enzymes (S9) and for 5 hours in the absence of S9 (Tyler 1982 – quoted from ATSDR 1998).

EGBE did not induce chromosomal aberrations in Chinese hamster V79 fibroblast cells. At high concentrations (from 8.5 mM, ca. 1 mg/ml), EGBE weakly induced aneuploidy (numerical chromosomal anomalies), SCEs and micronuclei, and potentiated the clastogenicity induced by methyl methanesulfonate. (Elias et al. 1996 – quoted from EPA 1999).

EGBE did not induce chromosomal aberrations in human lymphocytes when tested at concentrations up to 3000 ppm, but induced sister chromatid exchanges (SCEs) (Villalobos-Pietrini et al. 1989 – quoted from ECETOC 1995b).

83.6.2     In vivo studies

EGBE did not increase the incidence of micronuclei in the bone marrow cells of male rats or mice. Animals were given three intraperitoneal injections of EGBE 24 hours apart and sacrificed 24 hours after the last injection. Rats were dosed at 0, 7, 14, 28, 56, 112.5, 225, or 450 mg/kg, and mice were dosed at 0, 17, 34, 69, 137.5, 275, or 550 mg/kg. (NTP 1998 – quoted from EPA 1999, NTP 2000).

No increase in DNA adducts was detected by ³²P post-labelling in the brain, liver, kidney, testis, or spleen of rats following oral administration of 120 mg/kg EGBE (NTP 2000).

83.7     Carcinogenic effects

In a 2-year inhalation study (NTP 2000), F344 rats (50 animals/sex/group) were exposed to concentrations of 0, 31, 62.5, or 125 ppm of EGBE (0, 152, 307, or 614 mg/m³) 6 hours/day, 5 days/week. No effect on survival was observed. Benign and/or malignant pheochromocytoma of the adrenal medulla was observed in female rats (3/50, 4/50, 1/49, 8/49); the increase in high-dose animals relative to controls was not significant. Based upon these findings, NTP concluded “no evidence of carcinogenic activity in male F344 rats, and equivocal evidence of carcinogenic activity in female F344 rats”. Non-neoplastic findings are described in 4.4.1.1.

In a 2-year inhalation study (NTP 2000), B6C3F1 mice (50 animals/sex/group) were exposed to concentrations of 0, 62.5, 125, or 250 ppm of EGBE (0, 307, 614, or 1228 mg/m³) 6 hours/day, 5 days/week. Survival was significantly decreased in male mice at 125 and 250 ppm (54 and 53%, respectively). Significantly increased incidences of hepatocellular carcinomas (10/50, 11/50, 16/50, 21/50) and haemangiosarcomas (0/50, 1/50, 2/49, 4/49) were observed in high-dose male mice relative to controls. When hepatocellular adenomas and carcinomas were combined, no significant increase was observed in any exposure group. No significant increase in benign or malignant hepatocellular tumours or haemangiosarcomas was noted in female mice. Forestomach squamous cell papillomas and carcinomas (combined) were significantly increased (Trend Test = 0.017) in female mice relative to controls (0/50, 1/50, 2/50, 6/50) but not in male mice. Based upon these findings, NTP concluded “some evidence of carcinogenic activity in male and female B6C3F1 mice”. Non-neoplastic findings are described in 4.4.1.2.

84     Regulations

84.1     Ambient air

Denmark (C-value):        0.04 mg/m³ (MST 2002)

84.2     Drinking water

-

84.3     Soil

-

84.4     Occupational Exposure Limits

Denmark:                       20 ppm (98 mg/m³) H (At 2002)

ACGIH:                         20 ppm (98 mg/m³) skin (ACGIH 2001)

Germany:                        20 ppm (98 mg/m³ H (MAK 1998)

84.5     EU Classification

EGBE is classified for acute toxic effects (Xn;R20/21/22 – harmful by inhalation, in contact with skin and if swallowed) and for irritative effects (Xi;R36/38 – irritating to eyes and skin) (MM 2002).

84.6     IARC

-

84.7     US-EPA

Because of the uncertain relevance to humans of the tumour increases observed in the NTP inhalation studies, that EGBE is generally negative in genotoxic tests, and the lack of human data to support the findings in rodents, the human carcinogenic potential of EGBE, in accordance with the recent ’Proposed Guidelines for Carcinogen Risk Assessment’, cannot be determined at this time, but suggestive evidence exists from rodent studies. Under existing EPA guidelines, EGBE is judged to be a possible human carcinogen, Group C. (EPA 1999). 

85     Summary

85.1     Description

EGBE is a colourless liquid with a low vapour pressure (0.76 mmHg at 20°C); it is miscible with water.

85.2     Toxicokinetics

EGBE is rapidly absorbed and distributed throughout the body (humans and rats) following inhalation, oral administration and dermal contact. Following inhalation, peak blood levels were observed in humans within 1-2 hours following exposure; one study in human volunteers indicates an uptake of about 60% of the inspired amount during light physical exercise.

In humans, EGBE is eliminated primarily as 2-butoxyacetic acid (2-BAA) in the urine either free or conjugated to glutamine and, to a lesser extent, glycine. In rats, EGBE can also be converted to ethylene glycol or conjugated to glucuronide and sulphate to some extent. Excretion also takes place via expired air in form of carbon dioxide.

85.3     Human toxicity

85.3.1     Single dose toxicity, irritation

In human volunteers exposed by inhalation to EGBE at concentrations from 490-957 mg/m³ for 4-8 hours, irritation of the nose and throat, and eyes was noted; no haematological effects were observed. When seven male volunteers were exposed to EGBE (98 mg/m³) for 2 hours, none of the subjects complained of or showed any signs of adverse effects.

Poisoning cases are available reporting haematological changes and metabolic acidosis following acute ingestion of large doses of EGBE in household products and combined with other solvents.

85.3.2     Irritation, sensitisation

In a patch test, human volunteers showed no dermal effects following application of 10% aqueous EGBE.

85.3.3     Repeated dose toxicity

No differences between exposed and control workers (average airborne concentration of EGBE: 2.9 mg/m³) were observed for haematological parameters except for small changes for haematocrit (3.3% decrease) and mean corpuscular haemoglobin concentration (MCHC) (2.1% increase).

85.3.4     Toxicity to reproduction

No data have been located.

85.3.5     Mutagenic and genotoxic effects

No increases in micronuclei or sister chromatid exchanges were observed in workers exposed to both EGBE and to 2-ethoxyethanol (EGEE).

85.3.6     Carcinogenic effects

No data have been located.

85.4     Animal toxicity

85.4.1     Single dose toxicity

LC₅₀-values (4-hour exposure) of 2200-2400 mg/m³ have been reported for rats and of 3440 mg/m³ (7-hour exposure) for mice. One of six guinea pigs died after 4 hours of exposure to saturated vapour (ca. 4900 mg/m³).

Oral LD₅₀-values of 530-3000 mg/kg have been reported for rats, of 1230 and 1519 mg/kg for mice, of 950-1415 for guinea pigs, and of 320-3100 for rabbits.

Dermal LD₅₀-values of 406 to 1804 mg/kg have been reported for rabbits, of 255-4800 mg/kg for guinea pigs, and of 2273 mg/kg for rats.

Following inhalation, increased erythrocyte fragility was seen in rats exposed to 305 mg/m³ for 4 hours and haemoglobinuria was observed in rats and mice exposed to about 1000 mg/m³ for 7 hours; no such effects were observed in monkeys exposed to about 1000 mg/m³ for 7 hours.

Following oral administration to rats, haemoglobinuria was observed at 3000 and 1500 mg/kg in male and female rats, respectively. Haemolysis of erythrocytes accompanied by haemoglobinuria have been observed following a single dose of 500 mg/kg, and at 125 mg/kg although to a lesser degree. Guinea pigs dosed once with 250 mg/kg showed no adverse haematological effects. The haematological effects in rats were found to be dose- and age-dependent, with older rats being more sensitive than younger rats.

Following dermal exposure, haemolytic effects have been observed in rats by application of 260 mg/kg to the shaved skin.

85.4.2     Irritation

In rabbits, EGBE has been considered a severe irritant by the Draize protocol and an irritant by the EU protocol. Several other studies have also reported EGBE to be a skin irritant in rabbits. Severe skin irritation has been noted in guinea pigs after application of EGBE.

Female rabbits exposed to EGBE (³72 mg/kg, 8 hours) developed cutaneous lesions accompanied by necrosis of epidermis and dermis on the 4^th day after exposure; skin lesions healed within a 2-week period.

EGBE has in several studies been reported to be severely irritating when instilled (undiluted) in the eyes of rabbits. Moderate corneal injury was observed in rabbits in which 0.5 ml of a 15% dilution of EGBE was placed in the conjunctival sac; no effects were observed with a dilution of 5%. In a Draize test performed in rabbits, undiluted EGBE caused severe irritation, dilutions at 70 and 30% caused moderate irritation, and dilutions of 20 and 10% caused mild irritation.

Male mice exposed to 750-8200 mg/m³ EGBE for 10-15 minutes exhibited a 20% decreased in respiratory rate at the lowest concentration and a 40% decrease at the highest concentration.

85.4.3     Sensitisation

EGBE did not result in dermal sensitisation when tested in the guinea pig maximisation test.

85.4.4     Repeated dose toxicity

Following repeated inhalation exposure to EGBE vapours, haematological effects (decreased RBC counts and Hgb levels, increased MCH) have been observed in rats exposed for 90 days to 378 mg/m³; these effects had either decreased or returned to the range of the control values. The NOAEC was 123 mg/m³.

In 14-week NTP studies in rats and mice, haematological evaluations showed an exposure concentration-related anaemia (in female rats and mice from 152 mg/m³; in male rats and mice from 614 mg/m³). Histopathological effects were observed in the spleen, bone marrow (rats only), liver, kidneys, and forestomach from 307 mg/m³ in female rats, and from 614 mg/m³ in male rats and mice (both sexes). The lowest concentration used in the study, 152 mg/m³, was a LOAEC for haematological changes in female rats and mice; the NOAEC for male rats and mice was 307 mg/m³.

In chronic NTP-studies in rats and mice, exposure-related  anaemia was observed in both species; female animals were generally more sensitive than male animals. The LOAEC for haematological changes was 152 mg/m³ for rats and 307 mg/m³ for mice, the lowest concentrations tested in the studies of rats and mice, respectively.

Following oral administration of EGBE by gavage to male rats for 6 weeks, dose-related changes (decreased RBC counts and Hgb concentration, and increased MCH and haemoglobinuria) were observed from 222 mg/kg b.w./day, the lowest dose level used in the study. Histopathological changes were noted in the spleen from 222 mg/kg b.w./day, and in the liver and kidneys from 443 mg/kg b.w./day.

In a 13-week NTP drinking water study, haematological effects were observed in female rats from 82 mg/kg b.w./day and in male rats from 281 mg/kg b.w./day. Histopathological changes were observed in the spleen from 129 and 151 mg/kg b.w./day in males and females, respectively; and in the liver from 69 and 82 mg/kg b.w./day in males and females, respectively. The lowest dose level tested (69/82 mg/kg b.w./day in males/females, respectively) was a LOAEL for haematological effects.

When EGBE was administered by gavage to male mice for 6 weeks, the LOAEL for haematological effects was 357 mg/kg b.w./day, the lowest dose tested.

In a 13-week study, occluded dermal administration of EGBE to rabbits at dose levels up to 150 mg/kg produced no observable haematological effects.

The immune system did not appear to be a sensitive target of EGBE in rats exposed to EGBE in the drinking water at doses up to 506/444 mg/kg b.w./day in males and females, respectively, for 21 days.

85.4.5     Toxicity to reproduction

The reproductive toxicity of EGBE has been studied in a variety of studies in rats, mice and rabbits following inhalation or oral administration. EGBE did not cause adverse effects in any reproductive organ, including testes, in any of the studies. In a two-generation reproductive toxicity study, fertility was reduced in mice only at maternally toxic doses (above 1000 mg/kg b.w./day) when EGBE was administered in the drinking water.

In addition, several developmental toxicity studies have addressed the potential of EGBE to induce embryo- and/or foetotoxicity following oral, inhalation, and dermal exposures to rats, mice, and rabbits. Toxicity to embryos and/or foetuses was observed only at dose levels, which also resulted in maternal toxicity. No malformations were observed in any of the studies.

85.4.6     Mutagenic and genotoxic effects

EGBE was negative for mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA102, TA1535, and TA1537 both with and without metabolic activation. A weak mutagenic response was seen in one test in strain TA97a both with and without metabolic activation. When this test was repeated, a negative response was obtained.

No mutagenic activity was observed when EGBE was investigated using bacteriophage T4D with Escherichia coli B, CR63, and K12, and in a test with Escherichia coli WP2uvrA in the presence and absence of Arochlor-induced rat liver S9 mix.

EGBE or its metabolite 2-butoxyacetaldehyde (BAL) were not found to be mutagenic in an in vitro gene mutation assay using Chinese hamster ovary (CHO) cells, whereas both EGBE and BAL weakly induced gene mutations in Chinese hamster V79 cells at high concentrations.

EGBE did not induce sister chromatid exchanges (SCEs) or chromosomal aberrations in CHO cells with or without liver S9 mix or in Chinese hamster V79 fibroblast cells. At high concentrations, however, EGBE weakly induced aneuploidy (numerical chromosomal anomalies), SCEs and micronuclei, and potentiated the clastogenicity induced by methyl methanesulfonate.

EGBE did not increase the incidence of micronuclei in the bone marrow cells of male rats or mice given three intraperitoneal injections of EGBE.

85.4.7     Carcinogenic effects

NTP has performed 2-year inhalation studies in rats and mice. No evidence of carcinogenic activity was found in male rats and equivocal evidence of carcinogenic activity in female rats based on increased combined incidences of benign and malignant pheochromocytoma of the adrenal medulla. In mice, some evidence of carcinogenic activity was found based on increased incidences of forestomach squamous cell papillomas and carcinomas in female mice and increased incidences of hemangiosarcoma of the liver in male mice.

86     Evaluation

EGBE is absorbed and distributed throughout the body (humans and rats) following inhalation, oral administration and dermal contact. EGBE is metabolised to 2-butoxyacetic acid (2-BAA), which probably is responsible for the haemolytic effect identified as the critical end-point in toxicological studies on EGBE.

Only limited data on the toxic effects of EGBE in humans are available. These data comes largely from case reports dealing with accidental poisonings and/or workplace exposure. EGBE seems of low acute toxicity in humans. The available data show that after acute oral ingestion of large doses of EGBE (combined with other solvents), haematological changes and metabolic acidosis are the primary effects. The only indication of haemolysis (small changes for haematocrit and MCHC) following repeated inhalation was observed in workers exposed to an average airborne concentration of EGBE of 2.9 mg/m³; however, the changes were within the range of normal clinical values and are not considered as being adverse.

EGBE is of moderate acute toxicity whether animals are exposed via the oral, dermal, or respiratory routes with oral LD₅₀-values ranging from 320 to 3100 mg/kg (rats, mice, guinea pigs, rabbits), dermal LD₅₀-values ranging from 406 to 4800 mg/kg (rabbits, guinea pigs, rats), and LC₅₀-values of 2200-2400 mg/m³ (4-hour exposure) for rats and of 3440 mg/m³ (7-hour exposure) for mice.

Irritation of the nose and throat, and eyes has been noted in human volunteers exposed by inhalation to EGBE at concentrations from 490-957 mg/m³ for 4-8 hours, but not in volunteers exposed to EGBE (98 mg/m³) for 2 hours. The NOAEC for irritative effects of EGBE in humans is above 100 mg/m³.

In rabbits, EGBE is severely irritating when instilled (undiluted) in the eyes; dilutions at 70 and 30% caused moderate irritation, and dilutions of 20 and 10% caused mild irritation. In rabbits, EGBE has been considered a severe irritant by the Draize protocol and an irritant by the EU protocol. Severe skin irritation has also been noted in guinea pigs. Male mice exposed to 750-8200 mg/m³ EGBE for 10-15 minutes exhibited a 20% decreased in respiratory rate at the lowest concentration and a 40% decrease at the highest concentration.

Human volunteers showed no dermal effects of 10% EGBE in a patch test and EGBE did not result in dermal sensitisation when tested in the guinea pig maximisation test.

Intravascular haemolysis is the primary response elicited in sensitive animal species following inhalation, oral, and dermal exposure to EGBE.

Changes in haematological parameters have been observed in rats and mice following acute inhalation exposure (increased erythrocyte fragility in rats at 305 mg/m³ for 4 hours; haemoglobinuria in rats and mice at about 1000 mg/m³ for 7 hours). Following oral administration, haemolysis of erythrocytes accompanied by haemoglobinuria was observed in rats following a single dose at dose levels from about 125 mg/kg. Following dermal exposure, haemolytic effects have been observed in rats by application of 260 mg/kg to the shaved skin.

Following repeated inhalation exposure to EGBE vapours, the NOAEC for haematological changes was 123 mg/m³ in a 90-day study in rats; in the 2-year NTP-study, the LOAEC for haematological changes was 152 mg/m³ for rats and 307 mg/m³ for mice, the lowest concentrations tested in the studies of rats and mice, respectively. In the 14-week NTP-study, the lowest concentration used in the study, 152 mg/m³, was a LOAEC for haematological changes in female rats and mice; the NOAEC for haematological changes in male rats and mice was 307 mg/m³. Following oral administration of EGBE by gavage to male rats for 6 weeks, the LOAEL for haematological changes was 222 mg/kg b.w./day, the lowest dose level used in the study. In a 13-week NTP drinking water study, the lowest dose level tested (69/82 mg/kg b.w./day in males/females, respectively) was a LOAEL for haematological effects. When EGBE was administered by gavage to male mice for 6 weeks, the LOAEL for haematological effects was 357 mg/kg b.w./day, the lowest dose tested. In a 13-week study, occluded dermal administration of EGBE to rabbits at dose levels up to 150 mg/kg produced no observable haematological effects.

The available data indicate that certain species are more susceptible to the haemolytic effects of EGBE. The sensitivities range from that of guinea pigs, which displayed no haemolytic effects following a single oral administration of 1000 mg/kg b.w EGBE, to that of rats, which displayed increased osmotic fragility of erythrocytes at a single-inhalation exposure to 305 mg/m³ and haemolysis of erythrocytes following a single oral dose at dose levels from about 125 mg/kg. Humans appear to be less sensitive than rats are to the haemolytic effect of EGBE as haemolysis has not been observed in acute inhalation exposures of human volunteers to up to about 950 mg/m³; reversible haemolytic effects have been observed in cases where humans consumed single oral doses of 400 to 1500 mg/kg b.w. This difference in sensitivity between rats and humans is supported by in vitro studies, which have shown that erythrocytes from humans were unaffected by incubations with 2-butoxyacetic acid (2-BAA, the toxic metabolite of EGBE) at concentrations, which produced total rat erythrocyte haemolysis.

With respect to gender sensitivity, the available data consistently showed that female rats are more sensitive to EGBE-induced haemolysis than male rats are. Female mice also appeared to be slightly more sensitive than male mice. In vitro, female erythrocytes have shown a slightly greater sensitivity than male erythrocytes following incubation with 2-BAA.

Several studies have assessed the effect of age on the haemolytic effects in young and adult rats. Older rats appeared to be more sensitive than the younger rats following acute doses of EGBE; however, chronic exposures appear to impart a certain level of tolerance to rats and mice over time as apparent tolerance to EGBE-induced haemolysis in rats and mice was observed in the 2-year NTP inhalation studies.

The possibility that certain human subpopulations, including the aged and those predisposed to haemolytic disorders, might be at an increased risk from exposure to EGBE has been investigated using blood from the elderly, from patients with sickle cell disease, and from patients with spherocytosis. Erythrocytes from these potentially sensitive groups were unaffected by incubations with 2-BAA at concentrations, which produced total rat erythrocyte haemolysis.

Effects have also been observed in the liver (hepatocellular degeneration and haemosiderin pigmentation of Kupffer cells), kidneys (haemosiderin accumulation, renal tubular degeneration, and intracytoplasmic haemoglobin), bone marrow (hyperplasia), and spleen (haematopoietic cell proliferation, congestion, and increased haemosiderin pigmentation) following exposure to EGBE. These effects, which generally occur at higher exposure levels than those causing haemolysis, are secondary to the haematotoxicity of EGBE and a result of a compensatory response to the haemolysis.

The reproductive and developmental toxicity of EGBE has been studied in several studies in rats, mice and rabbits following inhalation, oral administration, or dermal application (developmental toxicity only). It can be concluded from these studies that EGBE does not affect the reproductive organs of parents (both males or females), and only results in adverse reproductive and developmental effects at dose levels, which also result in parental toxicity. No malformations were observed in any of the studies.

No data have been located regarding toxicity to reproduction in humans.

No increases in micronuclei or sister chromatid exchanges were observed in workers exposed to both EGBE and to 2-ethoxyethanol (EGEE).

EGBE has been tested for its potential to induce gene mutations in in vitro systems and cytogenetic damage in both in vitro and in vivo systems. In most of the tests, EGBE has given negative results. One laboratory, however, has reported weak genotoxicity responses, but only at toxic doses. Overall, the available data do not support a mutagenic or clastogenic potential for EGBE.

In 2-year NTP inhalation studies, no evidence of carcinogenic activity was found in male F344 rats, equivocal evidence in female F344 rats based on increased combined incidences of benign and malignant pheochromocytoma of the adrenal medulla, and some evidence in B6C3F1 mice based on increased incidences of forestomach squamous cell papillomas and carcinomas in female mice and increased incidences of hemangiosarcoma of the liver in male mice. These tumour increases are, according to EPA (1999), of uncertain relevance to humans. As EGBE is generally negative in the genotoxicity tests and as glycol ethers generally appear unlikely to be carcinogenic, the concern for a carcinogenic potential of EGBE is low.

No data have been located regarding carcinogenic effects in humans.

86.1.1     Conclusion

The critical effects following exposure to EGBE are the irritative effects on the respiratory tract and eyes observed in humans and in experimental animals and the haemolytic effect observed in experimental animals and probably also indicated by the sparse human data available.

Irritation of the nose and throat, and eyes was noted in human volunteers exposed by inhalation to EGBE at concentrations from 490-957 mg/m³ for 4-8 hours, but not in volunteers exposed to EGBE (98 mg/m³) for 2 hours. The NOAEC for irritative effects of EGBE in humans is thus above 100 mg/m³. Male mice exposed to 750-8200 mg/m³ EGBE for 10-15 minutes exhibited a 20% decreased in respiratory rate at the lowest concentration and a 40% decrease at the highest concentration. EGBE is irritating to the eyes and skin of experimental animals.

Haemolysis has been identified as the critical end-point in toxicological studies of EGBE. Certain species differences in sensitivity have been observed regarding the haemolytic effects of EGBE, with rats being particularly sensitive, mice sensitive, and guinea pigs appearing relative insensitive. The only indication of haemolysis (small changes for Hct and MCHC) following repeated inhalation was observed in workers exposed to an average airborne concentration of EGBE of 2.9 mg/m³; however, the changes were within the range of normal clinical values and are not considered as being an adverse effect.

Data indicate that humans are less sensitive to the haemolytic toxicity of EGBE than are rats as no or very slight haemolytic effects were observed in the poisoning cases after acute oral ingestion of large doses. This difference in sensitivity between rats and humans is supported by in vitro studies, which have shown that erythrocytes from humans were unaffected by incubations with 2-butoxyacetic acid (2-BAA, the toxic metabolite of EGBE) at concentrations, which produced total rat erythrocyte haemolysis.

87     References

ACGIH (2001). Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. Cincinnati, OH.

At (2002). Grænseværdier for stoffer og materialer. Arbejdstilsynets At-vejledning C.0.1, oktober 2002. http://www.at.dk/Overblik/atviden/vejled/c01/C01.htm#Indhold

ATSDR (1998). Toxicological Profile for 2-Butoxyethanol and 2-Butoxyethanol acetate. U.S. Department of Health & Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry.

ECETOC (1995a). The toxicology of glycol ethers and its relevance to man. Technical Report No. 64. Brussels.

ECETOC (1995b). Substance Profile. EGBE. In: The toxicology of glycol ethers and its relevance to man. Technical Report No. 64. Brussels. 109-116.

EPA (1999). Toxicological Review of Ethylene Glycol Monobutyl Ether (EGBE). In Support of Summary Information on the Integrated Risk Information system (IRIS). U.S. Environmental Protection Agency, Washington, DC.

MAK (1998). Maximale Arbeitsplatz-Konzentrationen gesundheits­schädlicher Arbeitsstoffe. MAK- un BAT-Werte-Liste, 1998.

MM (2002). The Statutory Order from the Ministry of the Environment no. 439 of Jun3 3, 2002, on the List of Chemical Substances.

MST (2002). B-værdivejledningen. Vejledning Nr. 2 2002, Miljøstyrelsen, Miljøministeriet.

NTP (2000). NTP Technical Report on the toxicology and carcinogenesis studies of 2-butoxyethanol (CAS NO. 111-76-2) in F344/N rats and B6C3F1 mice (Inhalation studies). NTP TR 484. NIH Publication No. 00-3974. U.S. Department of Health and Human Services, Public Health Service, national Institutes of Health.

http://ehp.niehs.nih.gov/ntp/members/tr484.

Appendix 12: 1-Methoxy-2-propanol (2PG1ME) 
88     General description

Most of the available toxicological data on 1-methoxy-2-propanol (2PG1ME) relate to the commercial compound methoxy-propanol (PGME), which consists of 95-99% 2PG1ME and less than 5% 2-methoxy-1-propanol (1PG2ME).

88.1     Identity

Molecular formula:          C₄H₁₀O₂

Structural formula:           CH₃-CH-CH₂-O-CH₃

                                                     OH

Molecular weight:            90.1

CAS-no.:                        107-98-2

Synonyms:                      1-Methoxy-2-propanol;

                                      1-Methoxypropan-2-ol;

                                      Methoxypropanol, alpha isomer;

                                      Propylene Glycol Monomethyl Ether;       

                                      PGME;

                                      2-Propylene glycol 1-methyl ether;

                                      2PG1ME;       

                                      methoxy ether of propylene glycol;

                                      alpha-propylene glycol monomethyl ether;

                                             propylene glycol 1-methyl ether;

                                      (+/-)-1-methoxy-2-propanol;

                                             1-Methoxy-2-hydroxypropane

88.2     Physical / chemical properties

Description:                    1-Methoxy-2-propanol (2PG1ME) is the a-isomer of propylene glycol monomethyl ether (PGME). It is a colourless liquid with a sweet ether-like odour.

Melting point:                  - 96 °C

Boiling point:                   120 °C

Density:                          0.924 g/ml    (at 20°C)

Vapour pressure:             9 mmHg (12 hPa) (at 20°C)

Concentration of

saturated vapours:           44348 mg/m³ (calculated at 20°C and 1 atm)

Conversion factor:           1 ppm = 3.75 mg/m³  (calculated at 20°C and 1 atm)

                                      1 mg/m³ = 0.267 ppm

Solubility:                        Water:  completely soluble

Partition coefficient:        log P_o/w = -0.49 to -0.43 (BUA 1997)

References:                                                          A&H (1990), ACGIH (1991), BUA (1997), DECOS (1993), ECETOC (1995).

89     Toxicokinetics

89.1     Absorption

The toxicity data of 2PG1ME indicate uptake by all routes of exposure.

In anaesthetised rats exposed to 1000 ppm 2PG1ME (3750 mg/m³), the respiratory uptake was 87% (Stott & McKenna 1984 – quoted from A&H 1990).

89.2     Distribution

Exposure by inhalation of rats over 6 hours to 300 -3000 ppm 2PG1ME (equivalent to 1125-11250 mg/m³) resulted in a continuous increase in blood  concentration, which ranged from 109-2133 mg/g. The blood/air partition coefficient of 2PG1ME was 12400 in one study and 403 in another study (Johanson & Dynesius 1988 and Stott & McKenna 1984, respectively – quoted from A&H 1990).

Oral administration to rats of 0.09 or 0.78 g/kg ¹⁴C-labelled 2PG1ME resulted in recovery of 92-98 % of the radioactivity. Forty-eight hours after dosing, the liver had the highest percentage (1.4 %) of the radioactive dose. The fat/blood partition coefficient was 0.09, indicating that 2PG1ME does not accumulate in adipose tissue. There were no indications of accumulation of radioactivity in other main body tissues, or in testes. (Miller et al. 1983).

89.3     Metabolism and elimination

Rats were given an oral dose of 0.09 g/kg b.w. ¹⁴C-labelled 2PG1ME. Sixty-six percent of the dose was recovered in the expired air, 63% of which as carbon dioxide. Urinary excretion accounted for 11%, while 0.9% of the radioactivity was found in faeces. The predominant urinary metabolite was the glucuronic acid conjugate of 2PG1ME, followed by the sulphate conjugate, propylene glycol and unchanged 2PG1ME. The urinary recovery increased to 19-25% after exposure to a dose of 0.78 g/kg b.w. (Miller et al. 1983). A metabolism scheme for 2PG1ME is given in Figure 1.

In rats exposed by inhalation to 300 - 3000 ppm 2PG1ME (1125-11250 mg/m³) for 6 hours, the kinetic was non-linear. The half-time in the blood was 2.4 hr after the lowest exposure level and 15.7 hr after the highest, indicating saturation of the metabolic pathways. (Morgott & Nolan 1987– quoted from A&H 1990).

Figure 1. Proposed metabolism for 2PG1ME (modified from A&H 1990).

89.4     Mode of action

Glycol ethers capable of giving rise to alkoxyacetic acids have been shown to exhibit bone marrow depression and developmental, testicular and immunological toxicity. 2PG1ME is a secondary alcohol which does not form methoxy propionic acid but is primarily metabolised to carbon dioxide. No propylene glycol ethers being secondary alcohols have been shown to cause developmental toxicity. (ECETOC 1995).

90     Human toxicity

90.1     Single dose toxicity

90.1.1     Inhalation

Two subjects exposed to rising concentration of PGME over two hours reported subjective signs of mild narcotic effects (headache, dizziness) at 300-400 ppm (corresponding to 1125-1500 mg/m³). When the concentration reached 1000 ppm, signs of CNS impairment occurred in one of the two subjects in a balancing test.  (Stewart et al. 1970).

90.1.2     Oral intake

No data were found.

90.1.3     Dermal contact

No data were found.

90.1.4     Irritation

Two male volunteers were exposed to steadily increasing PGME concentration from 1 to 2050 ppm (equivalent to 3.75 - 7700 mg/m³) over 2 hours. The odour was noted at less than 25 ppm (94 mg/m³) and reported as objectionable at 50-75 ppm (188 - 281 mg/m³). Mild eye irritation occurred at 300-400 ppm (1125 – 1500 mg/m³), eye, nose and throat irritation from 500 ppm (1875 mg/m³), and lachrymation and rhinorrhea from 750 ppm (2813 mg/m³). Severe eye irritation occurred in one of the two subjects at 1000 ppm (3750 mg/m³) and the man had to leave the chamber. The other man experienced severe lachrymation, blepharospasm, throat irritation and pain hindering him to breath through the nose at 2050 ppm (7700 mg/m³). The pain was reversible within 15 min. after ended exposure, while the eye irritation lasted for 1 hour and the nasal congestion was present for 24 hours. (Stewart et al. 1970).

Twenty-three men were exposed to 250 ppm (equivalent to 938 mg/m³) PGME for 1-7 hr. After 15-30 minutes, eight men experienced eye irritation, three had nose irritation, one had a headache and one had nausea. After 45-60 min, 20 men had developed eye irritation and increased blinking, 15 complained of nasal irritation, 2 of throat irritation and one had a headache. Ten men remained in the chamber after 2 hours, 8 of whom complained of eye and 5 of nasal irritation. All four subjects remaining in the chamber for 7 hr had eye irritation and lachrymation, while none of them could still detect the odour of PGME. (Stewart et al. 1970).

90.2     Repeated dose toxicity

No data were found.

90.3     Toxicity to reproduction

No data were found.

90.4     Mutagenic and genotoxic effects

No data were found.

90.5     Carcinogenic effects

No data were found.

91     Animal toxicity

91.1     Single dose toxicity

91.1.1     Inhalation

Groups of rats varying from 10 to 20 animals were exposed to single exposures of 7000, 10000 or 15000 ppm PGME (corresponding to 25500, 36400 and 54600 mg/m³, respectively ) for 1 to 8 hours.

Five out of 10 rats exposed to 54600 mg/m³ for 4 hours died, while 3/20 rats exposed to 36400 mg/m³ and 2/20 at 25500 mg/m³ died. Dose-related CNS depression was seen from 25500 mg/m³. At 54600 and 36400 mg/m³, there were signs of respiratory tract irritation with interstitial oedema, congestion and areas of emphysema in the lung.

Death occurred in 2/20, 15/25 and 15/20 rats exposed once for 6 hours to 25500, 36400 or 54600mg/m³ PGME, respectively. CNS-depression was seen at all exposure levels, and all rats at the highest exposure level, were unconscious. (Rowe et al. 1954).

Groups of 10 -15 rats exposed once to 10000 ppm (36400 mg/m³) for 6 hours, 6000 ppm (21800 mg/m³) or 3000 ppm (10900 mg/m³) for 7 hours were examined with respect to injury. At the highest exposure level, body weights were moderately depressed and liver, kidney and lung weights were increased. Microscopy of the animals at the highest concentration level  revealed slight irritation of the lungs, but there were no effects on other organs. The mid-concentration level caused slight histopathological effects including granulation of the cytoplasm of centrolubolar cells and non-fatty deposits in 5 out of 10 rats one day after exposure, while no effect was seen on the liver after 3 days. At the low exposure level, no effects were seen. (Rowe et al. 1954).

Five out of 10 guinea pigs exposed to PGME for 10 hours died.  Lethargy was reported at sublethal concentrations (Rowe et al. 1954).

Groups of 2 or 4 rabbits were exposed to one single exposure of 10000 or 15000 ppm (36400 or 54600 mg/m³) PGME for 4, 6 or 7 hours. One out of 4 animals died following 7 hours exposure to the high dose (Rowe et al. 1954).

One male monkey was exposed in a single exposure to 10000 ppm PGME (36400 mg/m³) for 6 hours. It showed eye and nasal irritation, ataxia and depressed respiration, followed by excitation and lethargy after the animal was removed from the exposure chamber. The effects were reversible. (Rowe et al. 1954).

91.1.2     Oral intake

An LD₅₀ – value of 6100 mg PGME/kg b.w. was reported on basis of administration of 9 dosage levels of  undiluted PGME to rats by gavage. Dyspnoe and symptoms of CNS depression, including somnolence, uncoordinated gait and ataxia, were seen. (Rowe et al. 1954).

For rats, LD₅₀ – values of  >5000, 5200, 5710 and 5900 mg/kg b.w. were reported without any details (IUCLID 2000).

An LD₅₀ – value for mice of 10800 mg/kg b.w. was reported without any details (IUCLID 2000).

For rabbits, LD₅₀ – values of  >1840 and 5300 mg/kg b.w. were reported without any details (IUCLID 2000).

One cat survived a single exposure to 2 ml/kg b.w. (corresponding to 1840 mg/kg b.w.), but showed behavioural changes for 2 days after exposure (IUCLID 2000).

For dogs, and LD₅₀ – value of 9200 mg/kg b.w. was reported. Signs of toxicity were nausea, vomiting and respiratory arrest. (Shideman and Procita 1951- quoted from ECETOC 1995).

91.1.3     Dermal contact

Groups of 5 or 10 rabbits were exposed to 5.0, 7.0, 10.0, 12.0 or 15.0 ml PGME/kg b.w. (equivalent to 4.6, 6.4, 9.2, 11.1 or 13.9 g/kg b.w.) under occlusive bandage for 24 hours. The exposed areas were then washed with soap and water, and the animals were observed for two weeks.  Four of 5 animals of the high dose group, and 2 out of 10 animals of the second highest dose group died within 5 days. All rabbits showed narcosis. (Rowe et al. 1954). An LD₅₀ – value of ca. 13000 mg/kg b.w. was reported from this experiment (IUCLID 2000).

Another study reported an LD₅₀ – value for rabbits of 14200 mg/kg b.w. (Smyth et al. 1962 – quoted from IUCLID 2000).

91.1.4     Irritation

Respiratory tract irritation was seen in rats and guinea pigs exposed by inhalation to high concentrations (from 10000 ppm – equivalent to 37500 mg/m³) of PGME for 4-10 hours (Rowe et al. 1954).

PGME was reported to be not irritating to the skin of rabbits. No details were given. (IUCLID 2000).

Repeated contact to rabbit skin over 13 weeks with2, 4 , 7 or 10 ml PGME/kg b.w. (equivalent to 1.85, 3.70, 6.47 or 9.24 g/kg b.w.) caused only occasional scaling and erythema, and no difference to controls could be demonstrated. (Rowe et al. 1954).

One drop of undiluted PGME was placed in the eyes of rabbits on five consecutive days. Mild transient irritative response of the conjunctiva was noted following each dose. No corneal injury was revealed by fluorescein staining. (Rowe et al. 1954).

PGME was reported to be not irritating or slightly irritating to rabbits eyes. No further details were given. (IUCLID 2000).

91.1.5     Sensitisation

No sensitisation was seen in Guinea pigs in an adjuvant test with topical applications described by Maguire (1973) (Carreon et al. 1984 – quoted from IUCLID 2000).

91.2     Repeated dose toxicity

91.2.1     Inhalation

Male and female Fischer 344 rats and B6C3F1 mice were exposed to 0, 300, 1000 or 3000 ppm PGME (equivalent to 0, 1125, 3750 or 11250 mg/m³) 6 hr/day for 9 days. At the high concentration, central nervous system depression and decrease in urine specific gravity was seen in rats, and liver weights were increased in male rats and mice. No effects were reported at any other concentration in rats or in mice and no gross pathological or histopathological effects were reported at any concentration in either species. (Miller et al. 1981).

Rats were exposed to 2500, 5000 or 10000 ppm PGME (9375, 18750 or 37500 mg/m³) 4 hours/day, 5 days/week for 2 weeks. No effects were seen in the low exposure group. At the two highest exposure levels, transient non-specific CNS-depression was reported, with tolerance development at the end of the exposure period. Body weights were decreased in the high dose group. (Goldberg 1964 – quoted from IUCLID 2000).

Groups of 5 male and 5 female rats were exposed to 10000 ppm (corresponding to 36400 mg/m³) PGME 30 minutes daily for 107 days (79 exposures), 1 hour daily for 106 days (78 exposures) or 2 hours daily for 116 days (84 exposures). Drowsiness and unsteadyness were seen after each 2 hour-exposure, and slight CNS depression was seen in the 1 hour-exposure group. The 2-hour group showed moderately increased liver and kidney weights. No other effects on organ weights haematology or histology were seen in this group or in the two other treatment groups. (Rowe et al. 1954).

In a 90-day-study, groups of 10 male and 10 female Fischer 344 rats were exposed to 0, 300, 1000 or 3000 ppm PGME (corresponding to 0, 1090, 3620 or 10900 mg/m³), 6 hours/day, 5 days/week. No effects were seen in the two lowest exposure groups. In the high exposure group, transient sedation in relation to the exposure was recorded during the first weeks of the study. Increased relative liver weights and, in females, slight hypertrophy of the liver in the centrilobular area were also reported at this concentration. No effects were seen in other organs or in haematology. (Landry et al. 1983).

A 90-day-study using the same exposure regime as used above for the rats was performed using 7 New Zealand White rabbits/sex/exposure concentration. No effects were seen at the two lowest exposure groups, while animals exposed to 10900 mg/m³ exhibited transient central nervous depression. No other effects were reported (Landry et al. 1983).

Exposure for 4 hours daily to 10000 ppm (corresponding to 36400 mg/m³) of two male monkeys 66 times over 91 days and of one female monkey 26 times in 38 days caused CNS depression after each exposure. Body weights were depressed and liver weights increased significantly. Histology revealed cytoplasma granulation in the central area and non-fatty degeneration. Slight congestion of the lungs was seen. No further adverse effects were reported. (Rowe et al. 1954).

Groups of rats were exposed 7 hours/day, to 6000 ppm (corresponding to 21800 mg/m³) for 114 days (81 exposures) or to 3000 or 1500 ppm (corresponding to 10900 or 5460 mg/m³) for 198 days (141 exposures). A group of controls was also used. At the high concentration, 4/10 males and 7/10 females died. The survivors exhibited narcotic effects after each exposure, although tolerance to the material developed during the last 2 months. Slight body weight depression and increased liver and kidney weights were reported, but no histopathological findings were seen. At the mid concentration, mortality was not higher than in the control group. Reversible, mild CNS depression was reported from exposure during the first week at this concentration. No effects were seen on body weights in this group, but the liver weights were significantly increased in both males and females. No microscopic changes were found. At the low dose, no adverse effects were seen. (Rowe et al. 1954).

Groups of at least 5 male and 5 female Guinea pigs were exposed 7 hours/day, 5 days/week to either 6000 ppm PGME (corresponding to 21800 mg/m³) for 113 days, to 3000 or to 1500 ppm  (corresponding to 10900 and 5450 mg/m³, respectively) for 184 days. In the high dose group, all animals survived, but marked narcotic effects were reported. Body weights were significantly depressed. Only very slight vacuolation and granulation in liver cells were seen. No adverse effects were seen at the two lower exposure levels. (Rowe et al. 1954).

One female rabbit was exposed 7 hours daily, 5 days/week to 6000 ppm PGME over a period of 113 days, two rabbits to 3000 ppm and 4 rabbits to 1500 or 800 ppm PGME over 184 days. The concentration levels correspond to 21800, 10900, 5450 or 2906.7 mg/m³, respectively. The one female treated at the high concentration showed narcotic effect and body weight depression. Liver and kidney weights were slightly increased, but no significant pathological effects were seen in the liver. Very slight local irritation and congestion and oedema were seen in the lungs. At 10900 and 5450 mg/m³, liver weights were slightly increased, microscopy revealing slight changes as described above for the guinea pigs. Slight congestion and oedema and areas of emphysema were seen in the lungs. No adverse effects were seen at the low concentration. (Rowe et al. 1954).

91.2.2     Oral intake

Groups of five male rats were given gavage doses of 0, 0.1, 0.3, 1.0 or 3.0 ml PGME/kg b.w. in olive oil (equivalent to 0, 0.09, 0.28, 0.92 or 2.77 g/kg b.w.) 26 times over 35 days. No animals died. In the high dose group, the liver and kidney weights were increased, while the lung, heart, spleen and testes weights were normal. No effects were seen in the 3 lower dose groups. (Rowe et al. 1954).

Dogs were exposed by gavage to 0, 0.5, 1 or 2-3 ml/kg b.w. (equivalent to 0, 0.46, 0.92 or 1.85-2.77 g/kg b.w.) PGME for 14 weeks. Mild central nervous system depression was observed at the two highest doses. Male dogs of the high dose group had spermiophages (possibly macrophages) in the epididymis and testis. No further information are given. (Stenger et al. 1972).

91.2.3     Dermal contact

Five male and 5 female New Zealand white rabbits were exposed dermally to 1000 mg/kg b.w., 5 days/week for 3 weeks. Slight scaling of the skin was reported. (Calhoun et al. 1984 - quoted from ECETOC 1995).

Groups of 5 rabbits were exposed to 0, 2.0, 4.0, 7.0 or 10.0 ml PGME/kg b.w. (equivalent to 0, 1.85, 3.70, 6.47 or 9.24 g/kg b.w.) under occlusion, 5 times /week for 13 weeks. Mortality is reported in Table 4.2.3.

Table 4.2.3 Mortality in dermal 90-day study with PGME

Decreased food consumption and body weight loss as well as narcosis were recorded prior to death in the two highest dose groups. In the lower dose groups, the deaths were associated with respiratory infections. Organ weights and gross pathology were normal, except for gastric retention and occasional haemorrhagic foci in the gastric mucosa in narcotic animals. Histological examination revealed renal tubular necrosis in 3 animals from the two highest dose groups who had died of the treatment, while other rabbits showed only a slight granular degeneration in the tubules. (Rowe et al. 1954).

91.3     Toxicity to reproduction

91.3.1     Inhalation

Ten male Wistar rats exposed for 10 days to 0, 200 or 600 ppm (equivalent to 0, 750 or 2250 mg/m³) PGME 6 hours/day showed no effects on testicular weights or histology. (Doe et al. 1983).

Pregnant Wistar rats were exposed to 0, 200 or 600 ppm PGME (equivalent to 0, 750, 2250 mg/m³) 6 hours/day on days 6-17 of gestation. No effects on dam weights, on number of pups, proportion of live pups or mean group pup weights were reported. (Doe et al. 1983).

Groups of 30-32 pregnant Fischer 344 rats were exposed to 0, 500, 1500 or 3000 ppm PGME (equivalent to 1875, 5625 or 11250 mg/m³) 6 hours/day from day 6-15 of gestation. The study was conducted in accordance with OECD guideline 414. Mild central nervous system depression was reported at the high exposure level at the beginning of the exposure period, but accommodation developed. In this group, food consumption was decreased the first 3 days, and body weight gain was depressed at the end of the exposure period. No differences on pregnancy rates, litter size, resorption rates or feral body weights were seen when compared to controls. Significantly increased delayed sternebral ossification was reported in pups of the high concentration group dams. There was no significant increase in incidence of malformations in any groups.

(Hanley et al. 1984).

Groups of 31-33 pregnant New Zealand white rabbits were exposed to 0, 500, 1500 or 3000 ppm PGME (equivalent to 1875, 5625 or 11250 mg/m³) 6 hours/day from day 6-18 of gestation, in a study in accordance with OECD guideline 414. Mild lethargy was seen in the high concentration group during the first two days of exposure. The overall weight gain was statistically reduced. Six rabbits died, four of which at the high concentration. No significant differences in relative number of pregnancies, litters,  resorptions or live foetuses or in foetal body weights were reported in any treatment group when compared to controls. No significant difference in incidence of malformations or variations in the pups of any treatment group was seen when compared to controls. (Hanley et al. 1984).

91.3.2     Oral intake

Male mice administred 2% PGME in the drinking water (approximately equivalent to 2.5 g/kg b.w.- assuming a water consumption of 3.5 ml/mouse/day and a mouse weight of 25 g)  for 25 days did not show any significant changes in testes or in seminal vesicle weights (Nagano et al. 1984 – quoted from ACGIH, 1991).

Male dogs exposed by gavage to 462 - 2772 mg/kg b.w. PGME for 14 weeks developed numerous spermiophages (macrophages) in the epididymis. The study is also described under point 4.2.2 (Stenger et al. 1972).

Female rats were dosed by gavage to 0, 0.05, 0.1, 0.2, 0.4 or 0.8 ml/kg b.w. PGME (equivalent to 0, 46, 92.4, 185, 370 or 739 mg/kg b.w.) on days 1-21 of gestation. There was no effect on the number of pups. The offspring showed significant delayed ossification of the skull at the highest dose. No information on maternal toxicity was given. (Stenger et al. 1972).

A continuous breeding study was performed in CD-1 mice. Twenty males and 20 females/group were dosed in the drinking water from 7 days before mating with 0, 0.5, 1.0 or 2.0 % PGME (according to DECOS 1993, the dose levels correspond to 0, 950, 1890 and 3330 mg/kg b.w. for males ); the offspring dosed continuously with 2.0 % PGME in the drinking water (approximately corresponding to 3330 mg/kg b.w.). In the F1- generation, birth weights of the pups of the high dose group were reduced. In the F2 – generation, reduction in birth weights and reduced epididymis and prostate gland weights, but no effect on sperm morphology, motility or density was reported. (Unpublished data by Dow, 1986 – quoted from IUCLID 2000 and DECOS 1993).

Female mice were exposed by gavage to 0, 0.5, 1 or 2 ml/kg b.w. (equivalent to 462, 924 and 1848 mg/kg b.w, respectively) PGME on days 1-18 of gestation. No evidence of foetotoxicity was seen. (Stenger et al. 1972).

Pregnant rabbits were exposed by gavage to 0, 0.25, 0.5 or 1 ml/kg b.w. PGME (equivalent to 0, 231, 462 or 924 mg/kg b.w.) on day 6-18 of gestation. No evidence of toxicity were seen in pups. (Stenger et al. 1972) 

91.3.3     Dermal contact

No data were found.

91.4     Mutagenic and genotoxic effects

91.4.1     In vitro studies

An Ames test conducted in  Salmonella typhimurium strains TA98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation using 2 - 6250 mg/plate was negative in all strains (unpublished report by Dow 1983a- quoted from ECETOC 1995).

A mammalian unscheduled DNA synthesis test in vitro using primary rat hepatocytes with PGME concentrations of 3.16 x 10 ^-5 to 0.1 M (2.85 x 10^-3 - 9.01 g/l) showed no effect on grain counts. (Mendrala 1983 - quoted from ECETOC 1995 and DECOS 1993).

A chromosomal aberration test conducted in Chinese Hamster ovary cells at PGME concentrations of 1.25, 2.5, 5.0 and 10.0 mg/ml was negative (unpublished report by Dow 1983b - quoted from ECETOC 1995).

91.4.2     In vivo studies

No data were found.

91.5     Carcinogenic effects

91.5.1     Inhalation

Groups of Fischer-344 rats were exposed to 0, 300, 1000 or 3000 ppm PGME (equivalent to 1125, 3750 or 11250 mg/m³) 6 hrs/day, 5 days/week for 3, 6 or 12 months (10 rats/sex/conc. + 5 males/conc. for investigation of a_2U-globulin nephropathy ), 18 months (20 rats/sex/conc.) or 24 months (50 rats/sex/conc.).

At the high exposure level, CNS-depression including decreased activity, incoordination and transient sedation was observed during the first week until 1-2 hours after each treatment. These symptoms disappeared, but reappeared at 12-18 months of exposure. At this exposure level,mortality increased in male rats from approximately 18 months of treatment, but the finding was not statistically significant. Body weights were significantly decreased in the animals treated for 12, 18 or 24 months at the high concentration level.

There was no effect on haematology in any group. In males of the high concentration group, serum creatinine was increased 78% and urea nitrogen 100% at 24 months. Serum alkaline phosphatase was significantly increased from 6 months at 11250 mg/m³ and at 24 months at 3750 mg/m³. Liver enzymes (alanine and aspartate amino-transferases) were mildly elevated in males of the high concentration group during the first, but not during the second year of the two year- treatment period.

Gross and microscopic pathology was performed on the animals treated for 2 years. Liver weights were increased in the high exposure group from 2 weeks to 2 years of dosing. The incidence of eosinophilic hepatocellular foci was increased in males of the two highest concentration groups exposed for 24 months. Male rats treated with the high concentration showed significantly elevated hepatic S-phase DNA-synthesis.

Kidney weights of high-exposure males were significantly increased in a time-dependent way from 12 months of exposure. Also female kidney weights were increased, although less consistently than in males. The kidneys of male rats treated with the highest concentration showed epithelium damage in the tubules with multifocal tubular basophilia, slight decrease of cell height and cell debris in the tubular lumen at one week. The effect was less clear at 13 weeks. Levels of a_2u-globulin in the epithelial cells of the cortical tubules were increased in all treated male groups (investigated until 12 months exposure period). The effect was most prominent during the first 2 weeks. At 13 weeks, a_2u- globulin staining involved about half the cortical tubules in the high concentration group, while only 2/5 males of the low concentration group showed slight staining. a_2u-globulin staining remained in males of the two highest dose groups at 6 and 12 months. No effect was seen in females at 1 and 2 weeks and the parameter was not investigated in females at later intervals. No difference in incidence of chronic progressive glomerulonephritis was seen compared to controls, but the effect was more pronounced in the affected animals in the high concentration group, especially in males. No significant increase in tumour incidence was seen in the 24 months-study. (Spencer et al. 2002).

Groups of B6C3F1-mice were exposed to 0, 300, 1000 or 3000 ppm PGME (equivalent to 1125, 3750 or 11250 mg/m³), 6 hrs/day, 5 days/week for 1 or 2 weeks, 3, 6, 12, 18 months (5-10 mice/sex/conc.) or 24 months (50 mice/sex/conc.).

Decreased activity, incoordination and transient sedation was reported at the high concentration for the first week of exposure. In the 24 months-study, mortality was non-significantly increased in male mice of the high exposure group from around 18 months when compared to controls, while no effect on mortality was seen in studies of shorter duration or in females. Body weights were significantly depressed 2-7% in mice exposed for 24 months to the high concentration. Also mice treated with the mid-concentration had significantly depressed body weight, although the effect was less marked.

Liver weights were increased in high exposure group animals following 2 weeks to 2 years. Hepatic S-phase DNA-synthesis was slightly, but significantly increased in males of the high concentration group.

No effects on kidney weight, gross appearance or histopathology were reported.

In the adrenal glands, pathological examination revealed atrophy of a female specific zone in all female mice exposed for 3 months to the highest concentration and in 2/10 females exposed to the mid-concentration level, but not in females treated for 24 months. The authors report that the toxicological significance of the effect is unknown.  (Spencer et al. 2002).

92     Regulations

92.1     Ambient air

Denmark (C-value):        0.03 mg/m³  (MST 2002)

92.2     Drinking water

-

92.3     Soil

-

92.4     Occupational Exposure Limits

Denmark:                       185 mg/m³ (50 ppm) (At 2002)

ACGIH:                         100 ppm (ACGIH 2001)

Germany:                        100 ppm (MAK 1998 )

92.5     Classification

Flammable with R10 (MM 2002)

92.6     IARC

-

92.7     US-EPA

-

93     Summary

93.1     Description

1-methoxy-2-propanol (2PG1ME) is a water soluble colourless volatile liquid with an ether like odour. 2PG1ME is the a-isomer of propylene glycol monomethyl ether (PGME) and makes out at least 95% of commercial PGME where the b-isomer is present in less than 5%.

93.2     Toxicokinetics

2PG1ME appears to be absorbed by all routes of exposure. Toxicological studies did not indicate that accumulation should occurr. The primary metabolic pathway was via demethylation and oxidation to CO₂, which is then exhaled. Conjugation and urinary excretion  played a minor role. The developmental toxicant methoxypropionic acid is not formed by 2PG1ME as it is a secondary alcohol, but only from the b-isomer 2-methoxy-1-propanol.

93.3     Human toxicity

93.3.1     Single dose toxicity

Groups of male volunteers were exposed by inhalation to different concentrations of PGME vapours for 1-7 hours.  Symptoms of slight CNS-depression (dizziness, headache) were reported from 1125-1500 mg/m³ in subjects exposed to rising concentration PGME. Overt CNS-impairment was seen in one of two subjects exposed to 3750 mg/m³

Volunteers exposed to 938 mg/m³ for 7 hours, complained of eye and nose irritation after 15-30 minutes, the irritation symptoms increasing and including complaints of throat irritation after 45 minutes. The symptoms were so strong that only four subjects remained in the chamber for 7 hours.

No reports of repeated dose toxicity, reproductive and developmental effects, mutagenic and genotoxic effects or carcinogenic effects in humans were found.

93.4     Animal toxicity

93.4.1     Single dose toxicity

Five out of 10 rats died following a 4 hour-exposure of 54600 mg/m³ PGME.. Dose-related CNS depression was reported in rats treated with single exposure from 25500 to 56600 for 1 to 8 hours, including unconsciousness in rats exposed to 54600 mg/m³ for 6 hours. Histopathological examination of rats dosed 10900-36400 mg/m³ for 6-7 hours showed slight granulation of liver cell cytoplasm and non-fatty liver deposits in half the rats exposed to 21800 mg/m³, while liver, kidney and lung weights increase.

Five out of 10 Guinea pigs died following 10 hours exposure to 54600 mg/m³PGME. One out of four rabbits died following 7 hours exposure to 54600 mg/m³.A monkey exposed to 36400 mg/m³ PGME for 6 hours showed CNS- symptoms including depressed respiration, excitation and lethargy.

Oral LD₅₀-values of 5000 to 6100 mg PGME/kg b.w. for rats, 10800 mg/kg b.w. for mice, 1840-5300 for rabbits and 9200 mg/kg for dogs were reported. In rats and dogs, general intoxication symptoms and symptoms of CNS depression were reported preceeding death.

Dermal application of 4600-13900 mg PGME /kg b.w. under occlusion for 24 hours caused narcosis in all animals, and 4 out of 5 rabbits at the high dose died. Dermal LD₅₀-values for rabbits of 13000 and 14200 mg/kg b.w were reported.

Respiratory tract irritation was reported from single 4 hour exposures of rats to 36400 or 54600 mg PGME /m³ . Slight irritation of the lungs was seen from a 7 hour exposure to 109000 mg PGME/m³, while no effects were reported at lower concentrations.

A monkey exposed to 36400 mg/m³ PGME for 6 hours showed eye and nose irritation. 

PGME was reported to be not irritating or slightly irritating to rabbits skin following single or repeated exposure. PGME was reported to be non-irritating or mildly irritating to the eyes of rabbits.

No sensitisation was seen in an adjuvant test in Guinea pigs.

93.4.2     Repeated dose toxicity

In an inhalation study including, rats, mice, rabbits and monkeys, the animals were exposed to PGME at different regimes, with concentrations from 300 to 10000 ppm (equivalent to 1125 - 37500 mg/m³), over 9 days to 6 months, 30 minutes to 7 hours daily. All species showed CNS-depression decreasing in studies of longer duration. Slight lung congestion and slight non-fatty degeneration and cytoplasma granulation of the liver in some of the studies. Overall, the effect level in rats and mice  was approximately 11000 mg/m³, while  rabbits showed effects in the liver and the lung at 5450 mg/m³..Guinea pigs appeared  slightly less sensitive, with showing CNS depression and slight liver effects at 22000 mg/m³, and monkeys showed CNS-depression at 36400 mg/m³. 

In a 90-day inhalation study in rats and rabbits exposed to 1090-10900 mg/m³, rats of the high concentration level exhibited CNS depression, increased liver weights and slight liver hypertrophy, while rabbits of the high exposure level showed CNS depression .

In dogs, oral ingestion of PGME over 14 weeks resulted in CNS-depression at 920 and 1850/2770 mg/kg b.w. Rats dosed 2.77 g/kg b.w. over 35 days showed increased liver and kidney weights.

Daily dermal doses of 6.47 and 9.24 g/kg b.w. over 13 weeks were toxic to rabbits. Occasional gastric retention and renal tubular necrosis were reported.

93.4.3     Toxicity to reproduction

Inhalation of up to 2250 mg/m³ PGME  for 10 days did not result in testicular effects in rats.

No effects on reproduction of female rats were reported from exposure up to 2250 mg/m³ PGME from days 6-17 of gestation.

No changes in testes or seminal vesicle weights were seen in male mice exposed to 2.5 g/kg b.w. PGME in drinking water for 25 days.Dogs exposed orally to 462-2772 mg/ kg b.w. for 14 weeks developed numerous spermiophages (macrophages) in the epididymis.

Delayed sternebral ossification was reported in pups of rats dosed 11250 mg/m³ from days 6-15 of gestation. Mild depression of the central nervous system and decreased body weight gain were seen in the dams of this treatment group, while no effect was seen on dams, reproduction parameters or pups at lower concentrations.

Pregnant rabbits exposed up to 11250 mg PGME/m³ had increased mortality, CNS depression and reduced weight gain at the high concentration, while there were no effects on reproduction or on development at this or lower concentration levels.

Delayed ossification of the skull was reported in pups of rats exposed orally to 739 mg/kg b.w. PGME on days 1-21 of gestation. No effect on the number of pups was seen. No details are given on maternal effects.

The F₁ and F₂-generation of mice exposed through the drinking water to approximately 3.3 g/kg b.w PGME in a continuous breeding study showed reduction in birth weights and reduced epididymis and prostate gland weights.

No evidence of foetotoxicity was seen from exposure of female mice by gavage to up to 1848 mg/kg b.w from days 1-18 of gestation.

No foetoxicity resulted from oral exposure of rabbits on days 6-18 of gestation to up to 924 mg/kg b.w.

93.4.4     Mutagenic and genotoxic effects

PGME was negative in an Ames test, in a unscheduled DNA synthesis test in rat hepatocytes and in a chromosome aberration test in Chinese hamster ovary cells. No in vivo studies were found.

93.4.5     Carcinogenic effects

Rats exposed by inhalation to 1125-11250 mg/m³ up to 24 months showed increased liver weights and incidence of eosinophilic heptocellular foci were increased in male of the high exposure group. Chronic progressive glomerulonephritis was more pronounced, and tubular epithelium damage in males treated at the high concentration compared to controls, but no there was no increase in tumour incidence. Levels of a_2u-globulin were elevated at 3 months in all the treated males compared to controls

Mice exposed to the same regime as the rats also had liver weights increase. No adverse effects on the kidney were seen, but females exposed for 3 months at the high concentration showed changes in the adrenal gland.

94     Evaluation

Most of the available toxicological data on 1-methoxy-2-propanol (2PG1ME) relate to the commercial compound methoxy-propanol (PGME), which consists of 95-99% 2PG1ME and less than 5% 2-methoxy-1-propanol (1PG2ME). On the basis of these data, it appears that 2PG1ME is of low systemic toxicity; irritation of the eyes, the mucous membranes and the respiratory tract as the critical effects. The substance is also a CNS-depressant.

2PG1ME appears to be absorbed by all routes of exposure. It is primarily metabolised via demethylation and oxidised to CO₂, a minor part being excreted through the urine in conjugated form. The toxic metabolite methoxyacetic acid is not formed by 2PG1ME, but only by its  b-isomer 2PG1ME.

PGME is of low acute toxicity. No poisoning cases in humans were found. LC₅₀-values in animals were approximately 54600 mg/m³ following 4 hours exposure in rats and10 hour exposure in  Guinea pigs, and higher in rabbits and monkeys. Oral LD₅₀-values of 5000-10800 mg/kg b.w. were reported for rats, mice, rabbits and dogs. The dermal LD₅₀ –value for rabbits was around 13000 mg/kg b.w.

PGME caused dizziness and headache in human volunteers from 1125 mg/m³, and one case of loss of ability to balance is reported at 3750 mg/m³.

CNS-depression was also the major symptom reported from acute studies in rats, mice, Guinea pigs, rabbits and monkeys from 25500 mg/m³. Oral and dermal exposure at sublethal doses also resulted in CNS depression in rats, rabbits and dogs.

In humans. The substance was irritating to the eyes, nose and throat at 938 mg/m³.

In animals, the substance was reported to be irritating to the respiratory tract of rats from 37500 mg/m³. PGME was not irritating or mildly irritating to the eyes of rabbits. No information on skin irritation in humans were available, and no or slight irritation occurred in rabbits following exposure to PGME. The substance was not sensitising in Guinea pigs.

No human data were available on effects of repeated exposure to PGME.

Animal data showed increased liver weights, hypertrophy of the liver and occasional slight non-fatty degeneration and granulation of the liver of rats, mice, guinea pigs, rabbits following repeated exposure to 5450 to 21800 mg/m³up to 6 months. In a two-year inhalation study in rats and exposed to 1125-11250 mg/m³, the liver weights and the incidence of eosinophilic hepatocellular foci were increased in the high dose animals.

Development of glomerulonephritis was significantly increased in male rats exposed to 11250 mg/m³ over 24 months compared to controls. There was no increase in tumour incidence. No increase was seen in females. The mechanism of nephrotoxicity in males was probably related to the measured increased levels of a_2u-globulin, which is specific to male F344-rats and considered not to be a relevant mechanism for humans.

No effect on testis resulted from inhalation exposure of rats up to 2250 mg/m³ PGME or oral exposure of mice to 2.5 g/kg b.w.. Dogs treated orally with 462 mg/kg b.w. had macrophages in the testes and epididymis. The finding was so scarcely described in the reference that it cannot be evaluated. However, as no similar pathological change were found in any other study, it is not considered to be toxicologically significant.

No foetotoxicity was seen from exposure by gavage of mice or rabbits during gestation at doses of 1848, respective 924 mg/kg b.w. Delayed ossification of the sternebrae and the skull occurred in pups of rats dosed 11250 mg/m³ by inhalation and orally to 739 mg/kg b.w., respectively. Maternal toxicity was seen in rabbits and rats at these levels. In a continuous breeding study in mice where PGME was given in the drinking water, reduced birth weights, epididymis and prostate weights were seen at 3.3 g/kg b.w. PGME while no effects were seen in the dams. On this basis, PGME is considered not to have any adverse effect on fertility or to cause developmental toxicity.

PGME was negative in three different in vitro mutagenicity tests. No in vivo tests were available.

No increased incidence of tumours was found in a 2-year inhalation study in rats and mice exposed up to 11250 mg/m³.

In conclusion, 2PG1ME is considered to be of low systemic toxicity and its critical effects  being irritative effects to the eyes, the mucous membranes and the respiratory tract and depression of the central nervous system.

95     References

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A&H (1990). Propylene Glycol Ethers and Their Acetates. Gunnar Johansson. Arbete och Hälsa 1990:32, Arbetsmiljöinstitutet.

At (2002). 1-Methoxy-2-propanol. In: At-Vejledning C.0.1. Grænseværdier for stoffer og materialer. Arbejdstilsynet oktober 2002.

BUA (1997). Methoxypropanol  In: BUA reports 173 and 174. German Chemical Society. GDCh -Advisory Committee on Existing Chemicals of Environmental Relevance (BUA). Hirzel Wissenschaftliche Verlagsgesellschaft Stuttgart 1997.

Chemfinder 2001 Propylene Glycol Monomethyl Ether. Http://www.chemfinder.com

DECOS (1993). Health based recommended occupational exposure limits for 1-methoxypropanol-2, 1-methoxypropylacetate-2. 2-methoxypropanol-1, 

2-methoxypropylacetate-1. Dutch Expert Committee on Occupational Standards. Labour Inspectorate, the Netherlands, RA5/93.

Doe JE, Samuels DM, Tinston DJ and de Silva Wickramaratne GA (1983). Comparative aspects of the reproductive toxicology by inhalation in rats of ethylene glycol monomethyl ether and propylene glycol monomethyl ether. Toxicol Appl Pharm 69, 43-47.

ECETOC (1995). The toxicology of glycol ethers and its relevance to man. ECETOC Technical Report 64.

Hanley Jr TR, Calhoun LL, Yano BL and Rao KS (1984). Teratologic evaluation of inhaled propylene glycol monomethyl ether in rats and rabbits 4, 784-794.

IUCLID (2000). 107-98-2. In: International Uniform Chemical Information Database. Existing Chemicals 2000. ECB, JRC, Italy.

Maguire HC Jr (1973). The Bioassay of Contact Allergens in the Guinea Pig. J Soc Cosmet Chem, 24, 151-162.

Miller RR, Ayres JA, Calhoun LL, Young JT and McKenna MJ (1981). Comparative short-term inhalation toxicity of ethylene glycol monomethyl ether and propylene glycol monomethyl ether in rats and mice. Toxicol Appl Pharm 61, 368-377.

Miller RR, Hermann EA, Langvardt PW, McKenna MJ and Schwetz BA (1983). Comparative metabolism and disposition of ethylene glycol monomethylether and propylene glycol monomethyl ether in male rats. Toxicol. Appl Pharm 67, 229-237.