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

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).

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