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

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.

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