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

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).

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