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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Principles of method if other than guideline:
New reproductive toxicology testing scheme which has been designated "Fertility Assessment by Continuous Breeding".
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): Diethylene glycol (CAS No. 111-46-6, Chemical Central, Kansas City, MO)
- Analytical purity: > 99% pure
- Other: infrared, ultraviolet/visible and nuclear magnetic resonance spectra, together with elemental analysis were consistent with the structure of the compound.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CD-1 (ICR) BR outbred swiss albino mice supplied by the Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: six week old upon receipt
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: initially, the animals were group housed (10 animals/group/sex) in solid bottom polypropylene or polycarbonate cages with stainless-steel wire lids and Ab-Sorb-Dri cage litter
- Diet: NIH-07 open formula pelleted chow (Zeigler Brothers, Gardners, PA), ad libitum
- Water: deionized/filtered water, ad libitum
- Acclimation period: 2 weeks (during this period two females and two males were euthanized and their sera analyzed for 11 viruses (Microbiological Associates, Bethesda, MD). All test results were negative for viral antibody titers.)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 °C
- Photoperiod (hrs dark / hrs light): 14-hr light/10-hr dark cycle

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Two separate standard solutions of diethylene glycol in deionized water were prepared at concentrations of 2.016 and 3.990 mg/mL. Portions of these solutions were diluted with water to make two additional standards at concentrations of 1.008 and 1.995 mg/mL. Water samples containing diethylene glycol at levels above 0.35% were initially diluted, in triplicate, with deionized water to concentrations of about 0.35%. The 0.0% (blank) and the 0.35% water samples were analyzed in triplicate without dilution.
Details on mating procedure:
On day 127 of the study, the following pairs (20 pairs per group) were established: high-dose-level males with control females, control males with high-dose-level females, and control males with control females. All pairs were housed for 1 week or until a copulatory plug was detected, whichever came first. DEG administration was discontinued during the cohabitation period, then reinstated to the individually housed anirnals. The resulting litters were assessed for the same end points. At the end of task 3, the F0 parents were killed and necropsied.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Exposure period: 7 days before, during and 21 days after a 98-day cohabitation stage
Premating exposure period (males): at least 7 days
Premating exposure period (females): at least 7 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0.35 other: % (nominal in water)
Remarks:
Equivalent to 612 mg/kg bw/day
Dose / conc.:
1.75 other: % (nominal in water)
Remarks:
Equivalent to 3063 mg/kg bw/day
Dose / conc.:
3.5 other: % (nominal in water)
Remarks:
Equivalent to 6125 mg/kg bw/day
No. of animals per sex per dose:
Task 1: 8 females and 8 males
Task 2: 40 males and 40 females (untreated control) and 20 males and 20 females into each of three dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose finding study: Eight-week-old mice were housed four per cage by sex, and eight females and eight males were assigned to each treatment group. The dose levels of DEG tested were 0, 1, 2.5, 5, 7.5 (0, 750, 1875, 3750, 5625 mg/kg bw/d), and 7500 mg/kg bw in the drinking water. All dosing solutions were available ad libitum for a period of 14 days after which the animals were euthanized. The endpoints measured were body weight gain, water consumption, and percentage mortality. The animals also were examined twice daily for clinical signs of toxicity. On the basis of these results, doses were selected for further studies.
- Breeding study: Eleven-week-old mice were als, while being continously exposed to the chemical. The pairs then were separated a located into four treatment groups as follows: 40 males and 20 females into each of three dose males and 40 females (untreated control) and 20 males per group of the chemical under test. For DEG these doses were 610, 3060, and 6130 mg/kg bw. Females and males from the same dose group were paired, housed one breeding pair per cage, and cohabited for 98 day and the male and female mice were housed individually and exposed to the chemical for a further three weeks. During this 119-day cohabitation/postcohabitation period, the day of delivery of each litter, the number of litters produced per breeding pair, the number and percentage of fertile pairs, the number, percentage, and sex of life pups per litter, and the mean body weights of live offspring were recorded. In addition, water consumption, parental body weights, mortality, and clinical signs of toxicity, were evaluated. The final litters were reared and weaned at day 21 (all other litters were euthanized at birth) for assessment in furter study (offspring assessment).
- Crossover mating: On day 127 of the study, the following pairs (20 pairs per group) were established: high-dose-level males with control females, control males with high dose females, and control males with control females. All pairs were housed for 1 week or until a copulatory plug was detected, whichever came first. DEG administration was discontinued during the cohabitation period, then reinstated to the individually housed animals. The resulting litters were assessed for the same end points as described above. At the end the F0 parents were killed and necropsied.
- Offspring assessment: The F1 generation from the breeding study for 0, 3060 mg/kg bw DEG was reared, continously treated, and at 74 +/- 10 days of age paired with nonsiblings from the same dose group. These animals continued on treatment and were cohabited either for 1 week or until a copulatory plug was detected. The litters produced were evaluated as described previously for the breeding study. The F1 parents were euthanized and necropsied.

Examinations

Parental animals: Observations and examinations:
Water consumption, parental body weights, mortality and clinical signs of toxicity were evaluated.
Oestrous cyclicity (parental animals):
yes
Sperm parameters (parental animals):
The sperm concentration, percentage of motile sperm and percentage of abnormal sperms were evaluated.
Litter observations:
The day of delivery of each litter, the number of litters produced per breeding pair, the number and percentage of fertile pairs, the number, percentage, and sex of life pups per litter, and the mean body weights of live offspring were recorded.
Postmortem examinations (parental animals):
At the end the F0 parents were sacrificed and necropsied.
Postmortem examinations (offspring):
The F1 parents were euthanized and necropsied.
Statistics:
The percentage body weight gain was analyzed by a two-way analysis of variance and Duncan's multiple range test. The percentage mortality was assessed by the X2 test.

The Cochran-Armitage test (Armitage, 1971) was used to evaluate any dose-related trends in fertility. The cumulative days between litters were assessed by williams' test.The X2 test of homogeneity was used to determine any difference in fertility among groups. Pairwise comparisons were made using Fisher' exact test (Fisher, 1934).

A Kruskal -Wallis analysis of variance (Kruskal and Wallis . 1952) on ranks was used to compare the number of litters per breeding pair and the number and sex of live pups among treatment groups . The Wilcoxon-Mann- Whitney U test (Mann and Whitney, 1947) was then used to make intergroup pairwise comparisons. Ordered differences were tested for by Jonckheere's test (Jonckeere. 1954).

To correct for the potential effect of the number of pups per litter on the average pup weight, an analysis of covariance (Neter and Wasserman, 1974) was (covariate = average litter size of live and dead pups). The least-squares group means generated this way were tested for overall and pairwise equalities by the F and t tests, respectively.

Similarly, organ weights were adjusted for body at the time of necropsy. The means estimated and tested for equality by the covariance methods described above for the pup body weights. Unadjusted organ weights were analyzed by the Kruskal-Wallis and Table I shows the effects of DEG on the Wilcoxon-Mann-Whitney U tests .

To control for possible sex differences, where applicable, the analyses were performed on males, females, and on the sexes combined. Pairs in which one or both partners died during the study were excluded from the statistical analysis. All differences were considered statistically significant at the p < 0 .05 level.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Dose finding study: No clinical signs of toxicity were observed in any animals in the control or 750 and 1875 mg/kg bw DEG groups. Piloerection, tremors, and lethargy were evident for the male mice at 5625 and 7500 mg/kg bw DEG and the females at the highest dose only.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Dose finding study: In both the 5625 and 7500 dose groups, 3/8 of the male died, whereas 2/8 of the females died in the 7500 mg/kg bw DEG group.
- Breeding study: During this test a total of 10 animals died: 2 control males and 2 control females; 1 female in the 3062 mg/kg bw group, and 2 and 3 males in the 3062 and 6125 mg/kg bw DEG groups, respectively. The random distribution of deaths suggests that they were unlikely to be treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Dose finding study: The percentage body weight gain for the sexes combined was significantly depressed for the 3750, 5625, and 7500 mg/kg bw DEG groups relative to all of the lower levels.
- Crossover mating: At the end of this test the parental animals (F0 of breeding study) were necropsied. For the male mice there were no significant differences in the body or organ weights, either absolute or adjusted for body weight. The mean body weight of the F0 females exposed to 3.5% DEG was significantly decreased relative to the control females. The magnitude of this decrease was approximately 7%.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- Dose finding study: The doses of DEG given in the drinking water were 0, 750, 1875, 3750, 5625, 7500 mg/kg bw. The daily water consumption was significantly decreased for the males in the 3750 and 7500 mg/kg bw DEG groups. The lowest dose level at which dehydration was noted was 3750 for males and 5625 mg/kg bw for females.
- Breeding study: The animals given 1.75 or 3.5% DEG consumed significantly more water than the controls. On the basis of the water consumption and body weight data, 0, 0.35, 1.75, and 3.5% DEG in the drinking water represented an average daily intake of 0, 612, 3062, and 6125 mg DEG /kg bw, respectively.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Crossover mating: At the end of this test the parental animals (F0 of breeding study) were necropsied. For the male mice there were no significant differences in the body or organ weights, either absolute or adjusted for body weight. Female animals exhibited significantly decreased absolute liver and pituitary weights, but their organ-to body weight ratios were not different from controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Crossover mating: There were no significant treatment-related gross or histopathological lesions in the organs examined from the male and female F0 mice.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- Crossover mating: There were no significant treatment-related gross or histopathological lesions in the organs examined from the male and female F0 mice.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- Crossover mating: Analysis of the cauda epididymal contents of F0 males at necropsy indicated that there were no effects of DEG (at 3.5%) on the sperm concentration or the percentage of motile or abnormal sperm.
Reproductive performance:
no effects observed
Description (incidence and severity):
- Crossover mating: There were no significant effects on the mating index or on the fertility of the 6125 mg/kg bw DEG-treated males or females compared with the control mice.

Details on results (P0)

Dose finding study: The doses of DEG given in the drinking water were 0, 750, 1875, 3750, 5625, 7500 mg/kg bw. No clinical signs of toxicity were observed in any animals in the control or 750 and 1875 mg/kg bw DEG groups. The daily water consumption was significantly decreased for the males in the 3750 and 7500 mg/kg bw DEG groups. The lowest dose level at which dehydration was noted was 3750 for males and 5625 mg/kg bw for females. Piloerection, tremors, and lethargy were evident for the male mice at 5625 and 7500 mg/kg bw DEG and the females at the highest dose only. The percentage body weight gain for the sexes combined was significantly depressed for the 3750, 5625, and 7500 mg/kg bw DEG groups relative to all of the lower levels. In both the 5625 and 7500 dose groups, 3/8 of the male died, whereas 2/8 of the females died in the 7500 mg/kg bw DEG group. On the basis of these data, drinking water concentrations of 0, 6102, 3062, and 6125 mg/kg bw DEG were selected for the 126 day exposure period.
Breeding study: The animals given 1.75 or 3.5% DEG consumed significantly more water than the controls. On the basis of the water consumption and body weight data, 0, 0.35, 1.75, and 3.5% DEG in the drinking water represented an average daily intake of 0, 612, 3062, and 6125 mg DEG /kg bw, respectively. During this test a total of 10 animals died: 2 control males and 2 control females; 1 female in the 3062 mg/kg bw group, and 2 and 3 males in the 3062 and 6125 mg/kg bw DEG groups, respectively. The random distribution of deaths suggests that they were unlikely to be treatment related. At 6125 mg/kg DEG, there was a significant decrease in the number of litters produced per pair, live pups per litter, proportion of pups born alive, and the absolute and adjusted live pup weight. A significant dose-related trend was also noted for the absolute live pup weight. Exposure of mice to 6125 mg/kg bw DEG also resulted in a significant increase in the cumulative days to litter. It is also of interest to note that at the highest dose of DEG fewer breeding pairs were able to produce litters; only 82, 76, and 59% of the pairs exposed to 6125 mg/kg bw DEG produced the third, fourth, and fifth litter, respectively, whereas 97-100% of the control mice produced such litters.
Crossover mating: There were no significant effects on the mating index or on the fertility of the 6125 mg/kg bw DEG-treated males or females compared with the control mice. The only end point in this task that was significantly affected was live pup weight, in which a 9% decrease was observed for the offspring of the control males and 6125 mg/kg bw DEG females. At the end of this test the parental animals (F0 of breeding study) were necropsied. For the male mice there were no significant differences in the body or organ weights, either absolute or adjusted for body weight. Analysis of the cauda epididymal contents of F0 males at necropsy indicated that there were no effects of DEG (at 3.5%) on the sperm concentration or the percentage of motile or abnormal sperm. The mean body weight of the F0 females exposed to 3.5% DEG was significantly decreased relative to the control females. The magnitude of this decrease was approximately 7%. These animals also exhibited significantly decreased absolute liver and pituitary weights, but their organ-to body weight ratios were not different from controls. There were no significant treatment-related gross or histopathological lesions in the organs examined from the male and female F0 mice.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
3 060 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Offspring assessment: The final litters from the 6125 mg/kg bw DEG group in the breeding study consisted of fewer live pups than the control litters. In addition, these pups, which had been exposed to 6125 mg/kg bw DEG in utero, had significantly depressed birth weights. For this litters from the breeding study, 12% (14/114) of the live-born pups and 95% (18/19) of the pups found dead on postnatal day 0 had craniofacial malformations including anencephaly and cleft palate. Furthermore, 50% (7/14) of the malformed live pups died by PN2. Similar malformations were also noted for live and dead pups in the other litters exposed to 6125 mg/kg bw DEG during breeding study. Hence, the mid dose (3062 mg/kg bw DEG) litters had to be used for evaluation of fertility and reproduction in the F1 generation. The mean body weights of the F1 generation after selection for this study at weaning (PN21) and mating (PN74) but not at birth, exhibited a significant decrease compared to controls. These percentages of decrease were larger at PN21 (14% for males and 11% for females) than at PN74 (11% for males and 8% for females). Continuous exposure of the F1 mice to 3062 mg/kg bw DEG in the drinking water indirectly, in utero and/or via lactation exposure, and directly from weaning to 74 +/- 10 days of age had no statistically significant effects on mating, fertility, or any of the reproductive parameters measured, relative to control animals. Three weeks after the end of the cohabitation period the F1 mice were subjected to necropsy. The body weights for the treated males and females were still significantly decreased compared with their control counterparts (11% and 7%, respectively). There were no significant differences in the absolute organ weights for either males or females. However, when adjusted for body weight there was a significant increase (11%) in liver weight for male mice. Sperm assessment indicated no significant differences in the sperm concentration or the percentage of motile or abnormal sperm in the cauda epididymis between male mice exposed to 0 or 3062 mg/kg bw DEG in the drinking water. In addition, there were no significant gross or histopathological changes detected in the organs examined from F1 male or female mice exposed to DEG.

Effect levels (F1)

Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Diethylene glycol as given in the drinking water to male and female mice in this experiment did not produce changes in the male or female reproduction tracts.