Reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Data on the components of the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade, diethylene glycol (DEG), triethylene glycol (TEG), and tetraethylene glycol (TTEG), were used to assess its reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: fertility

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study.

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

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

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system 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

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.

In Task 3, control and high dose parental animals were cross-mated

In task 4, the control and mid-dose offspring from the final Task 2 litters were mated.

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

Animals were not treated during cohabitation in cross-mating phases (Tasks 3 and 4).

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0%, 0.35%, 1.75% and 3.5% (= 0, 612, 3063, 6125 mg/kg bw)
Basis:
nominal in water

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
Task 3: 20 pairs/group
Task 4: 20 pairs/group

Control animals:

yes

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.

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.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

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 with parents from breeding study (Task 3): 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.

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

Crossover mating of F1 control and mid dose pups (Task 4): No significant effects were observed in the fertility or litter analysis. Body weight at necropsy was significantly decreased in the mid dose group of both sexes. Relative to controls, mid dose males weighed 11% less and mid dose females weighed 7% less. mid dose males were found to have significantly increased (by 11%) liver weights. There were no significant changes in female organ weights. No effect on any sperm parameters were observed.

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

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 exencephaly 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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 063 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

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.

Executive summary:

Diethylene glycol (DEG) was administered via the drinking water to 40 male and 40 female mice at concentrations of 0, 0.35, 1.75, and 3.5% (equivalent to approximately 0, 612, 3063, and 6125 mg/kg/day) for 7 days premating, a 98 day cohabitation period, and 21 days after cohabitation (Task 2). Task 3 was performed by cross-mating the control and high dose Task 2 parental animals. In Task 4, the control and mid-dose offspring from the final Task 2 litters were mated.

Exposure to diethylene glycol at levels up to 3.5% produced no significant adverse effects on fertility in Tasks 2, 3, or 4. Results from the Task 2 litter analyses show the high dose group to have a significantly decreased number of litters, with decreased litter size and adjusted pup weight. No significant differences were observed in the low or mid-dose groups.

In Task 3, the only significant result observed in the litter analysis was a decrease in mean pup weight in the control male-high dose female group. At necropsy, body weight in high dose females was decreased by 7% (p<0.01). No significant differences in adjusted organ weights were found for either sex.

In Task 4, no significant effects were observed in fertility or litter analysis. Body weight at necropsy was significantly decreased in the mid-dose group of both sexes. Relative to controls, mid-dose males weighed 11% less and mid-dose females weighed 7% less. Mid-dose males were found to have significantly increased (by 11%) liver weights. There were no significant changes in female organ weights. No effect on any of the sperm parameters was observed in Task 3 or 4.The NOAEL for fertility and reproductive toxicity was 6125 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 063 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

The database includes continuous breeding studies conducted on diethylene glycol and triethylene glycol and repeat dose toxicity data on tetraethylene glycol and is sufficiently robust to draw conclusions regarding reproductive toxicity of these compounds as a formulation.

Additional information

In the 2-generation key study, a Fertility Assessment by Continuous Breeding study (National Toxicology Program, 1984), diethylene glycol (DEG) was administered via the drinking water to 40 male and 40 female mice at concentrations of 0, 0.35, 1.75, and 3.5% (equivalent to approximately 0, 612, 3063, and 6125 mg/kg/day) for 7 days premating, a 98 day cohabitation period, and 21 days after cohabitation (study Task 2). Task 3 was performed by cross-mating the control and high dose Task 2 parental animals. In Task 4, the control and mid-dose offspring from the final Task 2 litters were mated. Exposure to DEG at levels up to 3.5% produced no significant adverse effects on fertility in Tasks 2, 3, or 4. Results from the Task 2 litter analyses show the high dose group to have a significantly decreased number of litters, with decreased litter size and adjusted pup weight. No significant differences were observed in the low or mid-dose groups. In Task 3, the only significant result observed in the litter analysis was a decrease in mean pup weight in the control male-high dose female breeding group. At necropsy, body weight in high dose females was decreased by 7% (p<0.01). No significant differences in adjusted organ weights were found for either sex. In Task 4, no significant effects were observed in fertility or litter analysis. Body weight at necropsy was significantly decreased in the mid-dose group of both sexes. Relative to controls, mid-dose males weighed 11% less and mid-dose females weighed 7% less. Mid-dose males were found to have significantly increased (by 11%) liver weights. There were no significant changes in female organ weights. No effect on any of the sperm parameters was observed in Task 3 or 4.The NOAEL for fertility and reproductive toxicity was 3063 mg/kg/day.

Several supporting studies describe no adverse effects of DEG, triethylene glycol (TEG), or tetraethylene glycol (TTEG) on fertility. First, Wegener (1953) reported no impairment of reproduction, fertility, litter size or embryotoxicity when 10 male and 10 female rats received 2 ml/kg bw or approximately 2200 mg/kg bw/day DEG by oral gavage over 8 weeks, were mated and 5 females were exposed through gestation and 5 others were exposed through delivery and weaning. Subsequently, 10 male and 10 female albino rats of the F1 generation were mated without observed effects on fertility, litter size or development of the offspring. The NOAEL for reproductive toxicity and embryotoxicity was 2200 mg/kg bw/day.

Second, in a Reproductive Assessment by Continuous Breeding study with TEG (Bossert et al., 1992), groups of Swiss CD-1 mice were exposed to 0, 0.3, 1.5, and 3% (equivalent to 0, 590, 3300, and 6780 mg/kg bw/day) TEG in the drinking water during a 98-day cohabitation period. No reproductive toxicity in either generation was observed at concentrations up to 3%. Reduced pup body weight was observed at the mid and high dose levels in F1 offspring only. No effect on pup body weight was seen in F2 offspring. No effect of treatment on the proportion of pairs with a litter, the number of litters per pair, the number of live pups per litter or the proportion of live born pups in the study. Therefore, the NOAEL for parental toxicity and for fertility was 6780 mg/kg bw/day.

In a 28 day drinking water study, 2000 mg/kg/day TTEG produced no evidence of reproductive effects in Wistar rats based on testicular and ovarian weights and gross and histopathological examination of reproductive organs (Schladt et al., 1998).

Additionally, TTEG was negative in the reproductive rodent dominant lethal assay performed by Bushy Run Research Center (1993). In the study, male rats were treated for five days with 5000, 25000 or 50000 ppm TTEG in the drinking water and subsequently mated to naïve females over ten weeks, with the females replaced weekly. Treatment produced evidence of toxicity in males at the high dose and urinary findings consistent with an osmotic diuresis at 25000 and 50000 ppm; however, no indications of subsequent reproductive or gestational effects were observed, including no significant pre-implantation loss over the ten week period. The NOEL for general toxicity was 25000 ppm.

Based on these component data, the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade is not expected to produce adverse effects on fertility and reproduction.

Justification for selection of reproductive toxicity – oral route
The NOAEL for reproductive toxicity is based on the murine continuous breeding study for DEG, the shortest component of this reaction mass. Although direct reproductive toxicity data are not available for TTEG, the lack of findings with the lower ethylene glycols supports conclusion of low potential of reproductive toxicity and effect on fertility across this class of compounds.

Effects on developmental toxicity

Description of key information

Data on the components of the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade, diethylene glycol (DEG) and triethylene glycol (TEG), were used to assess its developmental toxicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: the sexually mature, virgin Himalayan rabbits (Chbb: HM (outbred strain)) were supplied by Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 24 and 29 weeks old
- Weight at study initiation: mean body weight of pregnant animals only, ca. 2560 g (calculated from the means of the groups)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance was administered to the animals orally (by gavage) once a day during the period of major organogenesis (day 7 to day 19 p.i.) always at approx. the same time of day (in the morning). The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.). On day 29 p.i. all animals were sacrificed and examined macroscopically. The fetuses were dissected from the uterus, and further investigated with different methods. The test substance solutions were prepared only once for each study section because the stability of the test substance solution over a period of 21 days had been proven before treatment of the animals began. For the preparation of the solutions an appropriate amount of the test substance was weighed and subsequently dissolved in doubly distilled water. Due to technical reasons the study was carried out in 3 sections. Each dose group was represented in each section. A treatment interval of 7 days elapsed before the next section.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance solutions over a period of 21 days could be demonstrated. The results of the analyses of the solutions of test substance confirmed the correctness of the prepared concentrations.
On the basis of the duration of use and the analytical findings the rabbit food used was found to be suitable. Federal Register, Vol. 44, No. 91, of May 9, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable contaminants.
On the basis of the analytical findings, the drinking water was found to be suitable. German Drinking Water Regulation of May 22, 1986 served as a guideline for maximum tolerable contaminants.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination.
This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females.
The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).

Duration of treatment / exposure:

gestation day 7 - 19

Frequency of treatment:

daily

Duration of test:

30 days

No. of animals per sex per dose:

15

Control animals:

yes, sham-exposed

Details on study design:

Based on the results of a dose range-finding study, the following doses were selected for the prenatal toxicity study in rabbits:
- 100 mg/kg bw: should constitute the expected no observable effect level
- 400 mg/kg bw: with this dose marginal influences on dams and/or fetuses could not be ruled out
- 1000 mg/kg bw: with this dose signs of maternal toxicity (e.g. retarded body weight gain) may possibly have occurred. A higher dose level was not deemed to be necessary due to the recommendations in the test guidelines, which had to be taken into account.

Maternal examinations:

The consumption of food was determined daily during the entire study period. All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i.. The body weight change of the animals was calculated from these results. Furthermore, after terminal sacrifice the corrected body weight gain was calculated (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened). The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited. Mortality: A check was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).
Examinations of the dams at termination: On day 29 p.i. the dams were sacrificed by intravenous injection of a pentobarbital and the fetuses were dissected from the uterus. After the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded: - Weight of uterus before it was opened - Number of corpora lutea - Number and distribution of implantation sites classified as: --live fetuses --dead implantations: a) early resorptions (only decidual or placental tissues visible or from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy) b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible) c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Examination of the fetuses after dissection from the uterus: At necropsy each fetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded. Soft tissue examination of the fetuses: After the fetuses had been sacrificed by C02, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed . The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the fetuses was determined by internal examination of the gonads. If heads of fetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these fetuses were severed from the trunk, fixed in BOUIN´s solution and later processed and assessed according to WIISON´s method. About 10 transverse sections were prepared per head. Skeletal examination of the fetuses: After the soft tissue examination all fetuses were placed in ethyl alcohol for staining of the skeletons. The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed.

Statistics:

The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft (laboratory data processing, responsible:Dr. H.D. Hoffmann). Examinations of dams and fetuses: Dunnett's Test was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation loss, resorptions and live fetuses. Fisher´s Exact Test was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings. Significances resulting from these tests have been indicated in the tables (a for p < 0 .05, b for p < 0 .01).

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Food consumption of the substance-treated does was not influenced by the test substance administration. The observable differences between the control animals and the dams of test groups 1 - 3 (100, 400 and 1,000 mg/kg body weight/day), including the statistically significantly increased food consumption in the highest dose group on days 6 - 7 p.i. (pretreatment period), are without biological significance. There were no adverse effects on body weights or body weight changes which could be attributed to the test substance administration. All values are within the range of biological variation or of spontaneous nature, including the statistically significant decrease in mean body weight change of the intermediate dose females during days 0 - 29 p.i. (whole study period). The results of the corrected body weight gain (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened) do not clearly show any dose-related differences between the groups. The only statistically significant difference in comparison to the controls, the low value in test group 2 (400 mg/kg body weight/day), which is mainly caused by 2 does, is without biological relevance. Except one doe of test group 1 (100 mg/kg body weight/day), which showed a marked edema in the anogenital region during days 16 - 29 p.i. and one animal of test group 2 (400 mg/kg body weight/day), which had an accidental lesion of the left hindlimb (days 17 - 29 p.i.), there were no remarkable clinical observations in any doe of all groups. Both recorded clinical observations are spontaneous ones and are not related to the test substance administration. There were no mortalities.
There were no substantial differences concerning the uterus weights between the controls and the substance-treated groups. All values lie within the range of biological variation. Due to a technical error, the uterus weight of doe of test group 3 (1,000 mg/kg body weight/day) was not recorded.
The conception rate varied between 93 and 100%. Concerning all groups, there were no substance-related and/or statistically significant differences in the conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation loss, the number of resorptions or viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age. This includes the one doe of test group 1 (100 mg/kg body weight/day) which did not become pregnant due to a hydrometra in the one and a blind ending uterine on the other side.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The sex distribution in test groups 1 - 3 (100 - 1,000 mg/kg body weight/day) was comparable with the control group. The observable differences are without any biological relevance. The mean placental weights in the substance-treated groups (100, 400 and 1,000 mg/kg body weight/day) were not substantially influenced. The differences observed in comparison to the control are without biological relevance and lie within the range of biological variation. There were no clearly dose-related or statistically significant differences between the groups concerning the mean fetal weights. All values are still within the range of biological variation; therefore the observable differences between the control and the substance-treated groups are assessed as to be of spontaneous nature. The external examination of the fetuses revealed no malformations in any group and only one kind of variation (pseudoankylosis) in 2 fetuses each of the control group, of test groups 2 (400 mg/kg body weight/day) and 3 (1,000 mg/kg body weight/day). There were no so-called unclassified observations (like placentae fused) in any fetus. The examination of the organs of the fetuses revealed two types of malformations. One high dose fetus showed a septal defect, a rather common finding in the rabbit strain used; this malformation is also present in the historical control data to about the same extent. Furthermore, two control fetuses and one fetus of the 400 mg/kg group had an agenesia of the gallbladder. Variations were detected in each group including the control. The very common finding (separated origin of carotids) in the rabbit strain used in this study occurred without a clear dose-response relationship, but was more frequently seen in the substance-treated groups, the differences in relation to the control being statistically significant ; however, if the relevant values of the substance-treated groups are compared with the corresponding historical control values, these values are within the range of biological variation. Therefore, it becomes obvious, that the number of control fetuses with this finding is unexpectedly low. Thestatistically significantly increased number of fetuses of test groups 1 - 3 (100, 400 and 1,000 mg/kg body weight/day) is therefore assessed as to be of incidental nature. The other variations (hypoplasia of gallbladder, dilated renal pelvis, ovary bipartite) occur without a dose-response relationship and/or are to be found in the same kind of magnitude in the historical control data. Moreover, several fetuses out of test groups 0 - 3 (0, 100, 400 or 1,000 mg/kg body weight/day) showed focal liver necrosis or blood coagula around the bladder (socalled unclassified observations). Various malformations of the ribs and/or the vertebral column were seen in 1 fetus of the control and the 400 mg/kg group and in 3 fetuses of the 1,000 mg/kg group. No other skeletal malformations were recorded for any group. The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal and frontal bones), the ribs (accessory ribs, flying ribs, or rudimentary cervical ribs), the vertebral column (accessory thoracic vertebra) and the sternum (sternebra(e) of irregular shape, fused or bipartite) and were found in all groups without a clear dose-response relationship and/or without any statistically significant differences between the groups. In all groups signs of retardations (incomplete or missing ossification of skull bones, sternebra(e), and/or talus) were found; they occurred without any differences of biological relevance between the groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In conclusion, the oral administration of diethylene glycol to pregnant Himalayan rabbits by stomach tube on day 7 through day 19 p.i. in dosages of 100, 400 and 1,000 mg/kg body weight/day led to no adverse effects which can be causally related to the test substance administration in both the does and in the fetuses. The observable differences between the control group and the substance-treated groups appeared either without a clear dose-response relationship and/or were assessed as being without biological relevance, because the relevant values/findings are to be found in a similar range within the historical control data.
Summarized, diethylene glycol caused, under the conditions of this study, up to and including a dose of 1,000 mg/kg body weight/day no signs of maternal toxicity and no signs of embryo-/fetotoxicity; especially no teratogenic effects could be detected. As to the OECD GUIDELINE for testing of chemicals (No. 414, adopted May 12, 1981) in the case of substances of low toxicity (i.e. "if a dose level of at least 1,000 mg/kg body weight/day produces no evidence of embryotoxicity or teratogenicity") further embryotoxicity studies at higher dose levels may not be considered necessary.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The database includes developmental toxicity studies conducted on diethylene glycol and triethylene glycol and is sufficiently robust to draw conclusions regarding reproductive toxicity of these compounds as a formulation.

Additional information

In a developmental toxicity study by Hellwig et al. (1995), Himalayan rabbits were exposed to nominal concentrations of 100, 400, and 1000 mg/kg bw/day diethylene glycol (DEG) by oral gavage on gestation days 7 through 19. No adverse effects attributed to DEG exposure were observed in does or fetuses. Any observed differences between dose groups were without a clear dose response relationship or were within historical control values and, therefore, determined not biologically relevant. The NOAEL for maternal toxicity, embryotoxicity and fetotoxicity were the high dose of 1000 mg/kg bw/day. This is the key study because rabbits are considered the most relevant species for hazard assessment of this chemistry.

 

In several supporting studies, developmental toxicity was also assessed in mice and rats exposed to DEG during gestation. CD-1 mice received doses of 0, 0.5, 2.5, or 10 ml/kg/day (0, 559, 2795, 11180 mg/kg/day) DEG by oral gavage on gestation days 6 through 15 in a study by Bushy Run Research Center (1992; Ballantyne and Snellings, 2005a). Maternal toxicity at 10 ml/kg/day was characterized by mortality (21%) and clinical signs. No effects on body weight or food consumption were observed at any dose level. Water consumption was increased in the mid- and high-dose exposed animals. Increases in liver and gravid uterine weights at the mid and high doses were not statistically significant, but were considered to be treatment related, as was increased kidney weight at the high dose. Fetal body weights were reduced at 10 ml/kg/day. No treatment related increases in skeletal malformations or variations was observed. The NOEL was 559 mg/kg/day for maternal toxicity and 2795 mg/kg/day for developmental toxicity based on the reduced fetal body weights.

 

In a separate study by the National Toxicology Program (1991), Swiss CD-1 mice received 0, 1.25, 5, or 10 g/kg bw/day DEG by oral gavage on gestation days 6 through 15. Maternal body weights were not affected by any dose level of DEG. Water intake was significantly increased at the mid and high dose levels and food consumption was significantly decreased at the high dose only. Absolute and relative kidney weights were increased in mid and high dose animals at necropsy. Histopathologic examination revealed evidence of DEG-related renal degeneration and morbidity in one high dose animal and renal tubular degeneration in three pregnant high dose animals. No effects on pre- or post-implantation loss were observed. Fetal body weight was statistically significantly decreased at the high dose (85% of control). No increase in skeletal malformations was observed at any dose level. The developmental NOAEL was considered to be 5000 mg/kg bw/day, a dose level that produced significant maternal toxicity. The maternal NOAEL was 1250 mg/kg bw/day.

 

Sprague-Dawley rats received doses of 0, 1.0, 4.0, or 8.0 ml/kg/day (0, 1118, 4472, and 8944 mg/kg/day) DEG by oral gavage on days 6 through 15 of gestation in a study by Bushy Run Research Center (1992; Ballantyne and Snellings 2005a). Mortality (12%), decreased body weight and body weight gain, and clinical signs were observed in high dose dams. The three dams who died all showed evidence of moderate to severe microscopic kidney lesions consisting of tubular vacuolization and proteinosis. Food consumption was decreased and water consumption was increased at the 4.0 and 8.0 ml/kg/day dose levels. In high dose females, kidney and liver weights were increased at scheduled sacrifice on gestation day 21 and histopathologic examination showed an increased incidence of basophilia and interstitial nephritis indicating repair of damaged renal tubules. Fetal body weights were significantly reduced in the 8.0 ml/kg/day group. An increased incidence of individual skeletal variations was also observed in mid and high dose fetuses, but the pattern of delayed ossifications was consistent with reduced fetal body weight. The NOEL for both maternal and developmental toxicity was 1118 mg/kg bw/day.

 

Developmental toxicity has also been assessed in mice and rats exposed to TEG (triethylene glycol). In a supporting study, CD-1 mice received doses of 0.5, 5, and 10 ml/kg/day by oral gavage (equivalent to 563, 5630, and 11260 mg/kg bw/day) on days 6 through 15 of gestation (Bushy Run Research Center, 1990; Ballantyne and Snellings 2005b; Dow 2015). Clinical signs observed at 10 ml/kg/day included hypoactivity and audible and rapid respiration. A treatment related decrease in body weight gain was observed at the 10 ml/kg/day dose level and a treatment related decrease in body weight gain corrected for gravid uterine weight was observed at 5 and 10 ml/kg/day dose levels (Dow, 2015). Water consumption was increased at the mid and high dose (Dow, 2015). Relative kidney weight was significantly increased at 10.0 ml/kg/day, but no histopathologic changes were observed (BRRC, 1990). Decreased fetal weight and delayed bone ossification observed at 5 and 10 ml/kg/day represent a fetal developmental delay secondary to maternal toxicity (BRRC, 1990). The NOEL for both maternal toxicity and developmental toxicity was 0.5 ml/kg/day (563 mg/kg/day) (Dow, 2015).

 

In another supporting developmental toxicity study by Bushy Run Research Center (1991; Ballantyne and Snellings 2005b), Sprague-Dawley rats received doses of 1, 5, and 10 ml/kg TEG by oral gavage (equivalent to 1126, 5630, and 11260 mg/kg bw/day) on gestation days 6 through 15. Maternal toxicity was characterized by body weight reductions at 5 and 10 ml/kg/day, decreased body weight gain at 10 ml/kg/day, treatment associated clinical signs at 10 ml/kg/day, reduced food consumption at 5 and 10 ml/kg/day, increased water consumption at 5 and 10 ml/kg/day, and increased relative kidney weight with no correlated histopathologic changes at 10 ml/kg/day. Fetal body weights were reduced at 10 ml/kg/day and treatment-related reduced ossification of the thoracic region was observed at this dose level. The NOEL for maternal toxicity was 1 ml/kg/day (1100 mg/kg/day) and the NOEL for developmental toxicity was 5 ml/kg/day (5630 mg/kg/day).

 

Adverse developmental effects related to gestational exposure to DEG and TEG predominantly consisted of decreased fetal weight and related delays in skeletal ossification in rodents. These effects were found secondary to maternal toxicity and in all cases were observed at doses exceeding the current guideline limit dose for developmental toxicity studies. Delayed ossification is not considered adverse as these variations would be fully expected to ossify postnatally and would have no impact on the viability or function of the offspring (Carney and Kimmel, 2007; Marr et al., 1992). Pup weights would also be expected to reach the control levels postnatally (Marr et al., 1992). Because adverse reproductive effects are expected to decrease with increasing number of oxyethylene repeat units, similarly low potential for developmental toxicity is expected for tetraethylene glycol (TTEG). No effects on the development were seen in rabbit with DEG, the most relevant species for human hazard assessment for this chemistry. Therefore, based on these component data, the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade is not expected to produce adverse effects on development.

 

Justification for selection of developmental toxicity – oral route
Adverse reproductive effects are expected to decrease with increasing number of oxyethylene repeat units per molecule in rodents only. These effects seen in the lower ethylene glycols (DEG, TEG) were seen secondary to maternal toxicity and at doses exceeding current limit dose for guideline developmental toxicity studies. No effects on the development were seen in rabbit for this chemistry, the most relevant species for human hazard assessment. Therefore, the NOAEL for developmental toxicity of the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade is conservatively based on the rabbit NOAEL for diethylene glycol.

Justification for classification or non-classification

Classification for the reaction mass of 3,6,9-trioxaundecane-1,11-diol and 2,2'-oxydiethanol and 2,2'-(ethylenedioxy)diethanol and 3,6,9,12-tetraoxatetrade is not warranted based on data on the components, DEG, TEG, and TTEG, showing no concern for developmental and reproductive toxicity.