Registration Dossier

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
limited documentation; no examination of oestrus cycle or sperm parameters
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethane-1,2-diol
EC Number:
203-473-3
EC Name:
Ethane-1,2-diol
Cas Number:
107-21-1
Molecular formula:
C2H6O2
IUPAC Name:
Ethane-1,2-diol
Details on test material:
- Name of test material (as cited in study report): Ethylene glycol (EG) or 1,2-ethanediol
- Physical state: clear, odorless, colorless liquid
- Analytical purity: 99.82%
- Impurities (identity and concentrations): 0.18% diethylene glycol
- Purity test date: 1978-03-27
- Source: Union Carbide Corporation, Hahnville, Louisiana, USA

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, USA
- Age at study initiation: 7 weeks
- Housing: animals were housed in groups of 2 in stainless-steel wire cages.
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): fresh diet was prepared every 2 weeks with the percentage of ethylene glycol adjusted, based on the group mean body weight and food consumption, to maintain a relatively constant dosage level.

Details on mating procedure:
- M/F ratio per cage: at approximately 100 days of age, 10 males were mated to 20 females in each dosage group (1/2)
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
through 3 generations (not further information)
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
- F1 parental animals not mated until 100 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 100 days of age.
- Age at mating of the mated animals in the study: 100 days
- Brother and sister matings were avoided for each generation.
- The F1 and F2 rats were treated as described for the P animals until approximately 100 days of age, at which time the animals were cohabited.
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 200 and 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
10 males, 20 females
Control animals:
yes, concurrent no treatment
Details on study design:
- Other: the concentration of ethylene glycol in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and threefold during the second and third weeks of lactation, respectively, to adjust for increased food consumption by the dams. This change in concentration was based on earlier unpublished results from the laboratory. Increased food consumption during lactation has since been reported in another study performed at the laboratory (Yang et al., 1984).

- Two untreated diet control groups were included to estimate the variation between two groups treated alike.
Positive control:
In the dominant lethal study, 15 male F2 rats per dosage group from the reproduction study were removed from their ethylene glycol dosing regimen at 155 days of age and bred to three separate sets (one set per week) of 15 untreated females. Each female was killed on Day 12 of gestation, and the ovaries and uteri were examined for the number of live and dead fetuses. In addition, 15 diet control F2 males were given an ip dose of 0.50 g/kg of triethylenemelamine (TEM, Polysciences, Inc., Warrington, Pa.) on the day before mating (positive controls), and mated with three groups of females as previously described.

Examinations

Parental animals: Observations and examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: weekly except during gestation and lactation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly calculated dosage except during gestation and lactation
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1/F2/F3] offspring: the date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behaviour of dams and pups were observed daily. Offspring were weighed as litters at 4 and 14 days and individually at 21 days postpartum, the day they were weaned. Litter size was randomly reduced to 10, if necessary, on Day 4 postpartum. F1 rats were randomly selected within each dosage group for the next mating. Each litter was represented except for those conceived very late in the mating period.
Postmortem examinations (parental animals):
SACRIFICE
- Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings

GROSS NECROPSY
- Gross necropsy consisted of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examinations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries
Postmortem examinations (offspring):
SACRIFICE
- Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings

GROSS NECROPSY
- Gross necropsy consisted of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries

HISTOPATHOLOGY / ORGAN WEIGHTS
Microscopic examinations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries
Statistics:
Continuous data such as body weights were compared by analysis of variance validated by Bartlett’s test for homogeneity of variance. Duncan’s multiple range test was used to identify individual mean differences when indicated by a significant F value. Where Bartlett’s test indicated heterogeneous variances tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the x2 test and by exact test.
Reproductive indices:
The following reproductive indices were calculated and evaluated statistically by the previously described nonparametric methods: fertility index (male and female), days from first mating to parturition, gestation index, fraction of pregnancies that resulted in litters with live pups
Offspring viability indices:
Gestation survival index (fraction of newborn pups alive at birth), 0 to 4day survival index, 4 to 14 day survival index, 4 to 21-day survival index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No effects were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effects were observed.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The calculated dosages based on the nominal concentrations of EG in the diet were generally very close to the dosage goals. The weekly calculated dosages ranged from 1.0 to 1.3, 0.2 to 0.3,and 0.04 to 0.05 g/kg/day for males and from 0.9 to 1.2, 0.2 to 0.3, and 0.04 to 0.06 g/kg/day for females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No effects were observed.

OTHER FINDINGS (PARENTAL ANIMALS)
Ethylene glycol administration did not lead to statistically significant adverse effects on any of the parameters measured in the three mating intervals. Slight apparent increases in the dominant lethal mutation index, observed during the Week 2 mating for the high-dose (1000 mg/kg bw/day) group and during the Week 3 mating for the low dose (0.04 g/kg bw/day) group, were probably random occurrences unrelated to EG treatment. This interpretation is consistent with the absence of a dose-response relationship and the fact that a negative index of similar magnitude (-8.2%) was observed for the low dose group in the Week 2 mating. When compared to the combined control groups, significant decreases were observed as a result of TEM treatment for the number of females with implants, the total number of implants and the number of live implants.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
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)

VIABILITY (OFFSPRING):
There was no mortality of pups in any generation.

CLINICAL SIGNS (OFFSPRING):
No clinical signs had been observed.

BODY WEIGHT (OFFSPRING):
Treatment did not affect neonatal body weight at Days 4, 14 and 21 postpartum.

SEXUAL MATURATION (OFFSPRING):
The fertility inidces of any generation were not changed.

ORGAN WEIGHTS (OFFSPRING):
There were no treatment related findings in any weanlings.

GROSS PATHOLOGY (OFFSPRING):
No effects were observed.

HISTOPATHOLOGY (OFFSPRING):
There were no treatment related histopathological findings in F2 parents or F3 weanlings

OTHER FINDINGS (OFFSPRING):
The fertility index for male and female offspring, the days from first mating to parturition, the gestation index and the fraction of pregnancies that resulted in litters with live pups were not affected by treatment in any dose group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion