Glycollic acid

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Study published in 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Three-Generation Reproduction and Dominant Lethal Mutagenesis Studies of Ethylene Glycol in the Rat

Author:

L.R. DePass, M.D. Woodside, R.R. Maronpot & C.S. Well

Year:

1986

Bibliographic source:

Fundamental and Applied Toxicology 7, 566-572 (1986)

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The basis for the study design is the “NTP Fertility Assessment by Continuous Breeding Protocol"

GLP compliance:

not specified

Test material

Test material information

Constituent 1

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:

ethylene glycol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Corporation, Hahnville, Louisiana
- Date of receipt: 22/01/1975
- Description: the sample was 99.93% monoethylene glycol and 0.18% diethylene glycol
- Lot//batch number of test material: Not stated
- Expiration date of the lot/batch: Not stated
- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Assumed stable
- Stability under storage conditions:
Assumed stable
- Stability under test conditions:
Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Not applicable, test item administered as received, no preparation necessary.
- Reactivity of the test substance with the solvent/vehicle/test medium (if applicable): Not applicable, test item administered in diet

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Administered in diet

TYPE OF BIOCIDE FORMULATION (if applicable):

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: None stated

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 Laboratory, Wilmington, Mass.
- Age at study initiation: Young adult male and female nulliparous Fischer 344 rats
- Weight at study initiation: Range: Not stated
- Housing: Two per cage in stainless steel wire cage. During mating each male was hosed with two females. After mating and during lactation the female were housed individually in plastic shoebox cage with hardwood chips for nesting.
- Diet (e.g. ad libitum): Purina Formulab 5008 Chow (Ralston Purina Co. St. Louis, Mo)
- Water: ad libitum
- Acclimation period: Not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): Not stated
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: Not stated

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 2 weeks
- Mixing appropriate amounts with (Type of food): with the percentage of test item adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level.
- Storage temperature of food: Not stated

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: Not stated
- Proof of pregnancy: Not stated
- After days of unsuccessful pairing replacement of first male by another male with proven fertility: Not stated
- Further matings after two unsuccessful attempts: Not stated
- After successful mating each pregnant female was caged (how): Not stated
- Any other deviations from standard protocol: Not stated

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Administration of EG to the F0 rats of both sexes began at approximately 7 weeks of age. At approximately 100 days of age, 10 males were mated to 20 females in each dosage group.

The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister mating were avoided for each generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Untreated diet control B

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day

Remarks:

Untreated diet control A

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

40 mg/kg bw/day

No. of animals per sex per dose:

10 males
20 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not stated
- Rationale for animal assignment (if not random): Random assignment
- Fasting period before blood sampling for clinical biochemistry: Not stated
- Other: Administration of EG to the FO rats of both sexes began at approximately 7 weeks of age. At approximately 100 days of age, 10 males were mated to 20 females in each dosage group. 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. Litter size was randomly reduced to 10, if necessary, on Day 4 postpartum. Offspring were
weighed as litters at 4 and 14 days and individually at 21days postpartum, the day they were weaned. 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. The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister matings were avoided for each generation.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Not stated
- Time schedule: Not stated
- Cage side observations checked in table [No.?] were included. No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Not disclosed

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not stated
OTHER: Dominant lethal study was also performed, 15 males of F2 per dose from reproductive study were removed from their test item 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.

Oestrous cyclicity (parental animals):

No detail on oestrous cycle was reported.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: Testis weight & epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. Yes

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring: Yes

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE: Yes
- Male animals: All surviving animals
- Maternal animals: All surviving animals
GROSS NECROPSY: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

Postmortem examinations (offspring):

SACRIFICE: Yes
- Male animals: All surviving animals
- Maternal animals: All surviving animals
GROSS NECROPSY: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

Statistics:

Continuous data such as bodyweight 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 indicated heterogenous variances, t tests for equal or unequal variances were used to delineate difference between groups. Pup weight were compared by method of Weil (Weil, 1970). Discontinuous data such as implantation and reproductive indices were compared by multiple sum of ranks test. Frequency data were compared by the x2 test and by Fisher's exact test. Reproductive indices [ fertility index (male & female), days from matting to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pup alive at birth) 0 -4 day survival index, 4-14 survival index, 4- 21 day survival index] were calculated and evaluated statistically by nonparametric menthols

Reproductive indices:

Reproductive indices [ fertility index (male & female), days from matting to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pup alive at birth) 0 -4 day survival index, 4-14 survival index, 4- 21 day survival index]

Offspring viability indices:

No particular indices were indicated in the report. However, offspring were examine, weighted as litters at day 4 - 14 and individually at day 21. Survival was also recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The test item 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 (1.0 g/kg/day) group and during the Week 3 mating for the low dose (0.04 g/kg/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 result of TEM treatment for the number of females with implants, the total number of implants and the number of live implants.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

There were no effects on fertility index, mating, implantation, gestation or gross histopathology observed in the liver, kidney, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

other: Mild focal interstitial nephritist in high dose groups and well as control group. Also unilateral hydronephrosis observed in on F2 male high dose group.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the condition reported in this study, there were no reproductive or dominant lethal effects associated with dietary administration of ethylene glycol with no-observed-adverse-effect-level (NOAEL) considered as 1000 mg/kg/day.

Executive summary:

To assess the possible effects of ethylene glycol (EG) on reproductive performance and mutagenesis, three-generation reproduction and dominant lethal mutagenesis studies were performed in the Fischer 344 rat. EG was included in the diet at approximate dosages of 1.0, 0.2, and 0.04 g/kg/day during three generations of reproduction, Each generation was bred once. In a dominant lethal mutagenesis study, the F2 males from the reproduction study were bred to three consecutive lots of untreated females at weekly intervals. Concomitantly, another group of untreated F2 males that received a single ip injection of 0.50 mg/kg triethylenemelamine (TEM) were bred similarly to serve as a positive control group. There was no evidence of increased fetal death was observed in any of the groups receiving. EG produced no effects on fertility, fecundity, or reproductive performance. In addition, there was no evidence of a dominant lethal effect when EG treated males were mated with untreated females.