Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

Toxicity to reproduction

Administrative data

Endpoint:

three-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:

publication

Title:

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

Author:

DePass, L.R., Woodside, M.D., Maronpot, R.R., Weil, C.S.

Year:

1986

Bibliographic source:

Fundamental and Applied Toxicology 7: 566-572

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Version from 1983

Deviations:

not specified

GLP compliance:

no

Limit test:

no

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

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Age at study initiation: young adult nulliparous Fischer 344 rats
- Housing: two per cage in stainless-steel wire cages; during mating, each male was housed with 2 females; after mating and during lactation, the females were housed individually in plastic showbox cages with hardwood chips for nesting.
- Diet: Purina Formulab, ad libitum
- Water: city 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:

Administration of EG to the F0 rats of both sexes began at approximately 7 weeks of age. Fresh diet was prepared every 2 weeks with the percentage of test item (EG) adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level. However, the concentration of EG in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and three-fold 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.

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: individual

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Three generations

Frequency of treatment:

Daily, 7days/week

Details on study schedule:

At approximately 100 days of age, 10 males were added 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 matings were avoided for each generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

Two untreated diet control groups, designated 0.0A and 0.0B, were included to estimate the variation between 2 groups treated alike.

Examinations

Parental animals: Observations and examinations:

Body weights and diet consumption were recorded weekly except during gestation and lactation.

Litter observations:

The date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behavior 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 21 days 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.

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, t 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 Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described non parametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.

Reproductive indices:

yes

Offspring viability indices:

yes

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Based on the nominal concentrations, 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.

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)

Reproductive performance: No treatment-related effect was observed for any of the indices.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: There were no reproductive effects at the highest test dose

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment did not affect neonatal body weight at Days 4, 14 and 21 postpartum.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related histopathologic findings in F2 parents or in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male. In addition, mild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.

Other effects:

no effects observed

Description (incidence and severity):

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)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: There were no adverse effects at the highest test dose.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: There were no adverse effects up to the highest test dose.

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Reproductive Indices

		1.0 g/kg/d	0.2 g/kg/d	0.04 g/kg/d	0.0A	0.0B
F0 -> F1	Fertility index (%)	100	90	100	90	90
	Male	95	90	90	75	90
	Female	100	100	100	100	100
F1 -> F2	Fertility index (%)	100	100	90	90	90
	Male	85	95	85	90	85
	Female	100	100	100	100	94
F2 -> F3	Fertility index (%)	100	90	100	80	80
	Male	90	75	85	80	70
	Female	100	100	100	100	100

Neonatal body weight at day 21

		1.0 g/kg/d	0.2 g/kg/d	0.04 g/kg/d	0.0A	0.0B
F1 pups	males	30.6 +/- 4.5	30.9 +/- 4.9	30.7 +/- 6.4	30.6 +/- 3.6	27.9 +/- 4.3
	females	29.0 +/- 4.5	29.2 +/- 4.5	29.5 +/- 4.7	27.9 +/- 3.3	27.0 +/- 3.5
F2 pups	males	32.8 +/- 3.5	30.9 +/- 5.8	29.3 +/- 4.7	30.0 +/- 4.0	28.8 +/- 4.3
	females	30.8 +/- 3.4	30.2 +/- 4.9	28.8 +/- 3.8	28.5 +/- 3.1	27.5 +/- 3.4
F3 pups	males	30.2 +/- 4.0	30.9 +/- 4.0	30.9 +/- 4.0	32.0 +/- 3.9	30.2 +/- 4.6
	females	28.6 +/- 3.8	28.2 +/- 3.4	29.7 +/- 4.0	30.1 +/- 3.5	27.7 +/- 3.9

Applicant's summary and conclusion

Conclusions:

There were no reproductive effects associated with the inclusion of as much as 1.0 g/kg bw/day of test item in the diet.

Executive summary:

In a three-generation reproduction study in which F344 rats (both sexes) received 40, 200, or 1000 mg ethylane-1,2-diol/kg bw/d in the diet, there were no treatment-related parental effects (based on survival, body weight, food consumption, appearance, behaviour, and histopathology in major organs) or effects on fertility index, gestation index, gestation survival index, pup weight, appearance, behaviour, or histopathology in major organs (DePass et al., 1986).