Ethylene oxide

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

Effects on Reproduction in Fischer 344 Rats Exposed to Ethylene Oxide by Inhalation for One Generation.

Author:

Snellings, W.M., Zelenak, J.P., Weil, C.S.

Year:

1982

Bibliographic source:

Tox. Appl. Pharmacol. 63, 382 - 388

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene oxide

EC Number:

200-849-9

EC Name:

Ethylene oxide

Cas Number:

75-21-8

Molecular formula:

C2H4O

IUPAC Name:

oxirane

Specific details on test material used for the study:

- Physical appearance: gas

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Fischer 344 rats, 3 to 4 weeks of age, were used as the parent generation (F0). At the time of random selection of animals for the study, the only rats accepted were those for which no abnormal clinical signs were observed during the quarantine period and whose body weights were within plus or minus two standard deviations from the mean weight of all of the rats of the same sex. The F0 generation were housed in suspended, wire-mesh stainless steel cages. Pregnant females were placed in polycarbonate cages that were fitted with a wire-rod metal top and contained absorbent wood chip bedding. Food and water were available ad libitum during the non-exposure periods only. Rats were randomly assigned to one of five groups. Each group consisted of 30 males and 30 females. Three groups were exposed to EtO vapor at approximately 100, 33, or 10 ppm, and two control groups similarly maintained were exposed only to room air. Two control groups were used so that normal variability between similarly treated concurrent control groups could be evaluated. Exposures were initiated for both sexes after a 2-week quarantine period, for 12 weeks prior to the cohabitation period, exposures were for 6 hr per day, 5 days per week. The exposure regimen during cohabitation was 6 hr per day, 7 days per week. During the cohabitation period, one male was placed with each female. The day of observation of a vaginal plug was designated as day 0 of gestation. At the end of one week, a different male was placed with each female for which no vaginal plug had previously been observed. Any female that did not become pregnant after being mated with two different males was considered infertile. Likewise, any male that was unsuccessful in impregnating a female in two mating periods was considered infertile. After 2 weeks of mating, only the females were exposed. Exposure was 6 hr per day, 7 days per week.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Rats were randomly assigned to one of five groups. Each group consisted of 30 males and 30 females. Three groups were exposed to EtO vapor at approximately 100, 33, or 10 ppm, and two control groups similarly maintained were exposed only to room air. Two control groups were used so that normal variability between similarly treated concurrent control groups could be evaluated.

Details on mating procedure:

Premating exposure period for males and females: 12 weeks

Duration of treatment / exposure:

139 days / 14 weeks

Frequency of treatment:

6 h/d, 5 d/w

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.018 mg/L air (nominal)

Remarks:

10 ppm

Doses / concentrations 2

Dose / conc.:

0.054 mg/L air (nominal)

Remarks:

33 ppm

Doses / concentrations 3

Dose / conc.:

0.18 mg/L air (nominal)

Remarks:

100 ppm

No. of animals per sex per dose:

30

Control animals:

yes, concurrent no treatment

Details on study design:

Females for which a vaginal plug was observed were exposed from day 0 through day 19 of gestation. On day 20 of gestation, these females were placed in polycarbonate cages to have their litters, and exposure was stopped. Females for which a vaginal plug was not observed throughout the mating period were also placed in polycarbonate cages on the 20th day following the midweek of the mating period, and the exposure was stopped. 5 days after parturition, the dams were separated from their pups for approx. 6.75 h per day and exposed again to EtO vapor in wire-mesh cages for 6 h/d, 7 d/w, through day 21 post partum.

Examinations

Parental animals: Observations and examinations:

Body weights of all F0 animals were determined every 2 weeks until the cohabitation period. The uteri of all females of the F0 generation were fixed in 10% neutral buffered formalin and stained in equal parts of 20% solution of potassium ferricyanide and 1% solution of hydrochloric acid for 2 h to determine the number of implantation sites, and consequently if the animal were pregnant. Records were maintained to determine the following: male and female fertility indices, number of days from observation of the vaginal plug to parturition, number of implantation sites, ratio of number of pups born to number of implantation sites, number of litters, number of pups born dead ar alive, and F1a (offsping of F0) generation survival indices.

Oestrous cyclicity (parental animals):

-

Sperm parameters (parental animals):

-

Litter observations:

Each litter was weighed as a unit on days 4 and 14 post partum, and the average body weight of the pups in each litter was determined. On day 21 post paratum, the sex of each pup, its body weight and the mean of each litter were determined. On day 4 post partum, if the number of pups per litter were greater than 10, it was reduced to 10. These pups were culled randomly from each litter without regard to sex.

Postmortem examinations (parental animals):

-

Postmortem examinations (offspring):

-

Statistics:

The fiducial limit of 0.05 (two-tailed) was selected as the critical level of significance. Where appropriate, the litter was considered to be the exerpimental unit. All data of each exposure group were compared statistically to each air-control group separately, and the two air-control groups were compared to each other by using the following tests: continuous variable data were analyzed by Bartlett's test for homogeneity of variance, analysis of variance, and Duncan's multiple range test. Whenever the F value for analysis of variance was signfiicant, Duncan's multiple range test was used to denote which groups differed significantly from the controls. If Bartlett's test indicated heterogenous variance, the F test was employed to compare each exposure group with each air control group. The type of t test then used was selected according to the significance of the F value. The Student's test was used when the F value was not significant. The Cochran t test was used when the F value was significant. Contingency data were analyzed by Fisher's exact test. All other non-parametric data were compared using the multiple sum of ranks. The median and semi-interquartile range were reported for these non-parametric variables.

Reproductive indices:

-

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Mortality:

no mortality observed

Description (incidence):

No male or female rats of the F0 generation died or were moribund during this period. Furthermore, no dams died during gestation or lactation periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on body weight gain throughout the 12 weeks of exposure for males or females of any exposure group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The median number of implantation sites per pregnant female in the 100-ppm exposure group was 6, which is significantly lower than the median of 10 or 11 for the air-control groups. The median for the 33- and 10-ppm exposure group was 11. The ratio of the number of fetuses born to the number of implantation sites per female was determined for each litter. The median value of this ratio for the 100-ppm exposure group was statistically significantly lower than the value of either air-control group; 57 vs. 92 or 100.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Fertility indices indicated a statistical significance between the 100 ppm group and one or both air-control groups, however, statistical significant difference could not be confirmed.
There were statistically significantly more females in the 100-ppm exposure group whose gestation period was greater than 22 days than in either air-control group; however, no statistically significant differences in the length of the gestation periods were noted. All of the pregnant females in both air-control groups had a gestation period of 22 days, whereas, there were 7 of 14 in the 100-ppm exposure group that had longer gestation periods. Of these, 4 females had gestation periods of 23 days, 2 had 25 or 26 days, and one was undeterminable. All gestation periods in the 33- and 10-ppm EtO exposure groups were 22 days.

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

0.054 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: no mortality observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no statistically significant effects on the survival rate of the F1a generation when evaluated as the ratio of pups born alive to the number of pups born per pregnant female, the ratio of pups alive on day 4 post partum to the number of pups alive on day 0 per pregnant female, or the ratio of pups alive on day 14 or 21 post partum to the number of pups alive on day 4 per pregnant female.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant adverse effects were observed in the 100-ppm exposure group when the body weights of the pups per litter were determined at day 4, 14, or 21 post partum.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The major treatment-related adverse effect observed after mating was that the median number of pups born on day 0 post partum per litter for the 100-ppm exposure group was significantly (p < 0.001) lower than the medians for both air-control groups. The medians for the 33 ppm, 10 ppm, and the two air-control groups were 9 or 10 pups, whereas, the median was 4 for the 100-ppm exposure group. At parturition, no pups were found dead in the 100-ppm exposure group or in either air-control group, and there was no evidence of cannibalization.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

0.054 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of pups decreased

Remarks on result:

other: no mortality observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

0.054 mg/L air (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

In the present study, no treatment-related effects were noted in either the dams exposed to 33 or 10 ppm of ethylene oxide or in their litters.
Consequently, exposure of rats to 33 ppm of ethylene oxide under the stated experimental conditions is considered a no ill-effect exposure level for one generation of reproduction and thus was chosen as NOAEC.

Executive summary:

Male and female Fischer 344 rats were exposed for 12 weeks (6 hr/ day, 5 days/week) to either 10, 33, or 100 ppm of the test substance's vapor. The animals were mated. Female animals were continued to expose from Day 0 through Day 19 of gestation (6 hr/day, 7 days/week). At 100 ppm only, the major treatment-related adverse effect was significantly lower pups born per litter. Implantation sites per pregnant female were observed to be fewer and a smaller ratio of the number of fetuses born to the number of implantation sites per pregnant female was reported in the 100 ppm group. There were stastistically significant more females with a gestation period longer than 22 days in the 100 ppm group in comparison to control groups. No significant differences in body weight gain or any other signs of toxic effects from exposure were observed in the parent generation. There was no treatment-related adverse effect on survival or growth rate of the F1 generation during the lactation period, even when exposure was continued for dams.