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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Principles of method if other than guideline:
Studies were initiated to determine the teratogenic potential of three chemicals using inhalation as the route of exposure to simulate exposures that may be encountered in the workplace.
GLP compliance:
yes (incl. QA statement)
testing lab.
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene oxide
EC Number:
EC Name:
Ethylene oxide
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Physical appearance: gas
- Purity: minimum purity 99.7%

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
Sexually mature, New Zealand White rabbit does (5 to 6 mo of age; body weight, about 3 kg) were used. In addition to the 90 does required for the three experimental groups for each chemical (30 does/group), five does for replacements and two does for training bucks to the artificial vagina (AV) were obtained for each study. Eleven additional does were used as positive-control animals for the ethylene oxide study, and six does each were used as positive controls for propylene oxide and n-butyl acetate studies.
All rabbits were housed individually in stainless steel wire cages and provided with rabbit diet and water ad libitum, except during exposure. Following acclimation and quarantine, the does were divided into three exposure groups and one positive-control group by means of formal randomization (based on body weight) conducted by means of a computer program. One-third of each group (also randomly selected) was artificially inseminated in the afternoon of each day during a 3-day period.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Details on exposure:
Exposure to the test chemical or filtered air was initiated on the morning following artificial insemination (1 dg). Rabbits were placed in individual cages within the appropriate exposure chamber for the 7-hr daily exposure period.
Food consumption was measured for 2 wk prior to the initiation of exposure and at 5-day intervals during gestation. Body weights were measured in the morning (before exposure on 1-19 dg) on 1, 5, 10, 15, 20, 25, and 30 dg.
Rabbits assigned to the positive-control group received a single dose of 6-AN, a known teratogen.

Details on mating procedure:
Ten mature, naive bucks (6 to 7 mo old; body weight, 3 to 4 kg) of the same stock were purchased for breeding. Five of the original bucks were retained, and five were replaced for the second and for the third studies, so that a total of 10 bucks were available as semen donors for each artificial insemination period. All rabbits were identified by the supplier with a uniquely numbered, stainless steel ear tag.
Duration of treatment / exposure:
g.d. 1 - 19 or 7 - 19
Frequency of treatment:
7 h/d
Duration of test:
until day 30 of gestation
Doses / concentrations
Dose / conc.:
0.27 mg/L air (nominal)
150 ppm
No. of animals per sex per dose:
total: 64
Control animals:
yes, concurrent no treatment
Details on study design:
Since the exposure interval and the period of gestation during which the dam is exposed to the test chemicals may be significant in determining effects on reproduction and fetal development, these experiments were designed to provide exposures during critical periods of gestation and/or prior to breeding and implantation.


Maternal examinations:
Rabbits were killed at 30 dg. Liver, lungs, spleen, kidneys, ovaries and the gravid or non-gravid uterus were weighed and the weights recorded. Uteri of all apparently non-pregnant females were stained and examined for implantation sites. Observations of internal abnormalities of the pregnant and non-pregnant animals were recorded (e.g., adhesions, tumors, or evidence of infection). Samples (of appropriate size for proper fixation) were taken of ovaries, uterus, liver, lungs with trachea, spleen, and kidneys of each actual or potential parental female. Any abnormal tissues were preserved in 10% NBF. A randomized sampling of tissues from 25% of the females (a maximum of eight per group) and any grossly abnormal tissues were processed by routine techniques (paraffin embedding, hematoxylin and eosin staining) and subjected to histopathological examination. The residual tissues, and the tissues from the remaining 75% of the females, were preserved for possible future examination.
Data from adult animals, such as food consumption, body weight, and organ weights, are from pregnant animals only. Although formal randomization of body weights was used to select animals for the experimental groups, removal of data from nonpregnant animals from the group means tends to produce apparent deviations in initial body-weight values for some groups of animals. Results from histopathology studies are from a random sample of tissues from all females sacrificed at the termination of each study.
Ovaries and uterine content:
The uterus, with ovaries attached, was removed from each animal. The ovaries were excised, identified as to right and left, and the number of corpora lutea estimated by counting. The excised uterus was opened, the membranes and amniotic fluid were observed for abnormalities, and living and dead fetuses, and resorptions, were counted. Mortality in utero was classified and recorded as "early" (E, placenta and conceptus indistinguishable, or metrial gland), "mid" (M, placenta distinct, embryo partially to fully formed), and "late" (L, fully formed but not viable fetus). Beginning at the right ovary, numbers were assigned, in order, to each implantation site down the right horn to the cervix. Consecutive numbers for implantation sites in the left horn proceeded fron ovary to cervix.
Fetal examinations:
Live and recently dead fetuses were removed in serial order, blotted on a moist surface, freed of adherent material, and weighed. A fetus was designated as stunted when its size was below the normal range of variation of its littermates, as determined by a statistical test to reject extreme observations in
one direction. The crown-rump length of each fetus was measured and recorded. Concurrently, the placentas were removed, weighed and examined; abnormal placentas, if observed, were fixed for histological preparation and examination. Each fetus was examined for gross external abnormalities under an illuminated magnifier. The fetuses of both species were randomly divided into two equal groups for more detailed teratologic examination.
In one group, the heads were removed and placed in Bouin's fixative for subsequent examination of serial razor-blade-cut sections by the methods of Wilson (1965) and van Julsingha and Bennett (1977) for rabbits.
All fetuses were examined for internal abnormalities using Staples' (1974) technique, which is a modification of that of Barrow and Taylor (1969), and is similar to that described by Stertz (1977). The sex of each fetus was determined by external genitalia and visceral examination of the gonads. All fetuses were eviscerated; rabbit fetuses were skinned and air-dried prior to fixation. Following staining with alizarin red S, maceration with KOH, and clearing in glycerol, each skeleton was examined for abnormalities in size, shape, relative position, and degree of ossification. Results from fetal morphologic examinations were grouped into three categories (major malformations, minor anomalies, or morphologic variations) according to degree of severity, locus of fetal structural change, and incidence of these changes.
Binary response variables were compared among groups by chi-square tests for independence. Pairwise comparisons for significant findings used either a two-tailed chi-square test or a Fisher's Exact Test. Analysis of variance (ANOVA) method was used to analyze continuous variable data. Response proportions were analyzed by ANOVA with an arcsin transformation of the response proportion. Orthogonal a priori comparisons were made among treatment group means for rabbits. The orthogonal set of comparisons for rabbits was: Contrast I - Group 1 (control) versus Groups 2 and 3 (exposed to chemicals); and Contrast II - Group 2 (chemical exposure from 7 through 19 dg) versus Group 3 (chemical exposure from 1 through 19 dg). The orthogonal set of comparisons for rats was: Group 1 (control) versus Groups 2, 3, and 4 (exposed to chemicals); Groups 2 and 3 (exposed during gestation) versus 4 (exposed prior to mating and during gestation); and Contrast III - Group 2 (exposed from 7 through 16 dg) versus Group 3 (exposed from 1 through 16 dg). All orthogonal comparisons were two-tailed tests. Absolute maternal organ weights were analyzed by analysis of covariance using the terminal body wejght minus the weight of the gravid uterus (extragestational body weight) as the covariance. Relative organ weights were also analyzed
as a percentage of the extragestational body weight by analysis of variance. Body weights and crown-rump lengths for live male and female fetuses were analyzed by nested analysis of variance. The analysis takes into account the effects of treatment, litter, and sex on the body weight and crown-rump length measurements.
Repeated-measures data, such as maternal body weight, were analyzed by a multivariance repeated-measures analysis. Orthogonal polynomials were fit for each animal for which there were complete data, and a multivariate analysis of variance was performed an the coefficients to identify differences in growth patterns among exposure groups.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Fertility and reproductive status: Exposure to ethylene oxide had no significant effect on the percentage of pregnant animals surviving to 30 dg. The percentage of survivors which were pregnant and the percentage calculated from data which include animals dying during exposure was not decreased by exposure to ethylene oxide. Reproductive measures, including data for implantation sites, resorptions, and live and dead fetuses, were also unaffected by ethylene oxide exposure during pregnancy.
Food consumption and body weights: Although food consumption appeared to increase in all groups prior to exposure, and in groups 2 and 3 immediately following termination of exposure on 19 dg, there were no significant differences among treatment groups. Food consumption was diminished in all exposure groups during the interval from 25 to 30 dg. Comparisons of body weight values revealed no significant effect of the ethylene oxide exposures.

Organ weights and histopathology: Extragestational body weights, and weights of the pregnant uterus, liver, kidneys, spleen and ovaries, were similar for all treatment groups. Although a camparison of values for group 1 with those of groups 2 and 3 showed no significant differences for lung weights, both absolute and relative values for group 3 were higher than those for group 2. There were a variety of microscopic changes in the lungs, some of which could possibly be related to Pasteurella infections. Minimal suppurative bronchitis was observed in a group 1 rabbit; in group 3, one doe had minimal chronic suppurative pleuritis, one had moderate diffuse subacute pneumonitis, and one had severe suppurative bronchopneumania. The significantly higher lung weights in group 3 does were associated with the histopathalogic changes: doe 1893 (lung weight, 31.8 g) had severe chronic suppurative pleuritis; doe 2358 (lung weight, 25.2 g) had severe suppurative bronchopneumonia; doe 2952 (lung weight, 21.6 g) had diffuse moderate pneumonitis. No mortality was observed in group 3 during gestation; the deaths of two animals in group 1 and four in group 2 were attributed to pneumonia. Regressing corpora lutea were observed at necropsy in all rabbits that were not pregnant, as well as in rabbits (one from each exposure group) where pregnant was not grossly apparent and could only be detected by staining with ammonium sulfide. Corresponding uterine sections examined microscopically did not show evidence of pregnancy. In rabbits determined to be pregnant by uterine staining procedures, embryonic death presumably occurred soon after implantatian, thus the endometrial changes ordinarily associated with pregnancy were not observed. One group 1 rabbit and one group 3 rabbit had severe suppurative metritis.

Effect levels (maternal animals)

Dose descriptor:
Effect level:
0.27 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: no mortality observed

Maternal abnormalities

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Fetal measures and morphology: No significant effects among groups were observed tor fetal body weight, crown-rump length, sex ratio, or for placenta weights. The incidence of morphologic alterations in the rabbit fetuses was unaffected by exposure to ethylene oxide. One fetus in group 3 had multiple major defects. Supernumerary ribs were present in most of the fetuses. In addition to rudimentary ribs at the lumbar I position, ossification sites were observed in some fetuses at this locus.

Effect levels (fetuses)

Dose descriptor:
Effect level:
0.27 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Remarks on result:
other: no mortality observed

Fetal abnormalities

no effects observed

Overall developmental toxicity

Developmental effects observed:

Applicant's summary and conclusion

On balance, ethylene oxide is not considered to be teratogenic to rabbits.