Ethylene oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Snellings et al. (1982): chronic study in Fischer 344 rats, equivalent or similar to OECD 415, GLP not specified. Animals were exposed for 139 days 6 hr/day 5 days/week to 10, 33, and 100 ppm of ethylene oxide vapor. The NOAECs for parental and F1-animals was 33 ppm (=0.054 mg/l air).

Based on the adverse effect on the number of implantation sites and significantly lower number of pups, there is some evidence from experimental animals that the test substance may be a human reproductive toxicant.

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

- Physical appearance: gas

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.

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

Dose / conc.:

0.018 mg/L air (nominal)

Remarks:

10 ppm

Dose / conc.:

0.054 mg/L air (nominal)

Remarks:

33 ppm

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.

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:

-

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: 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.

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

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

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

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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

54 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The following is quoted from the RAC opinion proposing a harmonized classification and labelling at the EU level of ethylene oxide ( CLH-O-0000001412-86-164/F, adopted 22nd September 2017):

"In the case of ethylene oxide, the human data does not provide conclusive evidence on the effects of ethylene oxide on fertility. Therefore, the classification criteria for Category 1A for fertility effects are not fulfilled. The main evidence on the effects on fertility comes from the one-generation study in rats by Snellings et al. (1982c), in which significantly decreased number of

implantations and born foetuses per implantation site (indicating post-implantation losses) was observed without any signs of parental toxicity (e.g. decreases in weight gain) at 100 ppm. These

findings are supported by the studies by Generoso et al. (1987) and Hardin et al. (1983) showing increased incidences of resorptions and/or decreased incidences of implantations at 300 and 150 ppm, respectively. Additional support for the fertility effects comes from the studies reporting specific effects on spermatogenesis and sperm morphology. These include the studies by Mori et al. (1989, 1991) and Ribeiro et al. (1987) and are supported by the monkey study by Lynch et al., 1984 (reported by NEDO, 2004). Since these effects have been seen in the absence of clear signs of general toxicity in several studies, RAC considers that the available evidence is sufficient to meet the criteria of Category 1B for fertility. Although at higher dose levels GSH depletion in rats may have an impact on toxicity, decreases in implantations, increases in post-implantation losses and effects on spermatogenesis and sperm numbers and motility have been seen starting from the dose levels (50-100 ppm), at which no clear GSH depletion has been observed. Ethylene oxide is a well-established mutagen and it is possible that effects observed in one-generation studies are mediated by a genotoxic mechanism. Especially post-implantation losses observed after exposure during the pre-mating period may be due to dominant lethal effect caused by genotoxic insult. Genotoxic insult during the specific stages of spermatogenesis may also affect sperm quality by increasing the number of abnormal sperm as suggested by Ribeiro et al., 1987. However, other mechanisms cannot be excluded. Since there were clear effects on fertility, seen also as a decrease in sperm quality, these are not considered to be covered by a germ cell mutagenicity classification."

Effects on developmental toxicity

Description of key information

Snellings et al. (1982) conducted a study in Fischer 344 rats which was equivalent or similar to OECD 414. No treatment related effects on maternal survival, litter size, number of implantation and resorption sites and preimplantation losses were seen. Exposure to 100 ppm resulted in a statistically significant depression of body weight, No information on maternal weight gain was reported, the distinction of specific developmental effects or effects secondary to maternal toxicity is not possible.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental 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

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)

Remarks:

testing lab.

Limit test:

no

Specific details on test material used for the study:

- Physical appearance: gas
- Purity: minimum purity 99.7%

Species:

rabbit

Strain:

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.

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

Dose / conc.:

0.27 mg/L air (nominal)

Remarks:

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.

Statistics:

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.

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.

Dose descriptor:

NOAEC

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

Abnormalities:

no effects observed

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.

Dose descriptor:

NOAEC

Effect level:

0.27 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no mortality observed

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

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

Reference 2

Endpoint:

developmental 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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Physical appearance: gas

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

Twelve-week-old, male and female, Fischer 344 rats were acclimated for approximately 2 weeks prior to the initiation of mating. The only rats accepted for mating were those with a good health status and whose body weights were within plus or minus two standard deviations of the mean weight of all rats of the same sex. Animals were housed in a temperature (20 to 24°C) and relative humidity (40 to 60%) controlled room. The artificial fluorescent lighting was on a 12-hr light and 12-hr dark cycle. Food and water were removed during the exposure period and were available ad libitum at all other times. Pregnant rats were singly housed at all times in suspended wire-mesh stainless steel cages.
A stainless steel cylinder containing liquid EtO was heated to approximately 35°C. The vapor generated was metered into 4400-liter, stainless steel and glass inhalation chambers. Chamber atmospheric concentrations in both the test and control chambers were monitored by a gas chromatograph equipped with a flame ionization detector. The column packing material in this analysis was 30% Tergitol-TMN and 3% sodium methylate on Chromosorb W-NAW. Approximately eight analyses were obtained from each chamber within each 6-hr exposure period. Chamber temperature and relative humidity were controlled, and the values for these parameters were recorded during each exposure.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Appearance of a vaginal plug was considered as evidence of successful mating and as day 0 of the gestation period. Mated females were separated and assigned randomly to one of seven treatment groups. Three groups were exposed to EtO. The target concentrations were 100, 33, and 10 ppm. Two groups were handled in the same manner as the EtO-treated groups but were exposed to room air and served as negative controls. Two negative control groups were used so that normal variability between similarly treated concurrent control groups could be evaluated. The remaining two groups were treated by gavage with a single dose of aspirin, a known rodent teratogen, and served as positive controls. One group received 500 mg/kg of body weight on day 9 of the gestation period, and the other received 625 mg/kg on day 10. The aspirin was suspended in 0.2% carboxymethylcellulose.

Details on mating procedure:

A goal of 22 successfully mated females was established for each ethylene oxide exposure and air-control group and 11 successfully mated females for each positive control group. The inhalation exposure began on day 6 of the gestation period and continued through day 15. Each day animals were exposed for 6 hr during the first three quarters of the 12-hr light period.

Duration of treatment / exposure:

g.d. 6 - 15

Frequency of treatment:

6 h/d

Duration of test:

20 d

Dose / conc.:

10 ppm (nominal)

Remarks:

0.018 mg/m³

Dose / conc.:

33 ppm (nominal)

Remarks:

0.059 mg/m³

Dose / conc.:

100 ppm (nominal)

Remarks:

0.18 mg/l

No. of animals per sex per dose:

A goal of 22 successfully mated females was estalished for each ethylene oxide exposure and air-control group and 11 successfully mated females for each positive control group.

Control animals:

yes

yes, concurrent no treatment

Details on study design:

In preliminary studies with young, nonpregnant rats, depression in the rate of body weight gain has been observed for female Fischer 344 rats exposed to 100 ppm for approximately one to two weeks (unpublished data). All studies to date have been on the Fischer 344 rat and have incorporated three exposure levels of EtO: 100, 33, and 10 ppm.

Maternal examinations:

When killed, the number and position in the uterine horn of implantation sites, viable fetuses, dead fetuses, early resorption sites, and late resorption sites were recorded for all pregnant females.

Ovaries and uterine content:

The number of corpora lutea in the right and left ovaries was recorded for each female of all groups.

Fetal examinations:

On day 20 of the gestation period, females of all groups were anesthetized and the fetuses delivered by cesarean section. When killed, the number and position in the uterine horn of implantation sites, viable fetuses, dead fetuses, early resorption sites, and late resorption sites were recorded for all pregnant females. All fetuses were examined grossly for developmental defects. Any external abnormalities, along with the body weight, sex, and crown-to-rump length of each fetus, were recorded. The number of corpora lutea in the right and left ovaries was recorded for each female of all groups. One-half of each litter was fixed in 10% neutral buffered formalin for possible subsequent skeletal evaluation, and the remaining half was fixed in Bouin's fixative for possible subsequent examination of the soft tissues for anomalies.
All fetuses of the dams from the 100-ppm exposure group, the two air-control groups, and the two positive control groups were submitted for skeletal or visceral evaluation. Those fetuses that were examined skeletally were first macerated with potassium hydroxide and then stained with alizarin red, and those fetuses for soft tissue examination were sectioned by a razor slice technique. It was determined prior to the study that the fetuses of the 33- and 10-ppm exposure groups would be similarly evaluated if treatment-related teratologic effects were noted in the 100-ppm exposure group.

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 experimental unit (Weil, 1970). All data of each EtO-exposed and aspirin-treated group were compared separately to the data of each air-control group, and the two air-control groups were compared to each other. The same statistical procedures as those presented by Snellings et 01. (1982) were followed in this study for continuous variable and contingency data. All other non-parametric data were compared using the Wilcoxon sum of ranks test (Sokal and Rohlf, 1969). The median and quartile deviation (Beyer, 1968) were reported for these non-parametric variables.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related adverse effects on appearance or demeanor were observed for the adult females.

Body weight and weight changes:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant differences between any EtO exposure and air-control groups for any of the analyzed parameters associated with preimplantation loss or embryo and fetal resorption.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

One litter of the 33-ppm exposure group was totally resorbed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were found.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

In each EtO or air-control group, 17 to 22 females were actually pregnant in each group.

Other effects:

no effects observed

Description (incidence and severity):

The value for the mean number of fetuses for all EtO and air-control groups was eight or nine fetuses per dam. In general, the values of all groups were similar for the number of corpora lutea per dam and the number of implantation sites per dam.

Dose descriptor:

NOAEC

Effect level:

0.18 mg/L air (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no mortality observed

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Depression of body weights for both male and female fetuses in the 100-ppm exposure group. However, there were no significant differences in crown-to-rump length of either sex, and there were no gross abnormalities noted in the fetuses of any EtO exposure group or either air-control group.

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

The skeletal abnormalities observed in the 100-ppm EtO exposure group and the air-control groups were classified into two types: variations in ossification of sternebrae and distal thoracic vertebral centra. The variations in the sternebra were that the sternebrae were either split or poorly ossified and the variation in the vertebral centra was that they were bilobed. The percentage of litters (ratio of the number of affected litters to number pregnant x 100) and percentage of fetuses (ratio of the number of affected fetuses to number fetuses for the exposure group x 100) in the 100-ppm exposure group with distal thoracic vertebral centra variations in ossification were numerically elevated when compared to the air-control groups; however, there were no statistically significant differences noted. Furthermore, when the data for this alteration were expressed as a percentage of fetuses per litter, there were no significant differences noted between the 100-ppm and air-control groups. There were no significant differences observed for the incidence of variation in ossification of the sternebrae.

Visceral malformations:

no effects observed

Description (incidence and severity):

The visceral alteration most frequently observed in both the 100-ppm exposure group and air-control groups was renal pelvic dilatation (bilateral or unilateral). The renal papilla was present when this variation was observed. There were no significant differences between the 100-ppm exposure group and either air-control group for this alteration. All other alterations noted in skeletal and visceral development for these groups were found only in the air-control groups (at low incidences) and were not discussed.

Dose descriptor:

NOAEC

Effect level:

0.18 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: no mortality observed

Abnormalities:

no effects observed

Developmental effects observed:

no

In the aspirin-treated animals, especially the group administered 625 mg/kg on gestation day 10, statistically significant (p < 0.01) differences were observed for many of the parameters examined. These included fetal body weight and length, and number of fetuses with skeletal abnormalities. For the skeletal malformations, a dose response was indicated. Included in these malformations were ribs that were fused, split, crooked, missing, and extra (15th); and vertebrae that were fused and extra (two or more). The visceral abnormalities of both aspirin-treated groups were slightly increased in comparison to the air-control groups. These visceral malformations included hydrocephalus, hydromyelia, ureteral dilation, and undescended testes.

Conclusions:

The test substance was found to have no reproductive or teratogenic effect in rats under the consitions chosen.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

180 mg/m³

Study duration:

subacute

Species:

other: rats and rabbits

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The following is quoted from the RAC opinion proposing a harmonized classification and labelling at the EU level of ethylene oxide ( CLH-O-0000001412-86-164/F, adopted 22nd September 2017):

"Regarding developmental effects small decreases in foetal weights have been seen when pregnant females were exposed to 100-150 ppm. In the case of Snellings et al. (1982b), it is

uncertain if these were accompanied with decreased maternal body weights. However, in the study by Hackett et al. (1982), decreased foetal weights and skeletal variations were seen in the

absence of changes in maternal body weights. At higher doses more severe findings were found. Single high dose exposures during the critical periods of organogenesis resulted in foetal deaths

and malformations, especially eye disorders (Weller et al., 1999; Rutledge et al., 1989). These were accompanied by slight to severe maternal toxicity. However, it is not possible to conclude

that these malformations would have been in all cases secondary to maternal toxicity. Since ethylene oxide is a well-established mutagen, it can be hypothesised that malformations at high

doses in developing embryos could be caused by a genotoxic mechanism. On the other hand, it should be noted that at these high doses, GSH depletion may play a role in the foetotoxicity and

teratogenicity of ETO. There are only limited data available on the foetotoxicity of ethylene oxide in humans but in the few available studies suggestions on the increased incidence of spontaneous abortions have been obtained. Biases related to questionnaire based studies and/or the effects of confounders (e.g. other concurrent exposures) cannot be totally excluded.

Taking these together and applying a weight of evidence approach, it can be concluded that there are indications on the developmental effects of ethylene oxide. However, malformations have

been mainly seen at high dose levels in which GSH depletion may play a role. At lower dose levels, in the absence of maternal toxicity decreased foetal weights were observed. Additionally,

in one study skeletal variations were observed. These can be considered to support Category 2. Classification for developmental effects."

Toxicity to reproduction: other studies

Description of key information

Mori et al. (1989) performed a study in male Wistar rats using a concentration of 500 ppm and exposing the animals for 2, 4, 6, or 13 weeks 6 hours per day 3 days per week. Testicular toxicity and changes in glutathione metabolism in the testes were investigated. Results indicate that chronic inhalation of EtO induces testicular atrophy.

In addition, Mori et al. (1991) exposed male Wistar rats to concentrations of 50, 100, or 250 ppm for 6 hours per day, 5 days per week for 13 weeks. Dose effect relations of inhaled EO on spermatogenesis were evaluated. Results indicate that subchronic inhalation of EO at low concentrations affects spermatogenesis in rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

toxicity to reproduction: other studies

Adequacy of study:

other information

Reference 2

Endpoint:

toxicity to reproduction: other studies

Adequacy of study:

other information

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

Based on the criteria laid down in the aforementioned regulation the Committee for Risk Assessment (RAC) has proposed harmonising the classification and labelling at EU level to reproductive toxicant category 1B ( H360Fd) (RAC Opinion adopted 22 September 2017).