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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: basic information given; scientifically acceptable

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals 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.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: room 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
Doses / concentrations
Remarks:
Doses / Concentrations:
10; 33; 100 ppm (= about 0.018; 0.059; 0.18 mg/l)
Basis:
nominal conc.
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, 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.

Examinations

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
No treatment-related adverse effects on appearance or demeanor were observed for the adult females exposed to EtO. No body weight determinations were made during the gestation period. In each EtO or air-control group, 17 to 22 females were actually pregnant in each group. One litter of the 33-ppm exposure group was totally resorbed. The value for the mean number of fetuses for all EtO and air-control groups was eight or nine fetuses per dam, and no dead fetuses were found. 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. In general, the values of all groups were similar for the numher of corpora lutea per dam and the number of implantation sites per dam. Consequently, there were no significant differences calculated for percentage of preimplantation loss per dam. Furthermore, the mean values for the percentages of early, late, or total resorption sites per dam were not significantly different from the values of the air-control groups.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
0.18 mg/L air
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
The only significant effect observed in this study that was related to the exposure of EtO was 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. There were no gross external abnormalities noted in the fetuses from the EtO-exposed or air-control groups. 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. 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.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
0.18 mg/L air
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
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.