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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Species: Rat
Strain: Sprague-Dawley
Sub-strain: Crl:CD®(SD) lGS BR
Source of supply: Charles River (UK) Limited, Kent, UK
Number: 96
Sex: Time-mated females
Age: Young adult animals
Temperature: 22 ± 3 °C
Humidity: 30 to 70%
Lighting: Twelve hours of continuous artificial light in each twenty-four hour period
Ventilation: At least fifteen air changes per hour
Animals are housed in accordance with the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' and in alignment with the United Kingdom Home Office recommendations and requirements.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Time-mated females were dosed once daily by gavage, from Day 3 to Day 19 of gestation (the day prior to necropsy). The volume of test and control item administered to each animal was based on the most recent scheduled body weight and adjusted accordingly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing formulations were sampled on two occasions during the study and analyzed for concentration by Envigo Research Ltd., Shardlow, UK Analytical Services. Stability.
Details on mating procedure:
Females were time-mated with sexually mature proven males of the same strain by the animal supplier. The day of observation of positive evidence of mating was designated
Day 0 of gestation. The animal supplier identified mated females and provided identification details of their respective mating partners.
Duration of treatment / exposure:
Day 3 to Day 19 of gestation.
Frequency of treatment:
Once per day
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
Serial Observations
1 GeneralObservationslMeasurements

1.1 Morbidity/Mortality Inspection
Twice daily, early and late during the working period.

1.2 Clinical Observations
Once daily during the gestation period. During the dosing period, individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing.

1.3 Body Weights
Individual body weights were recorded on Day 5, 6, 7, 8, 11, 14, 17 and 20 of gestation.

1.4 Food Consumption
Dietary intake was recorded for individual animals on Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

1.5 Water Consumption
Monitored daily by visual inspection of water bottles.

Examinations

Maternal examinations:
Terminal investigations
1 Necropsy
Post mortem procedures were performed on all animals including any animal found dead or killed in extremis during the study. Animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of presumed gestation.

1.1 Gross Examination
Full external and internal macroscopic examination of adult females.
Ovaries and uterine content:
The uterus of any apparently non-pregnant female was immersed in 0.5% ammonium polysulfide solution to reveal evidence of implantation.
The uterus of each adult female was examined and the following recorded;
i. Pregnancy status
ii. Number of corpora lutea
iii. Gravid uterus weight
iv. Number, status (Live fetus, dead fetus, late intra-uterine death or early intra-uterine death) and intra-uterine position of implantations
Fetal examinations:
Fetuses will be removed and live fetuses were weighed before subcutaneous overdose of a barbiturate agent.
For fetuses from decedent females prior to Day 20 of gestation, examination of the fetuses was restricted to external appearance. Each individual fetus from all litters at Day 20 of gestation were examined and the following was recorded:
i. External fetal findings
ii. Fetal weight
iii. Fetal sex
iv. Placental weight
v. Skeletal and visceral fetal findings
After external examination, approximately half of each litter was fixed in 70% Industrial Methylated Spirit (lMS), processed for skeletal evaluation and finally stored in 50% glycerol. The remainder of each litter was fixed in bouins fluid for visceral evaluation by microdissection and finally stored in 10% neutral buffered formalin.
Statistics:
Quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Analysis was performed on the following parameters:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data and fetal litter and placental weights.
The data were first assessed using Shapiro-Wilk test for normality and Bartlett's test for homogeneity of variance. If the data are considered to be normal and homogeneous, parametric assessment, one way analysis of variance and, if significant, Dunnett's multiple comparison test was employed. Where the data are considered not to be normal or non homogeneous, non-parametric assessment, Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney 'U' test wsd used.
Indices:
All scheduled data was summarized in tabular form; additionally calculated data (i.e. body weight gain, adjusted body weights) was presented. Unscheduled data (i.e. arrival body weight, additional health monitoring body weights) was reported where this information is of assistance in the interpretation of the scientific outcome of the study. As the litter is the standard unit of assessment, mean values and incidences were calculated first within each litter and then group mean values calculated from these litter values.
Group mean values were calculated from all females with a live litter at Day 20 of gestation. Reproductive indices were calculated for all females/litters at Day 20 of gestation. For decedent animals, calculation of reproductive indices was restricted to pre-implantation loss. Non-pregnant animals are defined as having no implantations; corpora lutea for these animals are considered not to be associated with mating/pregnancy and therefore are not reported as part of the litter data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

There were no observations apparent at any dose level which were considered to be related to systemic toxicity of the test item.

Instances of increased post-dosing salivation were observed in females treated with 1000 mg/kg bw/day between Days 4 and 5 and then from Days 13 to 19. Such findings are commonly seen in this type of study and usually reflect unpalatability and/or an irritant nature of the test item formulation and are generally deemed to be of no toxicological importance. One female animal from this treatment group exhibited pilo-erection, tip-toe gait and a stained ano-genital region on Day 20 of gestation. Due to the isolated nature of these findings they were considered likely to be incidental.
Description (incidence):
There were no unscheduled deaths during the study.
Description (incidence and severity):
Group mean body weights and standard deviations are given in Table 3 and presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5. Individual data are given in Appendix 2, Appendix 3 and Appendix 4.

Animals treated with 1000 mg/kg bw/day showed general reductions in body weight gains from Day 5 of the treatment period, statistical significance was achieved from Days 8 to 11 and 11 to 14 (p<0.001) of gestation. These reductions in body weight gain resulted in lower mean body weights achieving statistical significance (P<0.01) on Days 14, 17 and 20 and cumulative body weight gain being statistically significantly reduced (p<0.001) from Day 11 until the end of the treatment period. This resulted in overall body weight gain for these animals being 24% lower than control. Final body weight and overall body weight gain when adjusted for the contribution of the gravid uterus weight remained statistically significantly reduced (p<0.01 - p<0.001 respectively) resulting in a 57% reduction in overall body weight gain in these females when compared to control.

No toxicologically significant effects were detected on body weights in females treated with 100 and 300 mg/kg bw/day.

Animals treated with 300 mg/kg bw/day exhibited a statistically significant reduction (p<0.05) in body weight gain from Days 11 to 14, however, at all other points during the study body weight gains were comparable to control. As overall body weight gain for these animals was only 3.7% lower than the control group and body weight gain when adjusted for gravid uterus weight was only 5.3% lower than control animals, this effect was considered to be of no toxicological significance and likely to be due to normal biological variation.
Description (incidence and severity):
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

General reductions in food consumption were evident in females treated with 1000 mg/kg bw/day throughout the treatment period which achieved statistical significance (p<0.05 - p<0.001) from Day 8.

At 100 or 300 mg/kg bw/day, there were no effects of treatment on food intake throughout the treatment period.
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Description (incidence and severity):
A summary incidence of female necropsy findings is given in Table 7. Individual data are given in Appendix 6.

At necropsy on Day 20 of gestation, treatment-related macroscopic findings included 3/24 females treated with 1000 mg/kg bw/day showing gaseous distension of the stomach. Two of these females also showed thinning of the glandular region of the stomach, and a further female exhibited thinning of the non-glandular and glandular region of the stomach which also had compacted contents. No such effects were evident in females treated with 300 or 100 mg/kg bw/day.

Other macroscopic findings were confined to one female from the 100 mg/kg bw/day dose group showing an increased pelvic space in the right kidney. Due to the isolated nature of this observation it was deemed unlikely to be related to treatment with the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Summary fetal data are given in Table 8. Individual data are given in Appendix 7 and Appendix 8.

There was no effect of maternal exposure at 100, 300 and 1000 mg/kg bw/day of the test item on litter data, as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and post implantation losses.

The number of corpora lutea (an event established prior to treatment) were similar in all groups, however, at 100 and 1000 mg/kg bw/day, due to statistically significant lower (p<0.05 and p<0.001 respectively) pre-implantation losses than control, the number of implantations were higher than control in these two groups but failed to achieve statistical significance. This was particularly reflected in animals treated with 100 mg/kg bw/day as a statistically significant increase (p<0.05) in the total number of fetuses was achieved. As these effects represent better survival for these treatment groups an effect of treatment is unlikely. The number of male fetuses born was statistically significantly increased (p<0.01) in females treated with 100 mg/kg bw/day. However, as this was not reflected in the two higher treatment groups this finding was considered to be incidental and due to normal biological variation.

At 1000 mg/kg bw/day, mean fetal weight for both sexes was statistically significantly lower (p<0.001) than control, resulting in statistically significantly lower (p<0.001) mean fetal weights. Placental weights were comparable to control in these females.

No adverse effects on fetal, litter or placental weights were detected in females treated with 100 or 300 mg/kg bw/day.

At 100 mg/kg bw/day, statistically significant increases (p<0.01) in gravid uterus weight and litter weight were considered to reflect the higher litter size at this dosage and are therefore considered to be unrelated to treatment with the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There were statistically significant increases (p<0.05) in the total number of fetuses exhibiting external findings from females treated with 1000 mg/kg bw/day. This was principally due to statistically significant (p<0.05) higher number of small fetuses and fetuses with the appearance of dark spots on the back.

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 100 or 300 mg/kg bw/day.

There were statistically significant decreases (p<0.05) in the total number of fetuses exhibiting external findings from females treated with 100 mg/kg bw/day. The finding noted which also achieved statistical significance (p<0.05) was a decrease in the number of small fetuses. However, this was not regarded as evidence of adverse developmental toxicity as a decrease is actually closer to the expected range for this parameter.
Description (incidence and severity):
Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

At 1000 mg/kg bw/day, there was a statistically significant (p<0.05) decrease in the number of fetuses showing incomplete ossification of the femur. In isolation, this finding is unlikely to indicate developmental toxicity and was considered to be incidental and unrelated to treatment. This finding was not observed at lower doses.

At 300 mg/kg bw/day there was an increase in the number of fetuses exhibiting a dumb-bell-shaped thoracic centrum of the vertebral column. In the absence of a similar finding at the high dosage, the occurrence of this isolated observation was considered incidental and not to represent an effect of treatment on fetal development.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day. Statistical analysis of the data did not reveal any significant intergroup differences.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]-bisoxirane (Eponex 1510) to pregnant rats from gestation Days 3 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 1000 mg/kg bw/day. No similar adverse effects were apparent at 100 or 300 mg/kg bw/day. Consequently, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) and 100 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL)for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day, although reduced fetal weights indicated an adverse effect on fetal growth. No changes in the measured fetal parameters or embryofoetal development were detected at 100 or 300 mg/kg bw/day. The `No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis. The study was designed to comply with the following guidelines:

US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by daily gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day.  A further group of twenty-four time mated females was exposed to the vehicle only (Arachis Oil) to serve as a control.  

Clinical signs, body weight change, food and water consumptions were monitored during the study.  

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents.  The number of corpora lutea, number, position and type of implantation, placental weights, fetal weights, sex and external and internal macroscopic appearance were recorded.  Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no observations apparent at any dose level which were considered to be related to systemic toxicity of the test item.

Body Weight

Animals treated with 1000 mg/kg bw/day showed general reductions in body weight gains from Day 5 of the treatment period.  These reductions in body weight gain resulted in cumulative body weight gain being reduced from Day 11 until the end of the treatment period.  This resulted in overall body weight gain for these animals being 24% lower than control.  Final body weight and overall body weight gain when adjusted for the contribution of the gravid uterus weight were reduced, resulting in a 57% reduction in body weight gain in these females when compared to control.  

No toxicologically significant effects on body weight were detected in females treated with 100 and 300 mg/kg bw/day.

Food Consumption

General reductions in food consumption were evident in females treated with 1000 mg/kg bw/day throughout the treatment period.

At 100 or 300 mg/kg bw/day, there were no effects of treatment on food intake throughout the treatment period.

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

At necropsy on Day 20 of gestation, treatment-related macroscopic findings included 3/24 females treated with 1000 mg/kg bw/day showing gaseous distension of the stomach, 3/24 showing thinning of the glandular region of the stomach, and 1/24 animals exhibiting thinning of the non-glandular region of the stomach which also had compacted contents.  No such effects were detected in females treated with 100 or 300 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal exposure at 100, 300 and 1000 mg/kg bw/day of the test item on litter data, as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and post implantation losses.  

At 1000 mg/kg bw/day, mean fetal weight for both sexes was lower than control, resulting in lower mean fetal weights.  Placental weights were comparable to control in these females.

No adverse effects on fetal, litter or placental weights were detected in females treated with 100 or 300 mg/kg bw/day.

Fetal Examination

External Findings

There were increases in the total number of fetuses exhibiting external findings from females treated with 1000 mg/kg bw/day.  This was principally due to higher numbers of small fetuses and fetuses with the appearance of dark spots on the back.  

Neither the type, incidence nor distribution of external finding apparent for fetuses at Day 20 of gestation indicated an effect of maternal treatment on fetal development at 100 or 300 mg/kg bw/day.

Detailed Visceral Examinations

Visceral examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

Detailed Skeletal Examinations

Skeletal examinations of fetuses on Day 20 of gestation did not indicate any obvious effect of maternal treatment on fetal development at 100, 300 or 1000 mg/kg bw/day.

Conclusion

The oral (gavage) administration of 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1 -diyloxymethylene)]-bisoxirane (Eponex 1510) to pregnant rats from gestation Days 3 to 19, at dose levels of 100, 300 or 1000 mg/kg bw/day was associated with lower maternal body weight gain during gestation and an effect on food consumption at 1000 mg/kg bw/day. No similar adverse effects were apparent at 100 or 300 mg/kg bw/day.  Consequently, 300 mg/kg bw/day was considered to represent the No Observed Adverse Effect Level (NOAEL) and 100 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL) for the pregnant female.

In-utero survival of the developing conceptus was unaffected by maternal treatment with 1000 mg/kg bw/day, although reduced fetal weights indicated an adverse effect on fetal growth.  No changes in the measured fetal parameters or embryofoetal development were detected at 100 or 300 mg/kg bw/day.  The `No Observed Effect Level' (NOEL) for developmental toxicity was therefore considered to be 300 mg/kg bw/day.