Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Jun 1978 to 7 Jul 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study predates GLP and modern guidelines, some restrictions in design and reporting, but otherwise acceptable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-cyclohexylethyl butyrate

EC Number:

264-158-4

EC Name:

1-cyclohexylethyl butyrate

Cas Number:

63449-88-7

Molecular formula:

C12H22O2

IUPAC Name:

1-cyclohexylethyl butyrate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: CD rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: Approximately 3 months
- Housing: Individually housed, except during mating, in wire mesh cages and maintained in a temperature-, humidity- and light-controlled room.
- Diet: Purina Laboratory Chow; ad libitum
- Water: Tap water; ad libitum
- Acclimation period: Approximately one month

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

The bred females were consecutively assigned to 4 groups in a block design, one control and 3 treatment groups of 25 rats each. After eleven rats had received treatment in the 3000 mg/kg/day dosage group, the remaining 14 rats were assigned to an added group of 100 mg/kg/day due to the excessive signs of toxicity observed at the 3000 mg/kg/day dosage level. The 100 mg/kg/day dosage group was then composed of 14 rats originally designated for the high dose exposure group which had not yet reached gestation day 6 and an additional 11 mated rats. The substance was administered undiluted at varying dosage volumes to permit the administration of dosage levels of 100, 300, 1000 and 3000 mg/kg/day. Eleven rats in the 3000 mg/kg/day dosage group had already received the compound and these rats remained on treatment at this dosage level. Compound was administered orally by gavage as a single daily dose on gestation day 6 through 15. The control females were sham-intubated on a comparable regimen. Individual dosages were based on body weights taken on gestation days 6, 9 and 12.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Mating was initiated on June 5, 1978 and the last cesarean section was on July 7, 1978. Male rats of the same strain were used for mating. One female and one male were placed together for mating. The day of mating was determined by daily inspection for the copulatory plug or by vaginal smear for sperm. The day a plug or sperm was found was designated day 0 of gestation and the female was returned to an individual cage.

Duration of treatment / exposure:

10 days

Frequency of treatment:

Daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

3 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25, except the 3000 mg/kg bw dose group which consisted of 11 rats.

Control animals:

yes, sham-exposed

Details on study design:

Groups of pregnant CD strain rats were treated orally with CP Butyrate at dosages of 100, 300, 1000, and 3000 mg/kg/day on days 6 through 15 of gestation. On day 20 of gestation, fetuses were delivered by cesarean section, and the uterine horns were examined for implantation sites, resorptions, and number of viable or nonviable fetuses. All fetuses were weighed and examined for external anomalies. One-third of the fetuses in each treatment group were examined for soft tissue anomalies. Two-thirds of the fetuses were stained and examined for skeletal anomalies.

Examinations

Maternal examinations:

Prior to treatment, the rats were observed daily for mortality and overt changes in appearance and behavior. The females were observed daily for mortality and clinical signs of toxicity from gestation days 6 through 20. Individual female body weights were recorded on days 0, 6, 9, 12, 16 and 20 of gestation.

Ovaries and uterine content:

On day 20 of gestation, all females were sacrificed by an overdose of carbon dioxide and the fetuses were delivered by cesarean section. The number and location of viable and nonviable fetuses, early and late resorptions, total implantations and corpora lutea were recorded.

Fetal examinations:

All fetuses were subjected to a gross examination to determine sex and any external abnormalities. Approximately one-third of the fetuses were placed in Bouin’s fixative and later sectioned by the method described by Wilson to examine viscera. The remaining two-thirds of the fetuses were fixed in alcohol, macerated with potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson, and examined for skeletal anomalies.

Statistics:

All statistical analyses compared the treatment groups with the control group, with the level of significance at p<0.05. Male to female fetal sex ratio, number of litters and number of fetuses with anomalies were compared using the Chi-square test criterion with Yates correction for 2 x 2 contingency tables and/or Fisher’s exact probability test as described by Siegel to judge significance of differences. The proportion of early and late resorbed fetuses and post-implantation losses were compared by the Mann-Whitney U-test to judge significance of differences. Mean number of corpora lutea, total implantations, and viable fetuses were compared by analyses of variances (one-way classification), Bartlett’s test for homogeneity of variances and the appropriate t-test (for equal or unequal variances) using Dunnett’s multiple comparison tables to judge significance of differences. Fetal body weights were compared by analysis of variance (hierarchal classification) and t-test using described by Steel and Torrie using Dunnett's multiple comparison tables to judge significance of differences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Conjunctivitis was frequently observed in all groups in this study and was not considered to be treatment related. Occasional observations of nasal discharge, soft stool and hair loss were noted throughout the study, and also were not considered to be treatment related.
There were no changes in appearance or behaviour attributable to treatment in the 100 and 300 mg/kg/day dosage group when compared to the control group. An increase in matting and staining of the hair in the anogenital region was noted in the 3000 mg/kg/day dosage group when compared to the control group. Slight ataxia was seen following treatment in two rats in the 1000 mg/kg/day dosage group on one day, but was not apparent 3 hours after treatment. Slight to severe ataxia was observed in all rats in the 3000 mg/kg/day dosage group from ½ to 4 ½ hours after treatment at various times throughout the treatment period. Ataxia was predominantly observed early in the treatment period in the 3000 mg/kg/day dosage group, and the frequency and severity of this observation decreased as the treatment period progressed. Ataxia was not present prior to the next daily dose, with the exception of one rat in the 3000 mg/kg/dosage group, which died on gestation day 8. Lethargy was also observed in a few rats in this group early in the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One rat in the 3000 mg/kg/dosage group died on gestation day 8. Survival was 100% in all other groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean maternal body weight gains in the 100 mg/kg/day dosage group were comparable to the control group. A slight reduction in mean body weight gain was noted during the latter portion of the gestation period in the 300 mg/kg/day dosage group when compared to the control group. This reduction may be attributed to a number of females in this group for which the number of live fetuses was considerably less than the group mean. There was a moderate reduction in mean body weight gains during the treatment period and a slight reduction after the treatment period in the 1000 mg/kg/day dosage groups. This resulted in slightly reduced mean body weight gains over the entire gestation period in this group compared to the control group. Mean body weight losses in the first 3 days of treatment and reduced body weight gains during the latter portion of treatment and after the treatment period were noted in the 3000 mg/kg/day dosage group. This resulted in a severely reduced body weight gain over the entire gestation period for this group when compared to the control group.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Two masses in the mammary region were reported in this study. One rat in the 100 mg/kg/day dosage group was found to have a mass at gestation day 20, and one rat in the 3000 mg/kg/day dosage group was found to have a mass at gestation day 18. Upon histological examination, these masses were found to be mammary adenocarcinomas. The occurrence of the masses is not considered to be treatment related as they occurred within a few days following the 10 day treatment period.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

A slight, though not statistically significant, increase in the mean number of early resorptions and corresponding post-implantation loss was noted for the 100-, 300- and 1000 mg/kg/day dosage groups when compared to the control group. These differences did not occur in a dose-related pattern. There was a slight though not statistically significant, increase in the mean number of late resorptions and a statistically significant increase in the mean number of early resorptions in the 3000 mg/kg/day dosage groups when compared to the control group. The increase in the mean number of post-implantation losses was also statistically significant in this group compared to the control group.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

A slight, though not statistically significant, increase in the mean number of early resorptions and corresponding post-implantation losses was noted for the 100-, 300- and 1000 mg/kg/day dosage groups when compared to the control group. These differences did not occur in a dose-related pattern. There was a slight though not statistically significant, increase in the mean number of late resorptions and a statistically significant increase in the mean number of early resorptions in the 3000 mg/kg/day dosage groups when compared to the control group. The increase in the mean number of post-implantation losses was also statistically significant in this group compared to the control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no statistically significant or biologically meaningful differences in the mean number of nonviable fetuses in all of the treatment groups when compared to the control group.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

One rat in the 100 mg/kg/day dosage group delivered prior to the scheduled sacrifice date. This was due to an inaccurate determination of copulation.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A slight decrease in the mean number of corpora lutea and implantations was seen in the 100-, 300- and 1000 mg/kg/day dosage groups. There was an increase in the mean number of corpora lutea and a statistically significant increase in the mean number of implantations in the 3000 mg/kg/day dosage groups when compared to the control group. Since ovulation and implantation occurred prior to treatment in this study, these differences are considered to be attributed to random occurrence.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

(General toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

2

Dose descriptor:

NOAEL

Remarks:

(Maternal developmental toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

early or late resorptions

pre and post implantation loss

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weights were comparable to the control group in the 100 and 1000 mg/kg/day dosage groups. There was a statistically significant increase in mean fetal body weights in the 300 mg/kg/day dosage group when compared to the control group. This is attributed to a number of litters in this group in which the number of fetuses per dam was considerably less than the group mean, with fetal body weights greater than the group mean. There was a statistically significant decrease in the mean fetal body weights in the 3000 mg/kg/day dosage group when compared to the control group.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

There was a decrease in the mean number of viable fetuses in all of the treatment groups when compared to the control group. This decrease was slight in the 100 mg/kg/dosage group and moderate in the 300, 1000 and 3000 mg/kg/day dosage groups. The decrease in mean number of living fetuses in the 1000 mg/kg/day dosage group was statistically significant. This decrease in the 100, 300 and 1000 mg/kg/day dosage groups may be partially due to the decrease in the mean number of corpora lutea and implantations described previously in these dosage groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant or biologically meaningful differences in the male to female sex ratio in all of the treatment groups when compared to the control group.

External malformations:

no effects observed

Description (incidence and severity):

Variations were comparable in type and frequency for all of the treatment groups and the control group. There were no statistically significant differences in the number of litters with malformations between any of the treatment groups and the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Variations were comparable in type and frequency for all of the treatment groups and the control group. There were no statistically significant differences in the number of litters with malformations between any of the treatment groups and the control group. However, there was a statistically significant increase in the number fetuses with malformations in the 100 mg/kg/day dosage group when compared to the control group. The malformation that was observed in the 100 mg/kg/day dosage group, bent ribs, was also observed in the control group and in the 300 and 1000 mg/kg/day dosage groups. A significant increase in the number of fetuses with reduced ossification of the skull were seen in the 100 mg/kg dosage group. However, the number of these effects present in higher dosage groups were comparable to controls. No malformations were observed in the 3000 mg/kg/day dosage group.

Visceral malformations:

no effects observed

Description (incidence and severity):

Variations were comparable in type and frequency for all of the treatment groups and the control group. There were no statistically significant differences in the number of litters with malformations between any of the treatment groups and the control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

(Developmental toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

3 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Treatment of pregnant rats with the test substance caused maternal and developmental toxicity at a dosage of 3000 mg/kg bw, respectively. The NOAELs were determined to be 1000 mg/kg bw for both maternal and developmental toxicity.

Executive summary:

In a teratology study conducted similar to OECD TG 414, Charles River CD rats were dosed (orally) at levels of 0, 100, 300, 1000 or 3000 mg/kg/day on days 6 through 15 of gestation. Maternal body weights were determined on days 0, 6, 15 and 20 of gestation, and observations of maternal behavior were made daily. Fetuses were delivered by caesarean section on day 20 of gestation. The number of viable fetuses, corpora lutea, implantation sites, still births and resorptions were determined. Fetuses were weighed and examined for external, skeletal and visceral anomalies.

All rats in the 3000 mg/kg dosage group showed signs of ataxia at various times during the treatment period. One rat in this dosage group died after the second treatment, which is unlikely to be related to treatment. Brief signs of ataxia were also observed in two rats of the 1000 mg/kg bw dose group. There was a moderate reduction in mean body weight gain during the treatment period and a slight reduction after the treatment period in the 1000 mg/kg bw/day group. Reduced body weight during the first three days of treatment and reduced mean body weight gains was seen in the 3000 mg/kg bw dosage group. These reduced body weight gains resulted in a decreased body weight. There were no significant differences in the number of nonviable fetuses, corpora lutea, implantations or in the male to female sex ratios. An increase in post implantation loss was noted in the 3000 mg/kg bw group. A significant decrease in mean fetal body weights was also seen in this group. A significant increase in the number of fetuses with bent ribs and with reduced ossification of the skull were seen in the 100 mg/kg dosage group. However, the number of these effects present in higher dosage groups were comparable to controls. No malformations were seen in the 3000 mg/kg dosage group.

In conclusion, treatment of pregnant rats with the test substance caused maternal and developmental toxicity at a dosage of 3000 mg/kg bw. The NOAEL was determined to be 1000 mg/kg bw for maternal and developmental toxicity.