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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
Study period:
June-July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
cis-4-tert-butylcyclohexyl acetate
EC Number:
233-881-7
EC Name:
cis-4-tert-butylcyclohexyl acetate
Cas Number:
10411-92-4
Molecular formula:
C12H22O2
IUPAC Name:
4-tert-butylcyclohexyl acetate
Constituent 2
Chemical structure
Reference substance name:
trans-4-tert-butylcyclohexyl acetate
EC Number:
217-598-6
EC Name:
trans-4-tert-butylcyclohexyl acetate
Cas Number:
1900-69-2
Molecular formula:
C12H22O2
IUPAC Name:
4-tert-butylcyclohexyl acetate
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 65 days
- Weight at study initiation: 221-257 g
- Housing: Rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat’s cage.
- Diet: Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, MO) in individual feeders.
- Water:Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C to 26°C
- Humidity (%): 30% to 70%
- Air changes (per hr): a minimum of ten changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The oral (gavage) route was selected for use because, in comparison with the dietary route, the exact dosage can be accurately administered.

PREPARATION OF DOSING SOLUTIONS:
Prepared formulations were stirred continuously during administration of the control and test article formulations.
Concentration in vehicle: 0, 4, 16, 64 mL/kg)

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test article was considered 100% active/pure for the purpose of dosage calculations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male rat per female rat
- Length of cohabitation: a maximum of five days.
- Proof of pregnancy: Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be DG 0 and assigned to individual housing.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Rats were administered the test article and/or the vehicle once daily on DGs 7 through 20.
The rats were administered the test article and/or the vehicle at approximately the same time each day.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
160 mg/kg bw/day
Dose / conc.:
640 mg/kg bw/day
No. of animals per sex per dose:
25 rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages were selected on the basis of findings from a dosage-range study (Protocol TIF00021) in which 4-tBCHA dosages of 37.5, 50, 150 and 300 mg/kg/day were administered to pregnant rats once daily for 14 consecutive days (DGs 7 through 20). No mortality occurred at dosages as high as 300 mg/kg/day. At 50 mg/kg/day and higher, slight excessive salivation and urine-stained abdominal fur were observed. In the 50, 150 and 300 mg/kg/day dosage groups, increased numbers of rats lost weight after the first dosage (DGs 7 to 8), as compared with the vehicle control group incidence. A dosage-dependent reduction in maternal body weight gain was observed after the second dosage (DGs 8 to 9). Body weights and body weight gains were comparable for the entire dosage period between the 4-tBCHA and vehicle control dosage groups. Absolute and relative feed values were reduced in the 50 and 300 mg/kg/day dosage groups for the first three days of treatment, as compared to the vehicle control group value. One rat in the 150 mg/kg/day dosage group had a very high feed consumption value during this period, which, when removed, resulted in a dose-dependent reduction in feed consumption for the first three days of treatment. Absolute and relative feed consumption values were comparable between the 4-tBCHA and control groups for the entire dosage period. Dosages of 4-tBCHA as high as 300 mg/kg/day did not adversely affect Caesarean-sectioning, litter, or fetal gross external evaluations.
Because 300 mg/kg/day, the highest dosage of 4-tBCHA tested in the dosage-range finding study, produced only transient reductions in maternal body weight gain and feed consumption values, a higher dosage, 640 mg/kg/day, was selected as the high dosage for the full study. This dosage was expected to reduce maternal body weight and feed consumption for the entire dosage period.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS:
The rats were observed for viability at least twice each day of the study. Observations for clinical signs and general appearance were made at least weekly during the acclimation period and on DG 0. The rats were also examined for clinical observations, abortions, premature deliveries and deaths before and between one and two hours after dosage and once daily during the postdosage period

BODY WEIGHT: yes
- Time schedule for examinations: recorded at least weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: gross lesions. Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.
The 640 mg/kg/day dosage group rat that was sacrificed on DG 20 was examined for gross lesions, pregnancy status and uterine contents. The lungs, trachea and esophagus were perfused with neutral buffered 10% formalin; these perfused tissues, along with the heart, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Clinical observations and other proportional data were analysed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male foetuses, percent resorbed conceptuses, foetal body weights and foetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyse the data. Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The 160 and 640 mg/kg/day dosages of 4-tBCHA were associated with dosage-dependent, statistically significant increases (p≤0.05 or p≤0.01) in the incidences of excess salivation (total) and slight excess salivation, as compared with the vehicle control group values. The 640 mg/kg/day dosage of 4-tBCHA also resulted in statistically significant increases (p≤0.01) in the incidences of moderate excess salivation, red perioral substance and sparse hair coat on the limbs and increased incidences of extreme excess salivation, ungroomed coat, sparse hair coat (total incidence for underside and limbs) and localized alopecia of the limbs and/or neck. Excess salivation was observed after dosing, approximately one to two hours following intubation. Additional clinical signs observed only in the 640 mg/kg/day dosage group rat that was sacrificed on DG 20 for humane reasons included extreme excess salivation, scant feces, decreased motor activity, ptosis, coldness to touch and apparent dehydration, as previously described.
All other clinical observations were considered unrelated to treatment with 4-tBCHA because: 1) the incidences in the 40, 160 or 640 mg/kg/day dosage groups did not significantly differ from the vehicle control group values (total and slight excess salivation, urine-stained abdominal fur, ungroomed coat, sparse hair coat (total and/or underside), soft or liquid feces, misaligned incisor(s), a scab on the mouth, chromodacryorrhea and chromorrhinorrhea; or 2) the observation occurred only in the vehicle control group (a black colored tip of the tail).
Mortality:
no mortality observed
Description (incidence):
One rat (19476) in the 640 mg/kg/day dosage group was sacrificed on day 20 of gestation (DG 20) because it had adverse clinical observations, as fully described below. These observations were considered to be effects of treatment with 4-tert butylcyclohexyl acetate (4-tBCHA) because they occurred at the highest dosage level tested and some of these adverse clinical observations also occurred in surviving rats in this dosage group.
All other rats in this study survived to scheduled sacrifice on DG 21.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The 640 mg/kg/day dosage group had significantly reduced (p≤0.05 or p≤0.01) average maternal body weights on DGs 9 through 21 (with the exception of DG 11, when the reduction in body weight gain was not significantly less than the vehicle control group value). These values reflected a significant reduction (p≤0.01) in maternal body weight gain for the entire treatment period (calculated as DGs 7 to 21) to 32% of the vehicle control group value and weight losses or reduced weight gains within this period, as described in the following information. Significant body weight losses (p≤0.01) occurred on DGs 7 to 8 and 8 to 9, after the first two 640 mg/kg/day dosages of 4-tBCHA were administered, and significant reductions (p≤0.01) in body weight gains occurred on DGs
15 to 18 and 18 to 21, as compared with the vehicle control group values. Maternal body weight gains on DGs 7 to 10 and for the entire gestation period (DGs 0 to 21) were also significantly reduced (p≤0.01), as compared with the vehicle control group value.

Dosages of 4-tBCHA as high as 160 mg/kg/day did not affect maternal body weight gains or body weights. All values in the 40 and 160 mg/kg/day dosage groups were comparable to and did not significantly differ from those of the vehicle control group.
Average body weight gains in the 40 and 160 mg/kg/day dosage groups were 103% and 102% of the vehicle control group value, respectively, on DGs 7 to 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative feed consumption values were significantly reduced (p≤0.01) for the entire dosage period (calculated as DGs 7 to 21) and at all intervals within this dosage period in the 640 mg/kg/day dosage group, as compared with the vehicle control group values. Absolute feed consumption values in the 40, 160 and 640 mg/kg/day dosage groups were 100%, 102% and 71% of the vehicle control group value, respectively, on DGs 7 to 21. Relative feed consumption values in these same respective dosage groups were 100%, 102% and 74% of the vehicle control group value on DGs 7 to 21.
Absolute and relative feed consumption values were unaffected by dosages of 4-tBCHA as high as 160 mg/kg/day.

Maternal developmental toxicity

Number of abortions:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Caesarean-Sectioning and Litter Observations
Pregnancy occurred in 24 or 25 rats in each dosage group. As a result of the mortality in the 640 mg/kg/day dosage group, Caesarean-sectioning observations on DG 21 were based on 24, 24, 25 and 24 pregnant rats with one or more live fetuses in the four respective dosage groups.
Fetal body weights (combined sexes and male and female) were significantly reduced (p≤0.01) in the 640 mg/kg/day dosage group, as compared with the vehicle control group values. The average value for combined male and female fetal body weights in the 640 mg/kg/day dosage group was approximately 11% less than the vehicle control group value.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No other Caesarean-sectioning or litter parameters were affected by dosages of 4-tBCHA as high as 640 mg/kg/day. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, the percentage of resorbed conceptuses and the percentage of live male fetuses were comparable among the four dosage groups and did not significantly differ. No dam had a litter consisting of only resorbed conceptuses, and there were no dead fetuses. All placentae appeared normal.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Fetal gross external alterations were limited to a single occurrence of an absent tail in the 160 mg/kg/day dosage group (fetus 19458-7). Skeletal examination of this fetus confirmed the gross absence of the tail, which was evident as the presence of fewer than normal ossified lumbar, sacral and caudal vertebrae (5, 1 and 0, respectively; usually there are 6, 3 and 7 ossified lumbar, sacral and caudal vertebrae, respectively).
No other gross external fetal alterations occurred.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Vertebrae: Skeletal examination of the externally malformed fetus with no tail (19458-7) in the 160 mg/kg/day dosage group revealed expected absence of lumbar, sacral and caudal vertebrae, as previously described. No other alterations occurred in this fetus.
Sternum: Duplication of the manubrium and the 1st through 3rd sternal centra occurred in one fetus (19440-15) in the 40 mg/kg/day dosage group. No other alterations occurred in this fetus.
No other skeletal malformations occurred.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Genitalia: Three (19404-13, 19411-2 and 19417-15) fetuses from three control group litters and two (19438-6 and 19443-2) fetuses from two litters in the 40 mg/kg/day dosage groups, respectively, had malpositioned testes. No other alterations occurred in any of these fetuses.
No other soft tissue malformations occurred.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test material is not selectively toxic to embryo-foetal developmental and did not result in teratogenicity, even at maternally toxic dosage.
Executive summary:

A pre-natal / developmental toxicity study was conducted according to the a US FDA guideline, similar to OECD 414 and in compliance with GLP. The test material or the vehicle, corn oil, was administered orally (via gavage) once daily on days 7 through 20 of presumed gestation (GD 7 through 20) at dosage of 0, 40, 160 and 640 mg/kg bw/d to Crl:CD(SD) rats (25/group). DG 0 was the day when sperm were observed in a smear of the vaginal contents or a plug was observed in situ. The dosage volume (10 mL/kg) was adjusted daily on the basis of the individual body weights recorded before intubation.

All rats were examined for clinical observations of effects of the test article, abortions, premature deliveries and deaths before dosage, between one and two hours after dosage and once daily during the post-dosage period. Body weights were recorded on GD 0 and daily during the dosage and post-dosage periods. Feed consumption values were recorded on GDs 0, 7, 10, 12, 15, 18, 20 and 21. All surviving rats were sacrificed on GD 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. All foetuses were weighed and examined for sex and gross external alterations.

Approximately ½ of the foetuses in each litter were evaluated for soft tissue or skeletal alterations.

One rat in the 640 mg/kg bw/day dosage group had severe adverse clinical observations attributable to the test material and was sacrificed on GD 20. All other rats in this study survived to scheduled sacrifice.

The 160 and 640 mg/kg bw/day dosages were associated with dosage-dependent, statistically significant increases in the incidences of excess salivation (total) and slight excess salivation, as compared with the vehicle control group values. The 640 mg/kg bw/day dosage also resulted in statistically significant increases in the incidences of moderate excess salivation, red perioral substance and sparse hair coat on the limbs and increased incidences of extreme excess salivation, ungroomed coat, sparse hair coat and localized alopecia of the limbs and/or neck. Excess salivation was observed after dosing, approximately one to two hours following intubation. Additional clinical signs observed only in the 640 mg/kg bw/day dosage group rat that was sacrificed on GD 20 included extreme excess salivation, scant feces, decreased motor activity, ptosis, coldness to touch and apparent dehydration. This rat also had the only necropsy observations associated with treatment with the test material, distension of the stomach with gas and yellow fluid and a litter of 17 dead foetuses (the dead foetuses were otherwise normal for their developmental ages).

Maternal body weights were significantly reduced in the 640 mg/kg bw/day dosage group on GDs 9 through 21, with the exception of GD 11, as compared with the vehicle control group values. These reductions in maternal body weight reflected significantly reduced body weight gains for the entire treatment period (calculated as GDs 7 to 21), significant weight losses on GDs 7 to 8 and 8 to 9, after the first two dosages were administered, and significantly reduced body weight gains on GDs 15 to 18 and 18 to 21, as compared with the vehicle control group values.

The 640 mg/kg bw/day dosage group also had significantly reduced absolute and relative feed consumption values for the entire dosage period (calculated as GDs 7 to 21) and at all calculated intervals within this dosage period, as compared with the vehicle control group values.

Pregnancy occurred in 24 to 25 rats in each dosage group. Foetal body weights were significantly reduced at the 640 mg/kg bw/day dosage group, as compared with the vehicle control group values. The average value for combined male and female fetal body weights in the 640 mg/kg bw/day dosage group was approximately 11% less than the vehicle control group value.

No other Caesarean-sectioning or litter parameters were affected by dosages of test material as high as 640 mg/kg bw/day. The litter averages for corpora lutea, implantations, litter sizes, live foetuses, early and late resorptions, the percentage of resorbed conceptuses and the percentage of live male foetuses were comparable among the four dosage groups and did not significantly differ.

No dam had a litter consisting of only resorbed conceptuses, and there were no dead foetuses. All placentae appeared normal.

The 640 mg/kg bw/day dosage was associated with transient delays in fetal development including statistically significant increases in the foetal (but not the litter) incidences of moderate enlargement of the renal pelvis of one or both kidneys and reversible delays in ossification of the caudal vertebrae, fore- and hind-limb phalanges and hind-limb metarsals. The delays in foetal development were considered to be associated with the significant reductions in foetal body weight that also occurred at the 640 mg/kg bw/day dosage level and are frequent observations at dosages that produced reductions in maternal body weight gain and feed consumption, as occurred in this study.

On the basis of these data, the maternal NOAEL is 160 mg/kg bw/d. The only observation associated with treatment with the test material at this dose-level was moderate excess salivation occurring once in each of two rats. This finding is a common response to a strong tasting substance and was therefore not considered as adverse. The 640 mg/kg bw/d dose-level was associated with body weight losses, reduced body weight gains and feed consumption values.

One female in this dose group was sacrificed due to severe adverse clinical observation. It is unclear whether this death, occurring in a single animal, should be considered as an adverse effect of the test substance.

The developmental NOAEL is also 160 mg/kg bw/d. Transient retardations in foetal development associated with 640 mg/kg bw/d dosage included a significant reduction in foetal body weight and associated significant increases in moderate dilation of the renal pelvis and delayed ossification of the caudal vertebrae, fore- and hind-limb phalanges and hind-limb metatarsals.

Based on the results of this study, 4 -tert-butylcyclohexyl acetate is not selectively toxic to embryo-foetal developmental and did not result in teratogenicity, even at maternally toxic dosage.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for developmental toxicity / teratogenicity endpoint.