4-tert-pentylcyclohexanone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jun 2006 to 14 Jul 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

This information is used for read across to Orivone

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jun 2006 to 14 Jul 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The NOAEL of this study is used as a starting point for the NOAEL for Orivone for sub-chronic toxicity.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: FDA guideline ICH S5a

Version / remarks:

September 1994

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

female

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 at arrival
- 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. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals
- Diet: Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, MO) in individual feeders. Analyses were routinely performed by the feed supplier.
- 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
- Humidity (%): 30% to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12. Each dark period began at 1900 hours.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The Testing Facility prepared suspensions of the test article on a weekly basis. These formulated suspensions were stored at room temperature and were stirred continuously during administration to the animals, sampling and aliquotting.

VEHICLE
- Concentration in vehicle: Dose levels of 40, 140 and 640 mg/kg bw/day correspond to 4, 16 and 64 mg/mL concentrations in the dosing solution
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration samples from the first and last days formulations were prepared were within the acceptable limits of ±15% of target. All homogeneity results were within the acceptable range of ≤5% relative standard deviation (RSD). The values obtained were 0.4%, 1.2% and 0.3% RSD for the 4, 16 and 64 mg/mL formulations, respectively. Stability of the test article in the vehicle control article at a concentration of 64 mg/mL and under room temperature conditions was determined concurrently. The samples were found to be stable for a period of 14-days.

Duration of treatment / exposure:

Days 7 through 20 of presumed gestation (referred to as DG 7 to 20; 14 days).

Frequency of treatment:

Once daily on the basis of the individual body weights recorded before dosage.

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Dose / conc.:

160 mg/kg bw/day (actual dose received)

Dose / conc.:

640 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosages were selected on the basis of findings from a dosage-range study in which 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). Because 300 mg/kg/day, the highest dosage of the test substance 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.

Observations and examinations performed and frequency:

CAGE SIDE 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.

DETAILED CLINICAL OBSERVATIONS:
- 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:
- Body weights were recorded at least weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods.

FOOD CONSUMPTION:
Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21.

Sacrifice and pathology:

POST-MORTEM EXAMINATIONS:
- All surviving female rats were sacrificed by carbon dioxide asphyxiation on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

OTHER:
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.

Statistics:

- Clinical observations and other proportional data were analyzed 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 fetuses, percent resorbed conceptuses, fetal body weights and fetal 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 analyze the data.
- Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The 160 and 640 mg/kg/day dosages of the test substance 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 the test substance 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.

All other clinical observations were considered unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

One rat in the 640 mg/kg/day dosage group was sacrificed on day 20 of gestation (DG 20) because it had adverse clinical observations. These
observations were considered to be effects of treatment with the test substance 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.

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 the test substance 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 the test substance 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 the test substance as high as 160 mg/kg/day.

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only necropsy observations associated with treatment with the test substance were those observed in the 640 mg/kg/day dosage group rat that was sacrificed on DG 20 (distention of the stomach with gas and yellow fluid and a litter of 17 dead fetuses).

All other necropsy observations were considered unrelated to treatment with the test substance because the observation occurred in only one or two rats in any dosage group, without dosage-dependency. These observations included white caseous material present in the pericardium of one rat in the 40 mg/kg/day dosage group, a finding usually associated with a prior intubation accident, urinary tract findings (slight dilation of the pelvis of one or both kidneys, pitted areas in a kidney, a thick walled urinary bladder containing a calculus along with slight dilation of a ureter, which also contained a calculus), and a large sacral lymph node, observed in one or two rats in the 160 mg/kg/day dosage group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Critical effects observed:

no

Conclusions:

Oral administration of the test substance to pregnant Crl:CD(SD) female rats at dose levels of 0, 40, 160 and 640 mg/kg bw/day for 14 days revealed a no observed adverse effect level (NOAEL) of 160 mg/kg bw/day for maternal toxicity.

Executive summary:

A FDA guideline developmental toxicity study similar to OECDTG414 and GLP was performed. 25 pregnant Crl:CD(SD) rats were exposed orally (gavage) to 0 (vehicle), 40, 160 and 640 mg/kg/day during gestation days 7 through 20. 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 substance, 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 DG 0 and daily during the dosage and post dosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21. All surviving rats were sacrificed on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. All fetuses were weighed and examined for sex and gross external alterations. Approximately ½ of the fetuses in each litter were evaluated for soft tissue or skeletal alterations.

Results:Mortality: Except one rat. All other rats in this study survived to scheduled sacrifice.

One rat in the 640 mg/kg/day dosage group had severe adverse clinical observations attributable to the test substance and was sacrificed on DG 20. Additional clinical signs observed only in the 640 mg/kg/day dosage group rat that was sacrificed on DG 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 substance, distention of the stomach with gas and yellow fluid and a litter of 17 dead fetuses (the dead fetuses were otherwise normal for their developmental ages).

Clinical signs: The 160 and 640 mg/kg/day dosages of the test substance were associated with dosage dependent, statistically significant increases in clinical observations the incidences of excess salivation (total) and slight excess salivation (1 and 2 hours after intubation), as compared with the vehicle control group values. The 640 mg/kg/day dosage of the test substance also resulted in statistically significant increases in the incidences of moderate excess salivation, red perioral area 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. 

Body weight:Maternal body weightswere significantly reduced in the 640 mg/kg/day dosage group on DGs 9 through 21, with the exception of DG 11, as compared with the vehicle control group values. The 640 mg/kg/day dosage group also had significantly reduced absolute and relative feed consumption values for the entire dosage period. Pregnancy occurred in 24 to 25 rats in each dosage group. Fetal body weightswere significantly reduced at the 640 mg/kg/day dosage group, as compared with the vehicle control group values. No other Caesarean-sectioning or litter parameters were affected by dosages of as high as 640 mg/kg/day. The 640 mg/kg/day dosage of the test substance was associated with transient delays in fetal development including statistically significant increases in the fetal (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 fetal development were considered to be associated with the significant reductions in fetal body weight that also occurred at the 640 mg/kg/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. 

Overall: At the high dose of 640 mg/kg bw after dosing p-tert-butyl cyclohexyl acetate quite severe clinical signs, body weight and food reduction was seen in the dams at 640 mg/kg bw was seen with one dam that needed to be sacrificed on day 20. The next lower dose no such effects were seen. The maternal and developmental NOAEL were both 160 mg/kg bw/day. The maternal decrease in body weight and food consumption at the high dose resulted in transient delay of fetal development. These effects are therefore not considered to be selectively toxic to embryo-fetal development and did not result in teratogenicity. 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: FDA guideline ICH S5a

Version / remarks:

September 1994

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

4-tert-butylcyclohexyl acetate

EC Number:

250-954-9

EC Name:

4-tert-butylcyclohexyl acetate

Cas Number:

32210-23-4

Molecular formula:

C12H22O2

IUPAC Name:

4-tert-butylcyclohexyl acetate

Test animals

Species:

rat

Strain:

other: 1

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 at arrival
- 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. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals
- Diet: Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, MO) in individual feeders. Analyses were routinely performed by the feed supplier.
- 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
- Humidity (%): 30% to 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12. Each dark period began at 1900 hours.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The Testing Facility prepared suspensions of the test article on a weekly basis. These formulated suspensions were stored at room temperature and were stirred continuously during administration to the animals, sampling and aliquotting.

VEHICLE
- Concentration in vehicle: Dose levels of 40, 140 and 640 mg/kg bw/day correspond to 4, 16 and 64 mg/mL concentrations in the dosing solution
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration samples from the first and last days formulations were prepared were within the acceptable limits of ±15% of target. All homogeneity results were within the acceptable range of ≤5% relative standard deviation (RSD). The values obtained were 0.4%, 1.2% and 0.3% RSD for the 4, 16 and 64 mg/mL formulations, respectively. Stability of the test article in the vehicle control article at a concentration of 64 mg/mL and under room temperature conditions was determined concurrently. The samples were found to be stable for a period of 14-days.

Details on mating procedure:

After acclimation, 130 virgin female rats were placed into cohabitation with 130 breeder male rats, one male rat per female rat. The cohabitation period consisted of a maximum of five days. 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:

Days 7 through 20 of presumed gestation (DGs 7 through 20)

Frequency of treatment:

Once daily on the basis of the individual body weights recorded before dosage.

Duration of test:

All surviving rats were sacrificed on DG 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

160 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

640 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dosages were selected on the basis of findings from a dosage-range study in which 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). Because 300 mg/kg/day, the highest dosage of the test substance 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:

CAGE SIDE 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.

DETAILED CLINICAL OBSERVATIONS:
- 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:
- Body weights were recorded at least weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods.

FOOD CONSUMPTION:
Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21.

POST-MORTEM EXAMINATIONS:
- All surviving female rats were sacrificed by carbon dioxide asphyxiation on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

OTHER:
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:

- Uteri of apparently nonpregnant rats were examined while being pressed between glass plates, to confirm the absence of implantation sites.
- The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantation sites, live and dead fetuses and early and late resorptions.
- Placentae were examined for size, color and shape.

Fetal examinations:

- Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative.
- Each fetus was subsequently weighed and examined for sex and gross external alterations.
- Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations, using a variation of Wilson’s sectioning technique
- The remaining fetuses (approximately one-half of the fetuses in each litter) were eviscerated, initially fixed in alcohol, and then cleared, stained with alizarin red S(7) and examined for skeletal alterations.

Statistics:

- Clinical observations and other proportional data were analyzed 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 fetuses, percent resorbed conceptuses, fetal body weights and fetal 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 analyze 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 the test substance 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 the test substance 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.

All other clinical observations were considered unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

One rat in the 640 mg/kg/day dosage group was sacrificed on day 20 of gestation (DG 20) because it had adverse clinical observations. These
observations were considered to be effects of treatment with the test substance 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.

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 the test substance 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 the test substance 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 the test substance as high as 160 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only necropsy observations associated with treatment with the test substance were those observed in the 640 mg/kg/day dosage group rat that was sacrificed on DG 20 (distention of the stomach with gas and yellow fluid and a litter of 17 dead fetuses).

All other necropsy observations were considered unrelated to treatment with the test substance because the observation occurred in only one or two rats in any dosage group, without dosage-dependency. These observations included white caseous material present in the pericardium of one rat in the 40 mg/kg/day dosage group, a finding usually associated with a prior intubation accident, urinary tract findings (slight dilation of the pelvis of one or both kidneys, pitted areas in a kidney, a thick walled urinary bladder containing a calculus along with slight dilation of a ureter, which also contained a calculus), and a large sacral lymph node, observed in one or two rats in the 160 mg/kg/day dosage group.

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:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

All placentae appeared normal.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-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.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- Skeletal examination of the externally malformed fetus with no tail in the 160 mg/kg/day dosage group revealed expected absence of lumbar, sacral and caudal vertebrae. No other alterations occurred in this fetus.
- Duplication of the manubrium and the 1st through 3rd sternal centra occurred in one fetus in the 40 mg/kg/day dosage group. No other alterations occurred in this fetus.
- One or both of the arches in the 6th cervical vertebra had the appearance of an arch in the 7th cervical vertebra in 1, 1, 1 and 4 fetuses from 1, 1,1 and 2 litters in the 0 (Vehicle), 40, 160 and 640 mg/kg/day dosage groups, respectively. No other alterations occurred in these fetuses.
- A bifid centrum in the 11th, 12th or 13th thoracic vertebra occurred in 0, 2, 2 and 3 fetuses from 0, 2, 2 and 3 litters in the four respective dosage groups. No additional alterations occurred in these fetuses.
- Presence of a cervical rib at the 7th cervical vertebra, a common variation in this strain of rat, was observed in 1, 2 and 2 fetuses from 1, 2 and 2 litters in the 0 (Vehicle), 40 and 160 mg/kg/day dosage groups, respectively. Fetus 19417-14 in the vehicle control group also had a short 13th rib. No other alterations occurred in any of these fetuses.
- A short 13th rib occurred in one control group fetus and in two fetuses from a 640 mg/kg/day dosage group litter, respectively. Vehicle control group fetus 19417-14 also had a rib present on the 7th cervical vertebra, as previously described. No other alterations occurred in the fetuses in the 640 mg/kg/day dosage group.
- The average numbers of ossified caudal vertebrae, forelimb phalanges and hindlimb metatarsals and phalanges were significantly reduced (p≤0.05 or p≤0.01) in the 640 mg/kg/day dosage group, as compared with the vehicle control group values. The average value for ossified forelimb phalanges (7.76) was within the historical range of the Testing Facility; however, the litter averages for ossified caudal vertebrae (6.58), hindlimb metatarsals (4.48) and hindlimb phalanges (5.40) were below the historical range for the Testing Facility. In 50 studies conducted at the Testing Facility between June 2004 and June 2006 (1113 control group litters; 8312 fetuses), the litter averages were 7.47 per fetus (range 6.69-8.23 per study) for caudal vertebrae, 4.84 per fetus (range 4.67-5.00 per study) for metatarsals, and 6.23 per fetus (range 5.54-7.75 per study) for hindlimb phalanges.
- There were no other statistically significant or biologically important differences among the four dosage groups in the average num ers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar and sacral), ribs, sternum (manubrium, sternal enters and xiphoid), forelimbs (carpals and metacarpals) or hindlimbs (tarsals).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Three fetuses from three control group litters and two 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.
- One fetus in each of the 40 and 640 mg/kg/day dosage groups had a folded retina in the right eye, a finding that is usually an artifact of fixation and/or sectioning. No other alterations occurred in these fetuses.
- The umbilical artery descended to the left of the urinary bladder in 3, 1 and 1 fetuses from 3, 1 and 1 litter in the 0 (Vehicle), 40 and 160 mg/kg/day dosage groups, respectively. No other alterations occurred in these fetuses.
- Slight or moderate dilation of the pelvis of one or both kidneys, a reversible developmental delay, occurred in 1, 2, 0 and 8 fetuses from 1, 2, 0 and 2 litters in the four respective dosage groups. Although the fetal incidence of moderate enlargement of the pelvis of both kidneys was significantly increased (p≤0.01) in the 640 mg/kg/day dosage group, as compared to the vehicle control group value, the litter incidence (one), the more relevant parameter, was not. One vehicle control group fetus also had moderate dilation of the renal pelvis in the right kidney. No other alterations occurred in any of these fetuses. In 50 studies conducted at the Testing Facility between June 2004 and June 2006 (1112 control group litters; 7805 fetuses), 10 litters (0.9%) and 11 fetuses (0.14%) had slight dilation of the pelvis of one or both kidneys, with a maximum of 3 (13.6%) litters and 4 fetuses (2.6%) per study, and 3 litters (0.27%) and 3 fetuses (0.04%) had moderate dilation of the pelvis of one or both kidneys, with a maximum of 1 (4.5%) litter and 1 fetus (0.7%) per study.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

visceral malformations

Remarks on result:

other: visceral and skeletal effects are associated with reduced body weight

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: vertebra

skeletal: hindlimb

visceral/soft tissue: urinary

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

640 mg/kg bw/day (actual dose received)

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:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL of maternal and developmental toxicity is 160 mg/kg bw, based on decreases in body weight and food consumption in dams, resulting in lower body weights and delayed development in pups.

Executive summary:

A FDA guideline developmental toxicity study similar to OECDTG414 and GLP was performed. 25 pregnant Crl:CD(SD) rats were exposed orally (gavage) to 0 (vehicle), 40, 160 and 640 mg/kg/day during gestation days 7 through 20. 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 substance, 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 DG 0 and daily during the dosage and post dosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18, 20 and 21. All surviving rats were sacrificed on DG 21, Caesarean-sectioned and a gross necropsy of the thoracic, abdominal and pelvic viscera performed. All fetuses were weighed and examined for sex and gross external alterations. Approximately ½ of the fetuses in each litter were evaluated for soft tissue or skeletal alterations.

Results:Mortality: Except one, all 25 other rats in this study survived to scheduled sacrifice.

One rat in the 640 mg/kg/day dosage group had severe adverse clinical observations attributable to the test substance and was sacrificed on DG 20. Additional clinical signs observed only in the 640 mg/kg/day dosage group rat that was sacrificed on DG 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 substance, distention of the stomach with gas and yellow fluid and a litter of 17 dead fetuses (average is 14 +/- 3). These dead fetuses were otherwise normal for their developmental ages.

Clinical signs: The 160 and 640 mg/kg/day dosages of the test substance were associated with dosage dependent, statistically significant increases in clinical observations the incidences of excess salivation (total) and slight excess salivation (1 and 2 hours after intubation), as compared with the vehicle control group values. The 640 mg/kg/day dosage of the test substance also resulted in statistically significant increases in the incidences of moderate excess salivation, red perioral area 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. 

Body weight:Maternal body weightswere significantly reduced in the 640 mg/kg/day dosage group on DGs 9 through 21, with the exception of DG 11, as compared with the vehicle control group values. The 640 mg/kg/day dosage group also had significantly reduced absolute and relative feed consumption values for the entire dosage period. Pregnancy occurred in 24 to 25 rats in each dosage group. Fetal body weightswere significantly reduced at the 640 mg/kg/day dosage group, as compared with the vehicle control group values. No other Caesarean-sectioning or litter parameters were affected by dosages of as high as 640 mg/kg/day. The 640 mg/kg/day dosage of the test substance was associated with transient delays in fetal development including statistically significant increases in the fetal (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 fetal development were considered to be associated with the significant reductions in fetal body weight that also occurred at the 640 mg/kg/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. 

Overall: At the high dose of 640 mg/kg bw after dosing p-tert-butyl cyclohexyl acetate quite severe clinical signs, body weight and food reduction was seen in the dams at 640 mg/kg bw was seen with one dam that needed to be sacrificed on day 20. The next lower dose no such effects were seen. The maternal and developmental NOAEL were both 160 mg/kg bw/day. The maternal decrease in body weight and food consumption at the high dose resulted in transient delay of fetal development. These effects are therefore not considered to be selectively toxic to embryo-fetal development and did not result in teratogenicity.