Cyclohexanepentanol, .beta.-methyl-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-26 to 2013-02-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 1996-03-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-02-13

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol

EC Number:

700-146-1

Cas Number:

1141487-54-8

Molecular formula:

C12H24O

IUPAC Name:

(R,S)-5-cyclohexyl-2-methyl-pentan-1-ol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11-12 Weeks
- Weight at study initiation: males: 252.81 to 350.93 g; females: 188.74 to 240.78 g
- Housing:
premating: two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 175 mm), with stainless steel top grill
mating: two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill
post mating: males were housed with their former cage mates; females were housed individually in polysulfone cages
- Sterilised nesting material (paper shreds) was provided near-term.
- Steam sterilized clean Corn cob was used as bedding.
- Diet (ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified)
- Water (ad libitum): deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature: between 20 and 24°C
- Relative humidity: between 49 and 67 percent
- Air changes: 12.8 – 13.7 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item was weighed in beakers and mixed with a small quantity of corn oil and stirred using a glass rod. The mixture was
transferred into the measuring cylinder. The volume was made up with the vehicle to get the final concentration of 20, 60 and 200 mg/mL for the G2, G3 and G4/G4R groups, respectively.
Following procedure was followed when 50 mL of dose formulation prepared:
The quantities of 1.0, 3.0 and 10.0 g of test item was weighed and mixed with a small quantity of corn oil. The mixture was transferred into the measuring cylinder. The volume was made up to 50 mL with the vehicle to get the final concentration of 20, 60 and 200 mg/mL for the G2, G3 and G4/G4R groups, respectively.
The suspension was stirred during sampling/gavage.

VEHICLE
- Justification for use and choice of vehicle: corn oil was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study.
In the dose range finder it was mentioned that the test item forms clear solution in corn oil, while it could not be dissolved/suspended in water, 0.5% aqueous sodium carboxy methyl cellulose in Milli Q water with 0.1% tween 80.

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: until there is evidence of sperms in the vaginal smear and /or vaginal plug or for a maximum period of 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to Gestation Day 0
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility
- After successful mating each pregnant female was caged (how): individually
- pre-coital time was calculated for each female

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, the prepared dose suspension was sampled in duplicate sets (two samples were drawn from each of
the top, middle and bottom layers for each set) from each dose levels on Day 1 and during 2nd month of the treatment period. Similarly, two samples from
middle layer were drawn from vehicle control for each set.
The estimation of the test item in the dose formulations was carried out by injecting the prepared sample solutions into Gas Chromatograph equipped with Flame Ionization Detector (GC-FID).

Results:
The test item was found to be homogeneous in all the dose formulation samples which met the acceptance limits of variation (85 to 115%) from the claimed concentrations and % RSD less than 10 %.
Based on the results, the test item was found to be stable for up to 24 hours at 1 and 200 mg/mL concentrations when stored at room temperature.

Duration of treatment / exposure:

- Males: 2 weeks prior to mating, during mating and up to and including the day before sacrifice which was done after the completion of the mating process (but at least a minimum period of four weeks).
- Females: 2 weeks prior to the mating period, during mating, pregnancy and up to lactation day 4

Frequency of treatment:

Males and females: once daily at approximately the same time each day (varying by ± 2 hours)

Details on study schedule:

not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE RANGE FINDER STUDY (Malleshappa, 2012)
- Dose selection rationale: the dose levels of 100, 300 and 1000 mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats and in consultation with the Sponsor.

The test item was weighed and dissolved in corn oil and administered to rats at the graduated dose levels of 100, 300 and 1000 mg/kg/day via oral gavage once daily for a period of 14 consecutive days. The rats in the vehicle control group received vehicle alone. The dose volume administered was 5 mL/kg body weight. Each group in the experiment was comprised of five male and five female rats.
All animals were observed for clinical signs (once daily) and mortality/morbidity (twice daily, except on holidays). Ophthalmological examination was performed before the start of treatment period and at the end of treatment. Body weights were measured on Days 1, 4, 8, 11 and 14 of treatment period and food consumption was measured on Days 4, 8, 11 and 14. On Day 15 of treatment, blood samples were collected for haematology and clinical chemistry. The rats were fasted overnight (water allowed) and subjected to a detailed necropsy and study plan specified organs were collected and weighed.
Results:
- no clinical sign or mortality in any of the groups during the experimental period.
- no ocular abnormalities were observed in animals of either sex of test item treated groups.
- mean body weights, body weight gain and food consumption of test item treated groups were comparable to vehicle control group during the treatment period.
- no test item-related changes in the various parameters evaluated in haematology, coagulation analysis and clinical chemistry.
- no test item-related changes in the terminal fasting body weights, organ weights and organ weight ratios in treated group animals when compared to the vehicle control group.
- no test item-related gross pathological changes among the doses tested.

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (1 to 2 hours after dosing), except mortality and morbidity, which was observed twice daily (once on holidays)
- Cage side observations checked: appearance, behaviour, clinical/toxic signs, mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment and at weekly intervals during treatment and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations:
- Males: initially and at weekly intervals thereafter
- Females: initially and at weekly intervals thereafter till cohabitation with males; Gestation Days (GD) 0, 7, 14 and 20 and on lactation days 0 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

PARTURITION:
- The duration of gestation was calculated from day ‘0’ of pregnancy to the day of parturition (Gestation Length).
- Females were observed for signs of difficult or prolonged parturition.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis (5 males in the control and high dose groups).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: not applicable

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- external deformities (dead and alive births)
- number and sex of pups
- pup body weight (days 0 and 4 post-partum)
- daily observations of the number of alive, dead and cannibalized pups

Postmortem examinations (parental animals):

SACRIFICE
All rats in this study were sacrificed at term after overnight fasting. Rats were anaesthetized with isoflurane, weighed and exsanguinated and subjected to detailed necropsy.
- males: after mating process
- females: on lactation day 5

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following tissues were prepared for microscopic examination (all adult animals): all gross lesions, epididymides, ovaries, uterus (with oviducts and cervix), vagina, prostate, seminal vesicles and coagulating glands, and testes
- The following tissues were prepared for organ weighing (all adult animals): epididymides, ovaries, uterus (with oviducts and cervix), prostate, seminal vesicles and coagulating glands, and testes
- The following tissues were prepared for microscopic examination (5 males / 5 females from main groups and all animals from recovery group): axillary lymph nodes, adrenals, brain (cerebrum, cerebellum and pons), bone marrow smear, cecum, colon, duodenum, femur with marrow, heart, ileum with Peyer’s patches, jejunum, kidneys, liver, lungs, mandibular lymph nodes, mesenteric lymph nodes, rectum, sciatic nerve, spinal cord, spleen, sternum with marrow, stomach, thymus, thyroid with parathyroid, trachea, and urinary bladder
- The following tissues were prepared for organ weighing (5 males / 5 females from main groups and all animals from recovery group): adrenals, brain (cerebrum, cerebellum and pons), heart, kidneys, liver, spleen, thymus, and thyroid with parathyroid

Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups were examined microscopically for histopathological changes. The uterus, cervix and vagina were examined in the remaining females from control and high dose groups.

For apparently non-pregnant rats, the uteri were stained with ammonium sulphide solution to identify the pre-implantation loss of the embryos. The
number of implantation sites and corpora lutea was recorded for all the dams.

Postmortem examinations (offspring):

SACRIFICE
- All surviving pups were sacrificed on lactation Day 4.

GROSS NECROPSY
- All surviving pups were necropsied on lactation Day 4. Dead and moribund pups were examined for possible defects and/or cause of
death.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical
package ver.12.0. All quantitative variables like body weight, food intake, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found significant.
Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea and implantations, pre-coital interval and gestation length (days) were analysed after
suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.
Z test was performed for testing the differences in proportions for mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.

Reproductive indices:

- male mating index (%) = (number of males with evidence of mating/number of males cohabited) x 100
- male fertility index (%) = (number of males siring a litter/number of males cohabited) x 100
- female mating index (%) = (number of females mated/number of females cohabited) x 100
- fecundity index (%) = (number of pregnant females (confirmed at necropsy)/ number of females with confirmed mating) x 100
- female fertility index (%) = (number of pregnant females (confirmed at necropsy)/number of females used for mating) x 100
- mean number of corpora lutea (CL)/group = total number of CL/total number of pregnant animals
- mean number of implantations/group = total number of implantations / total number of pregnant animals
- implantation index = (No. of implantation sites/No. of corpora lutea) x 100
- percentage of pre-implantation loss per group = (number of CL - number of implantations / number of CL) x 100
- post implantation loss (%) = ((number of implantations - number of live foetuses/pups) / number of implantations) x 100
- gestation index = (number of females with live pups born / number of females with evidence of pregnancy (confirmed at necropsy)) x 100

Offspring viability indices:

- mean litter size per group = (total number of foetuses/pups) / total number of pregnant /littered animals
- live birth index (%) = (number of viable pups born (at first observation) / total number of pups born (at first observation)) x 100
- Day 4 survival index (%) = (number of viable pups on lactation Day 4 / number of viable pups born) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- no clinical signs or mortality was observed during the course of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- body weights and food consumption were not test item-related affected at all the doses tested; significantly decreased body weight on GD20 and weight changes during gestation (GDs 0-7, 14-20 and 0-20) were correlated with lower mean litter size and thus not a sign of systemic toxicity.
- maternal food consumption during gestation was not affected by the treatment at all the doses.
- maternal body weights and food consumption during lactation periods were unaffected at all the doses tested.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- spermatogenesis was normal.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- 100 and 300 mg/kg Bwt/day doses: no treatment-related effects on the mean pre-coital time and gestation length.
- 100 and 300 mg/kg Bwt/day doses: no treatment-related changes were observed in the uterine/implantation data.
- 1000 mg/kg Bwt/day dose: treatment resulted in significantly increased mean precoital time and mean gestational length.
- 1000 mg/kg Bwt/day dose: treatment significantly increased the post- implantation loss, and a significantly lower gestation index was found.
- no treatment-related changes were observed on the fertility indices of sires and dams.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- no test item related changes in the organ weights and organs weight ratios in both males and females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- no test item related gross changes in both males and females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- no test item related microscopic changes in both males and females.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No clinical signs, mortality and neurological dysfunctions; no effectson on body weight and food consumption; no changes in haematology, clinical chemistry and urinalysis; no effects on organ weights, pathology and histopathology.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
- 100 and 300 mg/kg Bwt/day doses: no treatment-related effects on the mean litter size and mean viable litter size at the .
- 1000 mg/kg Bwt/day: treatment significantly decreased the mean litter size, mean viable litter size and live birth index due to higher post-implantation loss.
- Day 4 survival index was significantly lower at all the tested doses. The decreased Day 4 survival index was mainly due to the total death/cannibalism of pups observed in two dams on Day 1 at the 100 mg/kg Bwt/day and cannibalism of 6/11 and 6/13 pups in two dams at the 300 mg/kg Bwt/day doses. The changes observed at 100 and 300 mg/kg Bwt/day doses were considered an incidental finding.

BODY WEIGHT (OFFSPRING)
- mean body weight of male and female pups per litter were not affected by the treatment at 100 and 300 mg/kg Bwt/day doses.
- mean weight of female and total pups was also decreased on LD 0, when compared to vehicle control.

GROSS PATHOLOGY (OFFSPRING)
- no gross pathology findings in both males and female pups.

OTHER FINDINGS (OFFSPRING)
- mean number of male and female (and total number) pups per litter were not affected by the treatment at 100 and 300 mg/kg Bwt/day doses. At 1000 mg/kg Bwt/day, the treatment significantly decreased the mean number of male, female and total pups on LDs 0 and 4.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

preliminary (screening study)

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Oral administration of the test item to male and female Wistar rats prior to mating, during mating and post mating periods (for males), during pregnancy and up to lactation day 4 (for females) at the dose levels 100, 300 and 1000 mg/kg bw./day had no treatment-related effects on general health, body weights and food intake, terminal body weights, organ weights and organs weight ratios. There were no test item-related gross and microscopic changes in both males and females.
At the 1000 mg/kg bw/day, the treatment resulted in significantly higher mean gestation length and pre-coital time, significantly lower body weights of dams during the gestation period, lower mean number of male, female and total pups on LDs 0 and 4. The mean weight of female and total pups was also decreased on LD 0, when compared to the vehicle control. The observation of lower mean litter size, mean viable litter size and live birth index was due to the higher postimplantation loss. Day 4 survival and gestation index were also significantly lower due to higher dead/cannibalism of pups observed. No effects on reproduction were observed at lower dose levels of 100 and 300 mg/kg bw/d.

NOAEL (P-generation, male and female): 1000 mg/kg bw/day
NOAEL (reproduction/development): 300 mg/kg bw/day (preliminary)