3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

28-days repeated dose toxicity study with extended reproductive parameters

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: According to OECD Guideline 407 with additional examinations for reproductive performance.

Version / remarks:

Adopted 3 October 2008

Principles of method if other than guideline:

Additional reproductive-related endpoints included serum testosterone, serum estrogen, and spermatogenesis stages during microscopic examinations.

GLP compliance:

yes

Limit test:

no

Justification for study design:

No Data Available

Test material

Test material information

Constituent 1

Reference substance name:

3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

EC Number:

222-294-1

EC Name:

3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Cas Number:

3407-42-9

Molecular formula:

C16H28O

IUPAC Name:

3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexanol

Test material form:

liquid

Details on test material:

- Name of the test material: 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol
- IUPAC name: 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexanol
- Molecular formula: C16H28O
- Moleclar weight: 236.396 g/mol
- Substance type: Organic

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.
- Age at study initiation: (P) x wks; (F1) x wks: (P) 7 to 8 weeks old
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: Yes, 2 hrs fasted before dose admonition.
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Animal was identified by a unique identification (ID) number. The animals were identified by individual numerical number written on the tail, also specified on individual cage tag.
- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed, ad libitum.
- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier) of Eureka Forbes was provided in polypropylene bottles with stainless steel sipper tubes, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3º C
- Humidity (%): 30-70 %
- Air changes (per hr): 25±5 air changes per hour
- Photoperiod (hrs dark / hrs light): 12hrs dark/ light cycle

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil prior to treatment.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 125, 375 or 1125 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

No mating was performed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item (125, 375 and 1125 mg) was weighed on balance and dissolved in 5 ml of corn oil. An aliquot was taken from three different layers of the dose mixtures and was extracted in methanol for GCMS analysis. The concentration of the test item in each layer was calculated using the standard curve (5 mg IBC / ml methanol).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once per day.

Details on study schedule:

No data available

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 125 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7 rats per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No positive control was included.

Examinations

Parental animals: Observations and examinations:

Mortality, clinical signs (once per day), detailed clinical signs (once per week), body weight (treatment days 1, 8, 15, 22, 28, 29) food intake (once per week), water intake (once per week), ophthalmology (4th week of treatment), locomotor activity (4th week of treatment), hematology (end of treatment), clinical chemistry (end of treatment)

Hematologic parameters: Haemoglobin, RBC, Total and differential leucocyte count, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), and Platelet count

Clinical chemistry parameters: Sodium, Potassium, Glucose, Total cholesterol, Blood urea, Creatinine, Total protein, Albumin, SGPT (Serum glutamic pyruvic transaminase), ALT, SGOT (Serum glutamic oxaloacetic transaminase), AST, Hormones analysis (testosterone and estrogen) and Total bile acids.

Postmortem examinations (parental animals):

The following organs were weighed: liver, adrenals, spleen, heart, kidney, brain, testes, epididymides, ovaries, thymus. The following organs/tissues were examined microscopically: brain, stomach, large intestine, small intestine, liver, kidney, adrenal gland, spleen, heart, thymus, lungs, testis, ovaries, uterus, lymph nodes, perihperal nerve, bone marrow, and gross lesions (if any).

Statistics:

Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version-20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p≤0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance between control and low dose group, control and mid dose group and control and high dose group. The statistical decision was taken by preparing the univariant GLM MODEL procedure was used to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs attributed to the test item. Some abnormalities like nasal discharge, slight dullness, hunched posture, fore paw stained red, sneezing, red crust around the nostrils, perineum wet were common to all the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in body weight were observed, except for a slight increase in male body weight at 1125 mg/kg on day 8 of treatment (mean, 248 g) compared to the control group (mean 237 g). This effect was considered incidental and not toxicologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant effects were observed in the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No significant effects were observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No significant effects were observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant changes in haematology were as follows: significant increase in % monocytes in males treated at 125 mg/kg; significant increase in % basophils in males treated at 125 mg/kg; significant increases in white blood cell counts in females treated at 375 and 1125 mg/kg; significant decreases in red blood cell counts in females treated at 375 and 1125 mg/kg; significant decrease in haematocrit counts in females treated at 1125 mg/kg; significant increase in MCV in females treated at 1125 mg/kg; and significant increases in MCH in females treated at 375 and 1125 mg/kg. The observed changes in haematology were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significant changes in clinical chemistry were as follows: significant increase in total cholesterol in male rats treated at 1125 mg/kg; significant increase in creatinine in male rats treated at 125 mg/kg; significant increase in glucose in male rats treated at 375 mg/kg; significant increases in total proteins in male rats treated at 375 and 1125 mg/kg; significant increase in potassium in female rats treated at 1125 mg/kg; significant increase in sodium in female rats treated at 1125 mg/kg; significant increase in SGPT in female rats treated at 375 and 1125 mg/kg; significant decrease in glucose in female rats treated at 1125 mg/kg; significant increase in total protein in female rats treated at 1125 mg/kg; significant increases in urea nitrogen in female rats treated at 375 and 1125 mg/kg; and a significant increase in bile acid in female rats treated at 375 mg/kg. No significant changes in serum testosterone or serum oestrogen were observed.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant changes in locomotor activity scores were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathology performed at 0 and 1125 mg/kg showed excess of lymphocytes in the lungs of 2/7 control males, 1/7 high-dosed males, 1/7 control females, and 1/7 high-dosed females and reactive spleens in 3/7 control males, 1/7 high-dosed males, 2/7 control females, and 1/7 high-dosed females. These histopathological findings were consistent with the historical control data of the test facility and were therefore not considered to be toxicologically relevant. The histopathologic examinations did not reveal any significant effects on spermatogenesis.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 125 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

Effect levels (F1)

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) Male and Female

Dose		Testes	Epididymides	Ovaries	Uterus
0 (mg/kg)	Mean	2.6556	1.0201	0.0944	0.5236
	SD	0.6146	0.1210	0.0322	0.1078
	SEM	0.2323	0.0457	0.0122	0.0407
125 (mg/kg)	Mean	2.7550	0.9950	0.0953	0.5293
	SD	0.2371	0.1474	0.0382	0.1607
	SEM	0.0896	0.0557	0.0144	0.0607
375 (mg/kg)	Mean	2.8762	0.9389	0.0725	0.4847
	SD	0.1901	0.1180	0.0209	0.0706
	SEM	0.0718	0.0446	0.0079	0.0267
1125 (mg/kg)	Mean	2.6205	0.9031	0.0693	0.5155
	SD	0.4577	0.2122	0.0290	0.1438
	SEM	0.1730	0.0802	0.0110	0.0543

 

 SUMMARY OF RELATIVE ORGAN WEIGHTS Male and Female

Dose		Testes	Epididymides	Ovaries	Uterus
0 (mg/kg)	Mean	0.9291	0.3560	0.0383	0.2180
	SD	0.2331	0.0436	0.0136	0.0385
	SEM	0.0881	0.0165	0.0051	0.0146
375 (mg/kg)	Mean	0.9504	0.3462	0.0384	0.2135
	SD	0.1070	0.0756	0.0158	0.0687
	SEM	0.0405	0.0286	0.00601	0.0260
1125 (mg/kg)	Mean	0.9617	0.3139	0.0297	0.1994
	SD	0.0908	0.0451	0.0083	0.0375
	SEM	0.0343	0.0171	0.0031	0.0135
1000 (mg/kg)	Mean	0.9235	0.3176	0.0295	0.2213
	SD	0.1438	0.0678	0.0116	0.0614
	SEM	0.0544	0.0256	0.0044	0.0232

Clinical chemistry:

Dose		Testesterone nmol/L	Estrogen ng/L
0 (mg/kg)	Mean	14.11	9.01
	SD	2.94	0.87
	SEM	1.11	0.33
125 (mg/kg)	Mean	15.19	9.14
	SD	1.07	1.14
	SEM	0.40	0.43
375 (mg/kg)	Mean	12.30	10.00
	SD	1.80	2.03
	SEM	0.68	0.77
1125 (mg/kg)	Mean	13.64	10.39
	SD	1.94	1.35
	SEM	0.73	0.51

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1125 mg/kg bw/day in both male and female SD rats.

Executive summary:

The test chemical was given to 7 rats per sex per dose level at 0 (vehicle; corn oil), 125, 375 and 1125 mg/kg/day. The study was performed according to GLP and according to OECD 407 with some additional reproductive-related endpoints included. Results: All animals survived to planned death and there were no clinical signs attributed to the test chemical. No significant changes in body weight were observed, except for a slight increase in male body weight at 1125 mg/kg on day 8 of treatment (mean, 248 g) compared to the control group (mean 237 g). This effect was considered incidental and not toxicologically significant. No significant changes in food intake or water intake were observed. No significant changes in locomotor activity were observed. No abnormalities were found during the ophthalmic examinations. Significant changes in haematology were as follows: significant increase in % monocytes in males treated at 125 mg/kg; significant increase in % basophils in males treated at 125 mg/kg; significant increases in white blood cell counts in females treated at 375 and 1125 mg/kg; significant decreases in red blood cell counts in females treated at 375 and 1125 mg/kg; significant decrease in haematocrit counts in females treated at 1125 mg/kg; significant increase in MCV in females treated at 1125 mg/kg; and significant increases in MCH in females treated at 375 and 1125 mg/kg. The observed changes in haematology were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. Significant changes in clinical chemistry were as follows: significant increase in total cholesterol in male rats treated at 1125 mg/kg; significant increase in creatinine in male rats treated at 125 mg/kg; significant increase in glucose in male rats treated at 375 mg/kg; significant increases in total proteins in male rats treated at 375 and 1125 mg/kg; significant increase in potassium in female rats treated at 1125 mg/kg; significant increase in sodium in female rats treated at 1125 mg/kg; significant increase in SGPT in female rats treated at 375 and 1125 mg/kg; significant decrease in glucose in female rats treated at 1125 mg/kg; significant increase in total protein in female rats treated at 1125 mg/kg; significant increases in urea nitrogen in female rats treated at 375 and 1125 mg/kg; and a significant increase in bile acid in female rats treated at 375 mg/kg. No significant changes in serum testosterone or serum oestrogen were observed. The observed changes in clinical chemistry were considered not toxicologically relevant due to lack of dose relationship and due to lack of associated histopathological findings. No significant changes in absolute organ weight were observed, with the exception of significant increases in liver weight in female rats treated at 375 and 1125 mg/kg. Significant changes in relative organ weight included a significant decrease in relative heart weight in male rats treated at 125 mg/kg and two significant increases in relative liver weight in female rats treated at 375 and 1125 mg/kg. In male rats, no gross pathological findings were observed. In female rats, gross pathological findings were limited to a slightly increased stomach at 1125 mg/kg and an enlarged uterus with uterine fluid at 1125 mg/kg. Histopathology performed at 0 and 1125 mg/kg showed excess of lymphocytes in the lungs of 2/7 control males, 1/7 high-dosed males, 1/7 control females, and 1/7 high-dosed females and reactive spleens in 3/7 control males, 1/7 high-dosed males, 2/7 control females, and 1/7 high-dosed females. These histopathological findings were consistent with the historical control data of the test facility and were therefore not considered to be toxicologically relevant. The histopathologic examinations did not reveal any significant effects on spermatogenesis. Conclusion: NOAEL was considered at 1125 mg/kg bw/day.