4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg body weight/day for the test chemical when administered orally by gavage to male and female Sprague-Dawley rats.

Repeated dose toxicity: Inhalation

The repeated dose inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 0.00162 Pa (1.21509975e-5 mm Hg). Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely.  Therefore this study for repeated dose toxicity by inhalation route of exposure is considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for 3-(2,3,3-trimethyl-6-bicyclo[2.2.1]heptanyl)cyclohexan-1-ol (CAS no 66068-84-6) (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

2. - Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: Males: 200.20-241.22 g; Female: 194.30-220.28 g
- Fasting period before study: Yes, 2 hrs fasted before dose administration.
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with bedding of clean paddy husk in a controlled environment.
- 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, 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): 12-hrs dark/12-hrs light

3. TEST ANIMALS
- Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, Punjab, India.
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: Male 189.35-233.46 g, Female 192.86-227.63 g
- Fasting period before study: No data available
- Housing: Four rats per sex per cage were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with bedding of clean paddy husk. The cages were suspended on stainless steel racks in a controlled environment.
- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed (Provimi Animal Nutrition India Pvt. Ltd, Bangalore, India), ad libitum
- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier), ad libitum.
- Acclimatization period: 3 days

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

IN-LIFE DATES:
From: 17.09.2014 to 14.10.2014 (Male) 19.09.2014 to 16.10.2014 (Female)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

2. 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

3. PREPARATION OF DOSING SOLUTIONS: 0, 125, 375 and 1125 mg/Kg/day was weighed and dissolved in corn oil. Dose of the test item was freshly prepared prior to dosing on each day

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. As the test item was insoluble in water, the corn oil was used as vehicle to deliver the desired dose levels as it is also recommended in the toxicological evaluation guidelines.
- Concentration in vehicle: 0, 125, 375 and 1125 mg/Kg/day
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2. 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).

3. An aliquot was taken from three different layers of the dose mixtures after dosage preparations 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 VAC / ml methanol).

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Total : 56 rats
Control: 7 males, 7 females
125 mg/kg/day: 7 males, 7 females
375 mg/kg/day: 7 males, 7 females
1125 mg/kg/day: 7 males, 7 females

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Observations and examinations performed and frequency:

2. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included: Mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22, 28 and 29 (before sacrifice)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes

- Time schedule for examinations: In the fourth week of treatment
- Dose groups that were examined: All groups of rats.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy on the day of termination.
- Anaesthetic used for blood collection: Yes, a slight anesthesia was used.
- Animals fasted: Yes, overnight
- How many animals: All groups of rats
.- Parameters examined: Haemoglobin (Hb), RBC, Count Total and differential leucocyte count (TLC / DLC), Haematocrit (Hct / PCV), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), and Platelet count

.CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy on the day of termination.
- Animals fasted: Yes, overnight
- How many animals: All groups of rats.
- Parameters examined: Sodium and 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.

URINALYSIS: No data available

NEUROBEHAVIOURAL EXAMINATION: No data available

2. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.: Visibility and mortality, general cage side clinical examinations, behavioral changes or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The clinical signs were recorded daily and detailed clinical signs were recorded weekly on the prescribed formats.

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22, 28 and on day 29 before sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was recorded weekly once during the entire course of treatment
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly once

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Fourth week of chemical treatment.
- Dose groups that were examined: All 56 animals were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On completion of 28 days of treatment and prior to necropsy.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, overnight fasting.
- How many animals: All 56 animals were examined.
- Parameters checked in table [No.?] were examined.: Haemoglobin (Hb) , RBC Count, Total and differential leucocyte count, Haematocrit (Hct /PCV), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and Platelet Count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On completion of 28 days of treatment and prior to necropsy.
- Animals fasted: Yes, overnight fasting.
- How many animals: All 56 animals were examined.
- Parameters checked in table [No.?] were examined.: Sodium and 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

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined.: No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes, Locomotor activity was observed.

OTHER:
Organ weight were measured.

Sacrifice and pathology:

2. GROSS PATHOLOGY: Yes
Complete necropsy was carried out on all the animals to score the gross lesions and organ weight (inkl. epididymides, ovaries and testes). Tissues were collected from all animals and preserved in 10% formal saline. However, testes, ovaries and uterus were first fixed in Bouin’s fixative for short duration then transferred to 10% formal saline. The following tissues were collected at necropsy for histological analysis: Brain, Stomach, Large intestine, Small intestine, Liver, Kidneys, Adrenal gland(s), Spleen, Heart, Thymus, Lungs, Testis / Ovary, Uterus, Lymph nodes, Peripheral nerve (Sciatic), Bone marrow, and Gross lesions, if any.

HISTOPATHOLOGY: Yes The histological examination was conducted on the tissues ffom the control and the high-dose group animals based on double blind analysis by using Olympus Trinocular Microscope, (Model BX-51) at magnification of 10x, 20x and 100x.

3. GROSS PATHOLOGY: Yes, On completion of treatment, all surviving rats were sacrificed by CO2 asphyxiation. Complete necropsy was carried out on all the animals to score the gross lesions. Tissues were collected from all animals and preserved in 10% formal saline. However testes, ovaries and uterus were first fixed in Bouin’s fixative for two hours then transferred to 10% formal saline. Liver, Kidney, Spleen, heart, Kidney, Brain, Testis, Epididymidis, Ovaries, Thymus were weighed.

HISTOPATHOLOGY: Yes
Organ examined : Brain, stomach, small intestine, large intestine, Liver, Kidney, Adrenal Glands, Spleen, heart, Thymus, Lungs, Testis/ovaries, Uterus, Lymphnodes, peripheral nerves, bone marrow and gross lesions if any were examined

Other examinations:

The locomotor activity assessment was performed for all groups of rats in the fourth week of treatment. Each animal from different groups was placed in Digital Photoactometer for 5 minutes and the scores were recorded.

Statistics:

2. 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.

3. Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version 20.0. A ll 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. Paired t-value is dependent upon sample mean differences (mean difference between control and low dose, control and mid dose, control and high dose), standard deviations of sample differences (standard deviations difference between the groups) and number of sample differences. The values in all the tables are depicting only the individual means, standard deviation and standard errors .They do not indicate the paired differences but the absolute values. If the standard error is higher and paired t-value is not in range of confidence interval (minimum and maximum) it is considered to be insignificant. If it is outside the range and p-value is less than 0.05 it is significant. All results are presented at 95 percent.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

2. 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.

3. Some abnormalities like reddish nasal discharge, slightly lethargic, hunched posture, fore paw stain ed red, snuffling sound, nasal discharge, red crust around nostrils, perineum wet with urine and vocalization upon handling were common to all the groups.

Mortality:

no mortality observed

Description (incidence):

All animals survived throughout the treatment period of 28 days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

2. In male animals, a significant increase has been noticed in the body weight of 1125 mg/kg/day as compared to control animals on day 8. However, no change was noticed in the body weight of female animals taken at different time periods.

3. In male animals no significant change has been noticed in the body weight taken at the different per iods. In female rats, significant increase in the body weight by high-dose treatment has been observed on day 01, 08, 15, and 22.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No difference was observed among any groups of male as well as female animals.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

2. No difference was observed among any groups of male as well as female animals in water consumption as compared to control.

3. No difference in water consumption was observed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

2. No abnormalities were found in the ophthalmological examination (control vs. 1125 mg/kg/day treated group).

3. No difference was observed in Opthalmoscopic examination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

2. When treated with 1125 mg/kg/day, in male rat significant increased were observed in percentage of monocytes and basophils.

In female rat, significant increase were observed in WBC count, MCV and MCH levels and significant decreased in RBC count and Hct level as compare to control.

When treated with 375 mg/kg/day, in female rat significant increase were observed in WBC count and MCH level and significant decreased in RBC count as compare to control.

3. In male rats, platelet count was significantly decreased (p<0.05) in low and mid-dose treatment of verdyl acetate. The level of MCV was increased (p<0.05) in animals treated with mid-dose in comparis on to control. In female rats, high–dose treatment resulted into significant decrease in WBC count in comparison to control animals. The level of RBC and Hct were elevated significantly in all the dose-treated grou ps. Hgb has also shown a significant increase (p<0.05) in low and high-dose treated groups. An increase (p<0.05) was noticed in the level of MCV in mid and high-dose treated rats. A significant decrease in MCHC level was also noticed in the same group. Low and mid dose treatment resulted into significant increase in lymphocyte count. A significant decrease in neutrophil count was also observed in the same groups.

In females animals, Hgb has increased significantly in the sixth group i.e. 125mg/kg/day (p value is 0.013) and in eighth group i.e. 1125/mg/kg/day (p value is 0.043).Whereas in the seventh group i.e.375 mg/kg/day the significance level was found to be (0.142) hence it can be concluded that the third group is statistically insignificant due to higher standard deviation and standard error. In case of females rats eosinophils levels in seventh group i.e. 375 mg/kg/day attained the value of significance(0.056). As the p value is greater than 0.05, so we are unable to conclude our result at 95%. Hence itis statistically insignificant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

2. When treated with 1125 mg/kg/day, in male rat significant increase were observed in total protein and total cholesterol.

In female rat, significant increasewere observed in sodium, potassium, total proteins, BUN levels. And activity of SGPT and significant decreased were observed in Glucose level as compare to control.

When treated with 375 mg/kg/day, in male rat significant increase were observed in glucose and total proteins leve.
In female rat, significant increase were observed in SGPT activity, BUN level and decreased TBA level as compare to control.

When treated with 125 mg/kg/day, in male rat significant increase were observed in creatinine level as compare to control.

3. In male rats, potassium was increased significantly in mid-dose treatment. However, in the same group the levels of total proteins was decreased significantly (p<0.05). In animals treated with high-dose,a significant elevation in testosterone level was noticed as compare to control animals. In female rats, mid and high-dose treatment resulted in decrease (p<0.05, p<0.01) in the activity of SGOT respectively. An overall decrease in glucose level was also noticed in low and mid dose treated groups which was found to be statistically significant (p<0.05) as compared to control animals.

In male rats SGOT levels has increased in second i.e. 125mg/kg/day but they are not showing significant difference at 95 % level due to higher standard error of mean. In male rats glucose levels has decreased in third group i.e. 375 mg/kg/day but they are not showing any statistical difference due to higher standard error of mean. In male rats the total cholesterol increased for fourth group i.e. 1125/mg/kg/day in comparison to all rest of the male groups it achieved the p value 0.072 which is again greater than 0.05 hence it is found to be statistically insignificant.

In female rats SGPT level was increased in the sixth i.e. 125mg/kg/day .but it is statistically insignificant due to higher standard deviation. In female rats Glucose level has decreased in sixth i.e. 125 mg/kg/day (p value 0.028) & seventh group i.e. 375 mg/kg/day (p value 0.013) group significantly. The standard deviation and standard error was found to be higher in the eighth group i.e. 1125/mg/kg /day (p value 0.064) in comparison to fifth group i.e. control hence it was not found to be statistically insignificant as the paired t test depends upon both the differences in mean and standard errors.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No significant change was observed in the test compound treated groups as compared to control animals.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

2. In female rat, teatement with 375 and 1125 mg/kg/day absolut and relative liver weight were significantly increased as compared to control.

In male rats, treatement with 125 mg/kg/day significantly decreased in relaive heart weight were observed as compare to control.

The significant change in organ weight did not corroborate with the individual histological findings of the animals.

3. In male rats, the absolute weight of thymus was increased significantly in all the dose treated groups. The absolute weight of kidneys was increased significantly in mid and high–dose treated rats. However, the absolute weight of heart was decreased significantly (p<0.05) in lowdose treated animals,when compared to control group. The relative organ weight of brain was significantly decreased in low-dose group. However, thymus weight has shown a significant elevation all the groups.The relative weight of kidneys was found to be increased significantly in mid and high-dose treatment. The relative weight of testes was decreased to significant levels in the mid-dose treated rats. Low and mid-dose treatment reduced the weight of heart significantly. Epididymides have also shown significant reduction in mid-dose. In female animals, the absolute weights of liver and kidneys were found to be significantly increased
in high-dose treated groups in comparison to control. The relative weight of liver was increased (p<0.05) in the high-dose group. A significant elevation in the relative weight of kidneys was in mid and high-dose treated groups. The high dose also caused decrease (p<0.05) in the relative weight of heart.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. When treated with 1125 mg/kg/day, in female rat enlarged uterus with uterine fluid and Slightly enlarged stomach were observed in one of the rat.

Above changes produced in organs might be due to the adaptive metabolic and physiological changes, anoxic / hypoxic conditions during anesthesia and terminal sacrifice of the animals, which affect the normal structure of the organs and were considered to be dose independent. So, these findings were considered to be of no toxicological significance.

3. In terminally sacrificed rats, the incidence of necropsy findings include occurrence of mass (friable and hard in consistency) was observed in the urinary bladder of both control and treated groups. Slightly enlarged uterus with uterine fluid and a small follicular cyst was also observed in one of the female animals. The color of this mass varied from whitish to yellowish. In some animals urine was slightly viscous in consistency. Above mentioned changes produced in organs might be due to adaptive metabolic and physiological changes, anorexic / hypoxic conditions during anesthesia and terminal sacrifice of the animals. So, these findings were considered to be of no toxicological-biological significance.

Neuropathological findings:

not specified

Description (incidence and severity):

3. There was statistically significant increase (p<0.05) observed in the motor activity scores for high dose group of male animals when compared with the control animals. However, in female rats, a significant decrease was noticed in high-dose treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

2. No remarkable changes were observed in control and in 1125 mg/kg /day-treated animals.

A few microscopic findings were observed in 1125 mg/kg/day-treated animals included reactive spleen and excess of lymphocytes in lungs.

These types of findings may be considered to be within the range of normal background lesions which may be seen in rats of this strain and age of this study type and were considered incidental in nature with carbon dioxide inhalation and terminal changes at sacrifice, reflecting the usual individual variability.

However, 1125 mg/kg/day group animals did not reveal any toxic lesions on histological examinations when compared with the respective control group, hence further histological investigation was not extended to the two other lower dose (low-dose and mid-dose) treatment groups

3. No significant changes were observed in control as well as in high-dose-group animals. Few microscopic findings were observed in control as well as in high-dose treated animals which include interstitial pneumonia in lungs, focal lobular hepatitis in liver, reactive spleen with excess of lymphocytes and increased eosinophils in small intestine. The interstitial pneumonia may be due to use of corn oil (vehicle) for the administration of test item. However, these findings may not be linked with test item related effects as most of these have been observed in the control animals also. These types of findings may be considered to be within the range of normal background lesions which may be seen in rats of this strain and age of this study type and were considered incidental in nature with carbon dioxide inhalation and terminal changes at sacrifice, reflecting the usual individual variability. As high-dose group animals did not reveal any toxic lesions on histological examinations when compared with the respective control group, hence further histological investigation was not extended to the two other lower dose treatment groups (low-dose and mid-dose).

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

other: No effect observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Conclusions:

The no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg body weight/day for the test chemical when administered orally by gavage to male and female Sprague-Dawley rats.

Executive summary:

Data available for the various test chemicals was reviewed to determine the toxic nature of the target chemical. The studies are as mentioned below:

In a repeated dose oral toxicity study, the effects of the test chemical was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0, 125, 375 or 1125 mg/kg/day. The results showed that the test chemical significantly change the percentage of monocytes and basophils, WBC count, MCV and MCH levels in hematology, total protein and total cholesterol, sodium, potassium, total proteins, BUN levels in clinical biochemistry. Absolute and relative liver and heart weight was changed as compared to control. The significant change in organ weight did not corroborate with the individual histological findings of the animals.While no effects were observed in water consumption, opthalmoscopic examination or locomotor activity. At 1125 mg/kg/day, in female rat enlarged uterus with uterine fluid and Slightly enlarged stomach were observed in one of the rat.Above changes produced in organs might be due to the adaptive metabolic and physiological changes, anoxic / hypoxic conditions during anesthesia and terminal sacrifice of the animals, which affect the normal structure of the organs and were considered to be dose independent. So, these findings were considered to be of no toxicological significance. Histopathology performed on organs after treatment with 1125 mg/kg/day did not reveal any toxic lesions as compared to control. Hence, the no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg/day when male and female Sprague Dawley rats were exposed daily to the test chemical by oral route for 28 days.

In a 28 days repeated dose toxicity study, the effect of the test chemical was evaluated using male and female Sprague Dawley rats. Sprague Dawley (SD) rats of 7-8 weeks old were divided into four groups, in each sex  comprised of either 7 males or 7 females. Test item verdyl acetate was administered at the dose levels of 125, 375 and 1125 mg/kg/day for 28 consecutive days by oral route. Male animals were divided into four groups, group 1 served as control and group 2, 3 and 4 served as low, mid and high dose group respectively. Female animals were divided into four groups, group 5 served as control and group 6, 7 and 8 served as low, mid and high dose group. Corn oil was served as vehicle for the present study.  During the treatment period, detailed clinical signs, body weight, food and water consumption were recorded weekly. No abnormalities were found in the ophthalmological examination (control vs. high-dose treated group). Male animals treated with high dose test item exhibited significant (p<0.05) increase in the motor activity as compared to control animals. Whereas in female rats, a significant decrease was noticed at the same dose level. After the completion of dosing period, blood was collected for hematology and clinical biochemistry analysis. All animals were sacrificed on the day of termination and gross lesions as well as the weight of different organs were recorded. All organs were fixed in 10 % formal saline, except the reproductive organs, which were fixed in Bouin’s fixative for short duration and thereafter shifted to 10 % formal saline.  In the terminally sacrificed rats, the incidence of necropsy findings include occurrence of whitish to yellowish mass in the urinary bladder of both control and treated animals. In male rat, the relative organ weight of brain was significantly decreased in low-dose group as compared to control group. However, thymus weight was significantly elevated in all the groups. The relative weight of kidneys was increased significantly in mid and high-dose treated animals. The relative weight of testes was also decreased to a significant level in the mid-dose treated rats. Low and mid-dose treatment reduced the heart weight upto a significant level. Epididymides weight was significantly reduced in mid-dose treated animals as compared to control. In female rat, the relative weight of liver was significantly (p<0.05) increased in the high-dose group. A significant elevation in the relative weight of kidney was noticed in mid and high-dose treated groups. The high-dose treated animals exhibited significant (p<0.05) decrease in the relative heart weight.  Hematology of animals after treatment showed that platelet count was significantly (p<0.05) decreased with the treatment at low and mid-dose in male rat, while the MCV was significantly increased in the mid-dose as compared to control animals. In female rats, WBC count was significantly decreased after the exposure of test item at high–dose as compared to control animals. However, significant elevation in RBC and Hct level was noticed in all the treated groups as compared to control animals. Further, an increase (p<0.05) in the level of MCV was observed in the mid and high-dose treated animals. However, MCHC level decreased significantly in the same groups. A significant increase in   the lymphocytes was observed with low and mid-dose treatment. Whereas, the neutrophils count were significantly decreased in the same groups as compared to control animals. The level of Hgb was significantly increased with low and high-dose treatment in female rat as compared to control animals. In male rat, significant elevation in the level of potassium in serum was noticed with mid-dose treatment. However in the same group total proteins in plasma was significantly (p<0.05) decreased. High-dose treated animal exhibited a significant elevation in the testosterone level in plasma as compared to control animals. In female rat, decrease in the activity of SGOT in plasma was noticed only in the mid and high dose treated animals. Further, the level of glucose in plasma was also decreased in low and mid dose treated animals as compared to control animals. The microscopic histopathological findings include interstitial pneumonia and excess of lymphocytes in lungs, focal lobular hepatitis, reactive spleen, and increased eosinophils in small intestine. From the present investigations, it can be concluded that the changes in toxicological parameters were not dose dependent in nature in either of the sexes and most of the changes were in the category of background lesions. Therefore, it is concluded that the significant differences observed among the various toxicological end points may not have any toxicological relevance as all these parameters studied do not show a dose dependent toxicological response. Based on the observations made, the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1125 mg/kg body weight/day when administered orally by gavage to male and female Spargue Dawley rats.

Based on the data available, the no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg body weight/day for the test chemical when administered orally by gavage to male and female Sprague-Dawley rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is from K2 source

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Data available for the various test chemicals was reviewed to determine the toxic nature of the target chemical. The studies are as mentioned below:

In a repeated dose oral toxicity study, the effects of the test chemical was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0, 125, 375 or 1125 mg/kg/day. The results showed that the test chemical significantly change the percentage of monocytes and basophils, WBC count, MCV and MCH levels in hematology, total protein and total cholesterol, sodium, potassium, total proteins, BUN levels in clinical biochemistry. Absolute and relative liver and heart weight was changed as compared to control. The significant change in organ weight did not corroborate with the individual histological findings of the animals.While no effects were observed in water consumption, opthalmoscopic examination or locomotor activity. At 1125 mg/kg/day, in female rat enlarged uterus with uterine fluid and Slightly enlarged stomach were observed in one of the rat.Above changes produced in organs might be due to the adaptive metabolic and physiological changes, anoxic / hypoxic conditions during anesthesia and terminal sacrifice of the animals, which affect the normal structure of the organs and were considered to be dose independent. So, these findings were considered to be of no toxicological significance. Histopathology performed on organs after treatment with 1125 mg/kg/day did not reveal any toxic lesions as compared to control. Hence, the no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg/day when male and female Sprague Dawley rats were exposed daily to the test chemical by oral route for 28 days.

In a 28 days repeated dose toxicity study, the effect of the test chemical was evaluated using male and female Sprague Dawley rats. Sprague Dawley (SD) rats of 7-8 weeks old were divided into four groups, in each sex  comprised of either 7 males or 7 females. Test item verdyl acetate was administered at the dose levels of 125, 375 and 1125 mg/kg/day for 28 consecutive days by oral route. Male animals were divided into four groups, group 1 served as control and group 2, 3 and 4 served as low, mid and high dose group respectively. Female animals were divided into four groups, group 5 served as control and group 6, 7 and 8 served as low, mid and high dose group. Corn oil was served as vehicle for the present study.  During the treatment period, detailed clinical signs, body weight, food and water consumption were recorded weekly. No abnormalities were found in the ophthalmological examination (control vs. high-dose treated group). Male animals treated with high dose test item exhibited significant (p<0.05) increase in the motor activity as compared to control animals. Whereas in female rats, a significant decrease was noticed at the same dose level. After the completion of dosing period, blood was collected for hematology and clinical biochemistry analysis. All animals were sacrificed on the day of termination and gross lesions as well as the weight of different organs were recorded. All organs were fixed in 10 % formal saline, except the reproductive organs, which were fixed in Bouin’s fixative for short duration and thereafter shifted to 10 % formal saline.  In the terminally sacrificed rats, the incidence of necropsy findings include occurrence of whitish to yellowish mass in the urinary bladder of both control and treated animals. In male rat, the relative organ weight of brain was significantly decreased in low-dose group as compared to control group. However, thymus weight was significantly elevated in all the groups. The relative weight of kidneys was increased significantly in mid and high-dose treated animals. The relative weight of testes was also decreased to a significant level in the mid-dose treated rats. Low and mid-dose treatment reduced the heart weight upto a significant level. Epididymides weight was significantly reduced in mid-dose treated animals as compared to control. In female rat, the relative weight of liver was significantly (p<0.05) increased in the high-dose group. A significant elevation in the relative weight of kidney was noticed in mid and high-dose treated groups. The high-dose treated animals exhibited significant (p<0.05) decrease in the relative heart weight.  Hematology of animals after treatment showed that platelet count was significantly (p<0.05) decreased with the treatment at low and mid-dose in male rat, while the MCV was significantly increased in the mid-dose as compared to control animals. In female rats, WBC count was significantly decreased after the exposure of test item at high–dose as compared to control animals. However, significant elevation in RBC and Hct level was noticed in all the treated groups as compared to control animals. Further, an increase (p<0.05) in the level of MCV was observed in the mid and high-dose treated animals. However, MCHC level decreased significantly in the same groups. A significant increase in   the lymphocytes was observed with low and mid-dose treatment. Whereas, the neutrophils count were significantly decreased in the same groups as compared to control animals. The level of Hgb was significantly increased with low and high-dose treatment in female rat as compared to control animals. In male rat, significant elevation in the level of potassium in serum was noticed with mid-dose treatment. However in the same group total proteins in plasma was significantly (p<0.05) decreased. High-dose treated animal exhibited a significant elevation in the testosterone level in plasma as compared to control animals. In female rat, decrease in the activity of SGOT in plasma was noticed only in the mid and high dose treated animals. Further, the level of glucose in plasma was also decreased in low and mid dose treated animals as compared to control animals. The microscopic histopathological findings include interstitial pneumonia and excess of lymphocytes in lungs, focal lobular hepatitis, reactive spleen, and increased eosinophils in small intestine. From the present investigations, it can be concluded that the changes in toxicological parameters were not dose dependent in nature in either of the sexes and most of the changes were in the category of background lesions. Therefore, it is concluded that the significant differences observed among the various toxicological end points may not have any toxicological relevance as all these parameters studied do not show a dose dependent toxicological response. Based on the observations made, the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1125 mg/kg body weight/day when administered orally by gavage to male and female Spargue Dawley rats.

Based on the data available, the no observed adverse effect level (NOAEL) was considered to be 1125 mg/kg body weight/day for the test chemical when administered orally by gavage to male and female Sprague-Dawley rats.

Repeated dose toxicity: Inhalation

The repeated dose inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 0.00162 Pa (1.21509975e-5 mm Hg). Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely.  Therefore this study for repeated dose toxicity by inhalation route of exposure is considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for 3-(2,3,3-trimethyl-6-bicyclo[2.2.1]heptanyl)cyclohexan-1-ol (CAS no 66068-84-6) (as provided in section 7.2.3) is >2000 mg/kg body weight. Considering this, the end point for repeated dermal toxicity is considered as waiver

Based on the data available and applying the weight of approach, the test chemical is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route of exposure and hence it is not likely to classify as a toxicant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of approach, the test chemical is not likely to be toxic upon repeated exposure by oral, dermal and inhalation route of exposure and hence it is not likely to classify as a toxicant as per the criteria mentioned in CLP regulation.