Cyclohexanepentanol, .beta.-methyl-

Endpoint summary

Description of key information

Results from a GLP conform combined 28-day repeated dose oral toxicity/reproduction/developmental toxicity screening study (OECD 422) in rats are available indicating no systemic effects of toxicity of the test item up to the highest does tested of 1000 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

guideline study

Reason / purpose for cross-reference:

reference to same study

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

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 the 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
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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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.

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 determination 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
- Recovery animals (not mated animals): vehicle and test item were not administered to the animals for 14 days following the treatment period

Frequency of treatment:

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

Remarks:

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

No. of animals per sex per dose:

Main group: 10 males / 10 females
Recovery group (vehicle control recovery and high dose recovery): 5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 14 days

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

Observations and examinations performed and frequency:

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:
Main group:
- 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
- Recovery group: initially and weekly intervals thereafter

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

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period and at the end of recovery period
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: premating period: 5 males / 5 females of the main group; recovery period: all animals
- Parameters examined: haematocrit, haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, platelets, red blood corpuscles, reticulocytes, white blood corpuscles, differential leukocyte count (absolute)(neutrophils, lymphocytes, monocytes, eosinophils and basophils), prothrombin time analysis, and activated partial thromboplastin time analysis

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period and at the end of recovery period
- Animals fasted: Yes, overnight
- How many animals: premating period: 5 males / 5 females of the main group; recovery period: all animals
- Parameters examined: alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, albumin/globulin ratio [calculated values], blood urea nitrogen, bile acids, calcium, chloride, creatinine, gamma glutamyl transferase, glucose, globulin [calculated values], inorganic phosphorous, potassium, sodium, total bilirubin, total cholesterol, total plasma protein, and triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the pre-mating period (5 males / 5 females of main groups) and at the end of recovery period (all surviving rats)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, leukocytes, erythrocytes, appearance (colour and clarity), and volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: end of the dosing period for males (shortly prior to their scheduled sacrifice) and during
lactation period for females (shortly before the scheduled sacrifice); for recovery groups, neurological examination was carried out towards the end of recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / landing hindlimbs footsplay / home cage observations (posture, presence or absence of abnormal vocalizations and convulsions /observations during removal of animal from home cage and handling(subject's response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held) / open field observation / Physiological observations (body temperature, body weight)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All rats in this study were sacrificed at term after overnight fasting. These rats were anaesthetized with isoflurane, weighed and exsanguinated and subjected for detailed necropsy.

HISTOPATHOLOGY: Yes
- 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.

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, haematology, clinical chemistry, 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.
All analyses and comparisons were evaluated at the 5% (P≤0.05) level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

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

BODY WEIGHT AND WEIGHT GAIN
- body weights were unaffected by the treatment at all the doses tested.
- maternal body weights during lactation periods were unaffected at all the doses tested.
- the significantly decreased body weights (GD20) and weight changes (GDs 0-7, 14-20, 0-20) at 1000 mg/kg bw during gestation were correlated with a lower mean litter size in this group and thus not considered as a sign of systemic toxicity related to treatment with the test item.
- terminal fasting body weight was not treatment-related affected.

FOOD CONSUMPTION AND COMPOUND INTAKE
- food consumption were unaffected by the treatment at all the doses tested.
- maternal food consumption during gestation was not affected by the treatment at all the doses.
- maternal food consumption during lactation periods were unaffected at all the doses tested.

HAEMATOLOGY
- test item administration did not reveal any treatment related changes in the haematology and coagulation parameters of both males and females.

CLINICAL CHEMISTRY
- test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both males and females.

URINALYSIS
- no test item-related alterations in any urine parameters analysed in both male and female rats.

NEUROBEHAVIOUR
- no treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

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

GROSS PATHOLOGY
- no test item related gross changes in both males and females.

HISTOPATHOLOGY: NON-NEOPLASTIC
- no test item related microscopic changes in both males and females.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

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

Key result

Critical effects observed:

no

Conclusions:

Oral administration of 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 bwt/day had no effects on general health, body weights and food intake. Functional observations did not reveal any test item related changes at all the tested doses. The test item administration did not reveal any changes in haematology, clinical chemistry and urinalysis parameters. There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. There were no test item-related gross and microscopic changes in both males and females.

NOAEL (male and female rats): 1000 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Acceptable (RL=1)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity, oral, rat:

Oral administration of 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 over 28 days had no effects on general health, body weights and food intake. Functional observations did not reveal any test item related changes at all the tested doses. The test item administration did not reveal any changes in hematology, clinical chemistry and urinalysis parameters. There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. There were no test item-related gross and microscopic changes in both males and females.

 

Thus, the NOAEL for male and female rats was established at the highest dose level tested of 1000 mg/kg bw/day.

No repeated dose toxicity studies with longer duration or by different routes of administration are available so far.

 

Repeated dose toxicity, inhalation:

According to regulation (EC) 1907/2006 Annex IX, Section 8.6.2, Column 2, testing for sub-chronic inhalation toxicity is not considered to be required, for the following reasons:

 

- Repeated dose toxicity study via inhalation route does not need to be performed since the test substance is only slightly soluble in water (~18 mg/L (20°C/pH 7.4) and has a moderate vapour pressure of 0.14 Pa at 20°C and a boiling point of 267.5°C. Based on the logPow of 5.3 and a molecular weight < 200 g/mole (184.3 g/mole) the substance might be absorbed solely through aqueous pores. Based on the physico-chemical properties, the substance can safely be assumed to have a very low potential for human inhalation hazard during handling or application. Toxicological information on the elicitation of local effects in biological membranes caused by the test substance, indicate an absence of irritating or corrosive properties. Based on the above information, local effects upon inhalation can safely be excluded. Since the oral route of exposure allows animal being exposed with the highest systemic dose, compared with the inhalation route. Consequently, a repeated dose toxicity study via the oral route was conducted.

 

Repeated dose toxicity, dermal:

According to regulation (EC) 1907/2006 Annex IX, Section 8.6.2, Column 2, testing for sub-chronic dermal toxicity is not considered to be required, for the following reasons:

 

- Due to the low water solubility (18 mg/L at 20°C, pH 7.4) and the lipophilic character (log Pow 5.3), dermal adsorption at relevant rates can safely be excluded, thus the physicochemical properties suggest a significant rate of absorption through the skin.

Further, the lack of systemic and local toxicity in the acute dermal toxicity study up to a concentration of 2,000 mg/kg bw and the absence of any skin irritation effects in an in vivo skin irritation study may be seen as an indication that this is not a relevant route of entering human body.

 

The oral route is considered as most relevant route of exposure, since this route allows animals being exposed with the highest systemic dose, compared with the dermal route. Consequently, a repeated dose toxicity study via the oral route was conducted.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Recently conducted OECD 422 guideline study on repeated dose toxicity for 28 days in combination with a reproduction/developmental toxicity screening test according to GLP.

Justification for classification or non-classification

Based on the available results of combined 28-day repeated dose toxicity/reproduction/developmental toxicity screening (OECD 422) study in male and female rats, no classification as STOT-RE according to the CLP criteria is necessary, because the NOAEL for systemic toxicity is > 1000 mg/kg bw/d.