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Administrative data

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:
other: GLP guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Chemical name: 5-Cyclohexyl-2-methyl-pentanol
- Physical state: colourless to pale yellow clear liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, India
- 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

Administration / exposure

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)
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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

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