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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February – 09 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD guideline 407 without any deviation
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol
EC Number:
939-525-3
Cas Number:
1471313-03-7
Molecular formula:
C14H26O
IUPAC Name:
3-methyl-5-(2,2,3-trimethylcyclopent-3-en-1-yl)pentan-2-ol; 6-(2,2,3-trimethylcyclopent-3-en-1-yl)hexan-3-ol
Details on test material:
- Name of test material (as cited in study report): Sandalore
- Chemical name: 5-(2,2,3-trimethyl-3-cyclopentenyl)-3-methylpentan-2-ol
- CAS number: 65113-99-7
- Physical state: Clear colourless liquid
- Analytical purity: sum of C14H16O isomers 92.5 %
- Lot/batch No.: VE00061266
- Date received: 06 January 2010
- Expiration date of the lot/batch: 02 January 2012
- Stability under test conditions: At least 20 days
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: Wistar Han™:HsdHan™:WIST strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: Approximately 6-8 weeks
- Weight at study initiation: Males: 236-286 g; females: 161-194 g
- Housing: Animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: Pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 ºC
- Humidity: 55 ± 15 %
- Air changes: 15/h
- Photoperiod: 12 h dark / 12 h light (low intensity fluorescent lighting)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material was prepared at the appropriate concentrations as a solution in corn oil.
- The formulation was found to be to be stable for at least 20 days in a previous study conducted by Harlan Laboratories Ltd., UK. Analytical Services (Project Number: 0766-0156). Formulations were therefore prepared twice during the treatment period and stored at approximately 4 ºC in the dark.

VEHICLE
- Concentration in vehicle: 8.75, 81.3 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material formulation were analysed for concentration and results indicate that the prepared formulations were within 10 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
35, 325 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were chosen based on the results of previous toxicity work (Project Number: 0766-0156).
- Rationale for animal assignment: Animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were determined to ensure similarity between the treatment groups.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined immediately before dosing, up to 30 minutes post dosing and 1 and 5 h after dosing during the working week. Animals were observed immediately before and after dosing and 1 h after dosing at weekends and public holidays. During the treatment-free period, animals were observed daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter. Bodyweights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION:
- Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals

- Parameters checked for haematology: Haemoglobin, erythrocyte count, haematocrit, reticulocyte count, total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), platelet count, prothrombin time, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

- Parameters checked for clinical chemistry: Alkaline phosphatase (AP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl transpeptidase (γGT), total protein, albumin, albumin/globulin ratio, urea, creatinine, glucose, total bilirubin, total cholesterol, triglycerides, bile acids, calcium (Ca++), sodium (Na+), potassium (K+), chloride (Cl-) and inorganic phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: Investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6.
- Metabolism cages used for collection of urine: Yes, urine samples were collected overnight by housing the rats in metabolism cages.
- Animals fasted: Yes
- Parameters checked: Volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances and blood.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 26, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all non-recovery animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately 2 h after dosing on each occasion.
- Battery of functions tested: Behavioural assessments, sensory reactivity, forelimb/hindlimb grip strength and motor activity.

OTHER:
- Thyroid Hormone Assessment: At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored frozen at -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.
- Stage of oestrus: At termination, a vaginal smear was taken from all females and the stage of oestrus was recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- The following organs were isolated and weighed before fixation: adrenals, liver, brain, ovaries, epididymides, spleen,
heart, testes, kidneys, thymus, pituitary (post-fixation), thyroid/parathyroid (post fixation), prostate and seminal vesicles (with coagulating glands and fluids) and uterus with cervix

HISTOPATHOLOGY: Yes
- The following tissues from all non-recovery control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination: adrenals, ovaries, pituitary, bone & bone marrow (sternum), prostate, brain (including cerebrum, cerebellum and pons), rectum, caecum, sciatic nerve, colon, seminal vesicles (with coagulating glands and fluids), duodenum, epididymides, eyes, spinal cord (cervical, mid thoracic and lumbar), gross lesions, heart, spleen, ileum, stomach, jejunum, testes, kidneys, thymus, liver, thyroid/parathyroid, lungs (with bronchi), trachea, lymph nodes (cervical and mesenteric), urinary bladder, mammary gland uterus, cervix and vagina
- Any macroscopically observed lesions were also processed together with the liver and spleen from all 35 and 325 mg/kg bw/day dose group animals.
- In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Other examinations:
None
Statistics:
- All quantitative data were analysed by the Provantis™ Tables and Statistics Module and Statistical Package for the Social Sciences (SPSS).
- For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data.
- If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group.
- Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
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:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
- No mortality was observed.
- Increased salivation was evident in animals of either sex treated with 325 and 1000 mg/kg bw/day.
- Isolated instances of red/brown staining around the snout or mouth were evident in two males and one female at 1000 mg/kg bw/day and in one female treated with 325 mg/kg bw/day.
- Noisy respiration was also evident on Day 1 in one male treated with 1000 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN: No adverse effects on bodyweight development were detected for treated animals when compared to controls.

FOOD CONSUMPTION AND FOOD EFFICIENCY: No adverse effects on food intake or food efficiency were detected.

WATER CONSUMPTION:
- Water consumption was increased throughout the treatment period in animals of either sex treated with 1000 mg/kg bw/day and in recovery males throughout the treatment-free period.
- No such effects were detected in animals of either sex treated with 35 or 325 mg/kg bw/day or in recovery 1000 mg/kg bw/day females during the treatment-free period.

HAEMATOLOGY / CLINICAL CHEMISTRY: No toxicologically significant changes were detected in the haematological and blood chemical parameters measured.

URINALYSIS: There were no intergroup differences detected in the urinalytical parameters measured when compared to controls.

NEUROBEHAVIOUR (Functional observations):
Behavioural assessment:
- Open field assessments during Week 2 confirmed the clinical observation of increased salivation in three males and two females treated with 1000 mg/kg bw/day.
- No such effects were detected in animals of either sex treated with 35 or 325 mg/kg bw/day.
Functional performance tests: No toxicologically significant changes were observed in functional performance.
Sensory reactivity assessments: No treatment-related changes were detected in sensory reactivity.

ORGAN WEIGHTS:
- Animals of either sex treated with 325 and 1000 mg/kg bw/day showed a statistically significant increase in liver weight, both absolute and relative to terminal bodyweight. Following 14 days without treatment recovery animals continued to show an increase in absolute and relative liver weight.
- Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in absolute kidney weight and a statistically significant increase in relative kidney weight.
- Females from this treatment group also showed a statistically significant increase in absolute and relative thyroid weight.
- No such effects were detected in animals of either sex treated with 35 mg/kg bw/day.

GROSS PATHOLOGY:
- A dark liver was evident at necropsy in three males and two females treated with 1000 mg/kg bw/day.

HISTOPATHOLOGY:
The following treatment-related microscopic changes were identified:
- Liver: Centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 325 and 1000 mg/kg bw/day and in males treated with 35 mg/kg bw/day.
- Kidneys: Hyaline droplets nephropathy was evident in males treated with 1000 mg/kg bw/day.
- Thyroid: Follicular cell hypertrophy was noted in animals of either sex from all treatment groups.
All these conditions were observed to have mostly regressed among recovery high dose animals following an additional fourteen days without treatment. These were considered to be adaptive in nature and do not represent an adverse effect of treatment.

OTHER FINDINGS:

Stage of oestrus: Assessment of the stage of oestrus for females at termination showed no significant differences between treated and control groups.

HISTORICAL CONTROL DATA: Normal ranges for functional performance assessments, haematological, blood chemical values, and urinalytical findings, absolute and relative organ weight values in the Wistar Han™:HsdHan™:WIST strain rats were provided.

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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day in Wistar rats.
Executive summary:

In a repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, the substance was administered daily by oral gavage to groups of Wistar Han™:HsdHan™:WIST strain rats (5/sex/dose) for 28 days, at the dose levels of 0 (control), 35, 325 and 1000 mg/kg bw/day suspended with corn oil. Two recovery groups (5/sex/dose), were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for 28 consecutive days and then maintained without treatment for a further 14 days. Examinations during the study included: mortality, clinical signs, body weight change, food and water consumption, haematology, blood chemistry, urinalysis, macroscopic examination of main organs, measurement of organ weights and histopathology.

No mortality was observed. Clinical observations were confined to increased salivation detected in animals of either sex treated with 325 and 1000 mg/kg bw/day throughout the treatment period. Isolated instances of noisy respiration and fur staining around the snout or mouth were also evident in 1-3 animals at 325 and 1000 mg/kg bw/day. No treatment-related changes were detected in functional performance and sensory reactivity. No adverse effects on bodyweight development and food consumption were detected for treated animals when compared to controls. Increased water consumption was detected in animals of either sex treated with 1000 mg/kg bw/day throughout the treatment period and in recovery males during the treatment-free period. No toxicologically significant changes were detected in the haematological, blood chemical and urinalysis parameters measured. Macroscopic examinations revealed a dark liver, evident in three males and two females at 1000 mg/kg bw/day. A statistically significant increase in liver weight, both absolute and relative to terminal bodyweight was evident in animals of either sex treated with 325 and 1000 mg/kg bw/day and in recovery animals following 14 days without treatment. Microscopic examinations of liver sections revealed hepatocellular hypertrophy in animals of either sex treated with 325 and 1000 mg/kg bw/day and in males treated with 35 mg/kg bw/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature. Microscopic findings were also evident in the thyroid together with increased absolute and relative thyroid weights for females treated with 1000 mg/kg bw/day. Follicular cell hypertrophy was evident in animals of either sex from all treated groups. The liver and thyroid changes together with associated organ weight changes were considered to be adaptive in nature and do not represent an adverse effect of treatment. The microscopic kidney changes identified as increased severity of hyaline droplets in the proximal tubules of the kidneys were evident in males treated with 1000 mg/kg bw/day together with an increase in absolute and relative kidney weight. Accumulations of globular eosinophilic material in the tubular epithelium is a well documented effect, peculiar to the male rat. Female rats and other species do not develop nephropathy and for this reason, the effect is not indicative of a hazard to human health. The microscopic effects detected in non-recovery animals were observed to have generally or completely regressed among recovery high dose animals following 14 days without treatment.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day in Wistar rats.