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Toxicological information

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:
Jun 13, 2006 - Feb 07, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl
EC Number:
439-730-3
EC Name:
(trans(trans))-4'-vinyl-4-(4-methylphenyl)bicyclohexyl
Cas Number:
155041-85-3
Molecular formula:
Hill formula: C21H30 CAS formula: C21H30
IUPAC Name:
(trans(trans))-4'-Vinyl-4-(4-methylphenyl)bicyclohexyl
Details on test material:
Description: solid, off white, crystal mass with a yellowish tinge

Test animals

Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Age at delivery: 6 weeks
- Body weight range at acclimatization: Males: 129.2 – 159.0 grams (mean 145.1 grams), Females: 114.4 – 133.6 grams (mean 124.0 grams)
- Acclimatization: For 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRYCONDITIONS
- Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 30-70 %). Values outside of these ranges occasionally occurred, usually following roomcleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

ACCOMODATION
- In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

DIET
- Pelleted standard Provimi Kliba 3433 (batch nos 01/06, 36/06, and 23/06) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

WATER
- Community tap-water from Itingen was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSE FORMULATION
The dose formulations were prepared daily from 30-June-2006 to 06-July-2006 and weekly from 07-Juli-2006 after confirmation of dose formulation stability by the PI for dose formulation analysis.The test item was pestled, weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer. The mixtures were heated to 50°C to increase solubility, cooled down before application and stored at room temperature (15-25°C).Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Analyses were performed by the Principal Investigator of the analytical phase, according to a HPLC analytical method supplied by the Sponsor. Details of the analytical method are documented in the raw data generated by the Principal Investigator (and/or his/her staff) and the Principal Investigator provided an analytical phase report. Concentration and homogeneity of the dose formulations was determined in samples taken during week 3 of the treatment. Unless otherwise specified, the dose formulations were delivered under ambient conditions. Samples of dose formulations were not discarded without the written authorization of the study director.
Duration of treatment / exposure:
28 days (groups 1-3), 6 days (group 4), 14 days (recovery group)
Frequency of treatment:
daily for 28 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 10 Group 2: 40 mg/kg body weight: 5Group 3: 200 mg/kg body weight: 10 Group 4: 1000 mg/kg body weight: 10
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
The animals were observed in their home cages, outside their home cages in a standard arenaand in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

FOOD CONSUMPTION
The food consumption was recorded once during the pretest period and weekly thereafter,using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

BODY WEIGHTS
Body weights were recorded weekly during pretest, treatment and recovery and beforenecropsy, using an on-line electronic recording system consisting of a Mettler balanceconnected to the RCC computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results of the Functional Observational Battery are presented in the summary andindividual tables of the Detailed Clinical Observations (Weekly) under week 4. This method ofdata presentation permits a clear evaluation and assessment of weekly clinical signs observed during the study.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull straingauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangulargrasping ring and with the hind paws outside a triangular grasping ring. Using one hand, theanimals were held towards the base of the tail and steadily pulled away or towards the ring untilthe grip was broken. Each measurement was repeated three times, the means were calculatedand recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS FöhrMedical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.Low beams count was reported in 10-minute intervals as well as the total activity of themeasuring period.

CLINICAL LABORATORY INVESTIGATIONS
- Blood and urine sampling: after 4 weeks 28-July-2006 (Allocation A, groups 1-3 and B, group 1) after 4 weeks 18-Aug-2006 (Allocation B, group 3)after 6 weeks 11-Aug-2006 (Allocation B, group 1)after 6 weeks 01-Sept-2006 (Allocation B, group 3).
- Blood sampling:after 8 days 07-July-2006 (Allocation A and B, group 4). From group 4 animals (allocation A and B), blood samples were taken by heart puncture atnecropsy if possible. No blood for determination of coagulation parameters was collected fromthese animals. In case the amount of blood collected was not enough to determine allparameters listed (due to the bad condition of the animals), the person responsible for Clinical Laboratory Investigations decided, which parameters were determined.Blood samples for hematology and clinical biochemistry were collected from all other animalsunder light isoflurane anesthesia. The animals were fasted in metabolism cages forapproximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused bycircadian rhythms. Blood samples were drawn from the retro-orbital plexus using a microhematocritglass capillary tube.Urine was collected during the 18-hour fasting period into a specimen vial.The assays were performed at the testing facility under internal laboratory quality control conditions to assure reliable test results.

HEMATOLOGY
The following hematology parameters were determined: Erythrocyte coun, tHemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin Heinz bodies (slides were prepared but not evaluated since no changes in methemoglobin levels were noted), Total leukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase,Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, total Albumin, Globulin, Albumin/Globulin ratio, Bile acids

URINALYSIS
The following urinalysis parameters were determined: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes
Sacrifice and pathology:
NECROPSY
- Sacrifice: after 4 weeks 28-July-2006 (groups 1-3, Allocation A) after 8 days 07-July-2006 (group 4, Allocation A and B) after 6 weeks 11-Aug-2006 (group 1, Allocation B) after 6 weeks 01-Sept-2006 (group 3, Allocation B)

- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. From group 4 animals (allocation A and B), blood samples were taken by heart puncture at necropsy if possible. No blood for determination of coagulation parameters was collected from these animals. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (at least 3 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes w/optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland exorbital, Liver, Lungs filled w/formalin at necropsy, Lymph nodes - mesenteric, mandibular, Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl. coagulating gland), Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord - cervical, midthoracic, lumbar, Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder filled w/formalin at necropsy, Uterus, Vagina, Gross lesions

Additional tissues (such as ear tattoo) were retained in accordance with standard operating procedures but were not processed or examined further.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at terminal sacrifice from the animals of the control and group 3 were examined by the study pathologist. Since test item-related morphologic changes were detected in organs of the high-dose (group 3) animals, those same organs (liver, adrenal glands, and testes) from the low-dose group (group 2) were examined to establish a no-effect level.The same organs of group 4 animals which were terminated in extremis after 6 days of treatment on day 8 (July, 7th, 2006) were not examined.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,body weight, clinical laboratory data, organ weights, and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate wereapplied if the variables could be assumed to follow a normal distribution for thecomparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test whenthe data can not be assumed to follow a normal distribution.
-Fisher's exact-test was applied to the macroscopic findings.
-Student’s t-test was applied to grip strength and locomotor activity data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Please refer to the tables attached.

MORTALITY / VIABILITY
All animals treated with 1000 mg/kg/day were killed in extremis on July 7th, 2006, after 6 days oftreatment and two more days without.One female (No. 70) treated with 200 mg/kg/day died on the first day of the recovery period.The cause of death could not be established.One female (No. 47) treated with 200 mg/kg/day died spontaneously on treatment day 21. The animal died one hour after application without showing any symptoms previously. The cause of death could not be established. One female (No. 48) treated with 200 mg/kg/day and two females of the control group (No. 37 and 39) died on the day of scheduled necropsy after blood sampling. These deaths are attributed to the blood sampling procedure.

CLINICAL SIGNS
Almost all animals treated with 1000 mg/kg/day, which were killed in extremis, showed pale and soft feces, emaciation, sedation, prostration, piloerection, and hunched posture. Slight emaciation from day 7 to 10 in one female treated with 200 mg/kg/day is considered to be test item-related. All other findings are considered not to be test item-related.

FUNCTIONAL OBSERVATIONAL BATTERY
In addition to the other clinical signs noted during daily observations, salivation was seen in one male (no. 65) treated with 200 mg/kg/day in week 4.

- Grip Strength: No test item-related differences were noted in the mean grip strength when compared with the controls.
- Locomotor Activity: Significantly decreased locomotor activity (p<0.05) was seen from 20-30 min. in males treated with 40 or 200 mg/kg/day and from 10-20 min. in females treated with 200 mg/kg/day. A statistically significant increase was seen from 40-50 min. in males treated with 200 mg/kg/day (p<0.05).These observations were inconsistent across sexes and sporadic and are therefore considered not to be test item-related.

FOOD CONSUMPTION
In animals treated with 1000 mg/kg/day, which were killed in extremis on 7-July-2006, food consumption was extremely low. Mean food consumption during the treatment period was reduced at 200 mg/kg/day. A slight decrease in absolute food consumption was also recorded in males treated with 40 mg/kg/day. A decrease in relative food consumption was noted in males at both dose levels only. The decrease was compensated during recovery.

BODY WEIGHT
Body weight and body weight gain were clearly reduced in all animals treated with 200 or 1000 mg/kg/day.

CLINICAL LABORATORY INVESTIGATIONS
- Hematology: Except for decreased hematocrit and hemoglobin in both sexes treated with 200 mg/kg/day and increased hemoglobin concentration distribution width in females treated with 200 mg/kg/day, all changes were within the range of historical data for rats of this strain and age. After recovery, these changes, together with decreased erythrocytes in both sexes (in range for males) treated with 200 mg/kg/day were still present. All other changes were within the normal range of historical data for rats of this strain and age.

- Clinical Biochemistry: After 4 weeks of treatment, the following statistically significant changes were noted:Increased urea in both sexes treated with 200 mg/kg/day (p<0.01); Decreased glucose in males treated with 200 mg/kg/day (p<0.05); Increased creatinine in males treated with 40 mg/kg/day (p<0.05) and males and females treated with 200 mg/kg/day (p<0.01 in males, p<0.05 in females); Increased bilirubin in males treated with 200 mg/kg/day (p<0.05) and females treated with 40 mg/kg/day (p<0.05); Decreased phospholipids in males treated with 40 or 200 mg/kg/day (p<0.01); Increased aspartate aminotransferase in males treated with 200 mg/kg/day (p<0.01); Increased alanine aminotransferase, lactate dehydrogenase, and glutamatedehydrogenase in both sexes treated with 200 mg/kg/day (p<0.01); Slightly increased potassium (p<0.01 in males and p<0.05 in females) and decreased calcium (p<0.01) in both sexes treated with 200 mg/kg/day. After recovery, slightly increased creatinine (p<0.05) and decreased phospholipids (p<0.01) were still found in males treated with 200 mg/kg/day. Total bilirubin was decreased in males treated with 200 mg/kg/day (p<0.05). Alanine aminotransferase was increased in males and females treated with 200 mg/kg/day (p<0.01). Alanine aminotransferase showed a clear tendency towards recovery. In females treated with 200 mg/kg/day potassium was slightly decreased. The above findings were considered to be test item-related. All other changes (even though reaching statistical significance) were within the range of historical data for rats of this strain and age and/or without dose relation and inconsistent across sexes.

- Urinalysis: In urinalysis, slightly increased leukocytes were found after 4 weeks of treatment in females treated with 200 or 40 mg/kg/day. After recovery, a trend for reversibility was noted in the affected females treated with 200 mg/kg/day. All other findings (even if reaching statistical significance) were within the normal range for rats of this strain and age.

ORGAN WEIGHTS
- After 4 Weeks: A dose related increase in absolute and relative liver weights and a dose related decrease in absolute and relative adrenal weights was found in all test item treated rats. The changes were statistically significant in all females and in males treated with 200 mg/kg/day for absolute and relative adrenal weights and liver to body weight ratio.

- After 6 WeeksAbsolute and relative adrenal weights were decreased in all animals treated with 200 mg/kg/day. In females treated with 200 mg/kg/day, increased relative thymus weights were found. In males treated with 200 mg/kg/day, decreased absolute and relative epididymis weights were noted. The liver to body weight ratio was increased in all animals treated with 200 mg/kg/day.

MACROSCOPIC / MICROSCOPIC FINDINGS
After 6 days of treatment, reddish discoloration of the testes was found in 6/10 males treated with 1000 mg/kg/day. In 8/10 males and 9/10 females treated with 1000 mg/kg/day the thymus was reduced in size. After 4 weeks of treatment, all males and 3/5 females treated with 200 mg/kg/day had enlarged livers and reduced adrenal glands. After 4 weeks of treatment and 2 weeks recovery, all males and 4/5 females treated with200 mg/kg/day had reduced adrenal glands. All other macroscopic findings were considered incidental. Microscopically, centrilobular hepatocellular hypertrophy, at minimal severity, was recorded in 4 of 5 males and 2 of 5 females treated with 200 mg/kg/day of the test item. Hepatocellular hypertrophy was not observed after the recovery period. This finding was deemed to be of metabolic nature due to the absence of any further lesion. Cortical atrophy in adrenal glands often (in 3 males and 4 females) with focal/multifocal subacute inflammation was recorded in all males and 4 of 5 females treated with 200 mg/kg/day. Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg/day. These findings were also present after the recovery period. Hemangiectasis in adrenal cortices was recorded in one female treated with 200 mg/kg/day after recovery.Focal/multifocal multinucleated spermatid giant cells at minimal severity were observed in two animals treated with 200 mg/kg/day. This finding was not persistent after recovery. The presence of focal/multifocal multinucleated spermatid giant cells is often associated with early stages of atrophic seminiferous tubules.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands

Any other information on results incl. tables

Please refer to the tables attached.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 40 mg/kg body weight/day of the test item could be established as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

Purpose

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for toxicological risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

In the subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 40 and 200 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 200 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

A fourth group of 10 males and 10 females was treated with 1000 mg/kg body weight/day of the test item. These animals had to be killed in extremis on day 8 after 6 days of treatment and 2 days without treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose (200 mg/kg) animals, and due to findings in the high dose group from all animals of the low dose group (40 mg/kg).

Results

Oral administration of the test item to Wistar rats at doses of 40 and 200 mg/kg/day, for 28 days resulted in 5 unscheduled deaths, only one of which was considered to be test item related.

Treatment related findings were generally restricted to reduced food consumption and reduced body weight and body weight gain in all animals treated with 200 or 1000 mg/kg/day.

Decreased hematocrit and hemoglobin in both sexes treated with 200 mg/kg/day and increased hemoglobin concentration distribution width in females treated with 200 mg/kg/day were present after 4 weeks of treatment as well as after recovery. Decreased erythrocytes in both sexes (in range for males) treated with 200 mg/kg/day were also present after recovery.

In clinical biochemistry, the main changes seen were increased creatinine, decreased phospholipids and increased alanine aminotransferase with alanine aminotransferase showing a tendency towards recovery. Other affected parameters, e.g. aspartate aminotransferase were also indicating changes in the liver and kidneys.

A slightly increased number of leukocytes in the urine was found after 4 weeks of treatment in females treated with 200 or 40 mg/kg/day with a tendency towards reversibility during recovery. In the absence of macroscopic or microscopic correlating findings, this is considered test item related but non-adverse.

Histologically, alterations in the liver, adrenal glands and testes were found in animals treated with 200 mg/kg/day. Hepatocellular hypertrophy correlated with increased liver weights and enlarged livers. It was not present after recovery and was considered to be of metabolic nature due to the absence of any further lesion. Cortical atrophy in adrenal glands often with focal/multifocal subacute inflammation was recorded in both sexes treated with 200 mg/kg/day.

Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg/day. These findings were also present after the recovery period. Cortical atrophy corresponds to macroscopically reduced adrenal glands and decreased adrenal weights. Findings in the adrenals are considered to be test item-related.

Focal/multifocal multinucleated spermatid giant cells at minimal severity were observed in two animals treated with 200 mg/kg/day. This finding was not persistent after recovery. The presence of focal/multifocal multinucleated spermatid giant cells is often associated with early stages of atrophic seminiferous tubules.

Conclusions

Based on the results of this study, 40 mg/kg body weight/day of the test item could be established as the no-observed-adverse-effect-level (NOAEL). Based on this study no classification or labelling is warranted.