Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-11 to 2010-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study reliable without restrictions Minor deviations from the guidelines OECD 408 (1998) and EU Method B.26 (2008) - according to the guidelines, blood urea nitrogen (clinical chemistry) should be measured. This was not done in this study . - according to the guidelines, the cerebrum should be investigated during histopathology, but instead the cerebral cortex was investigated.
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998-09-21
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-
- Molecular formula: C10H20O
- Molecular weight: 156.3 g/mol
- Physical state: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - Rat, RccHan TM: WIST(SPF)
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation (treatment): 8 weeks
- Weight at acclimatisation: males: 143.9 to 203.8 g ; females: 102.8 to 166.7 g
- Housing: in groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK).
- Diet (ad libitum): pelleted standard Harlan Teklad 2914C (batch no. 20/10) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water from Itingen
- Acclimation period: 7 days; under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12; music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 10% ethanol in corn oil
Details on oral exposure:
DOSE FORMULATION:
The dose formulations were not corrected for purity and were prepared with test item as supplied by the Sponsor.
The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study C84885), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

The test item was delivered as solid block in aluminium flasks. Prior to the preparation of the dose formulations, the test item was molten in the flask at 80 °C for 24 h to ensure homogeneity. Thereafter, the test item was allowed to cool down and solidify. Small flakes of test item were scraped from the block and weighed into a tared glass container on a suitable precision balance. Then ethanol was added (the volume of ethanol equalled 10% of the final volume of the dosing solution) and the mixture was stirred until the test item was completely dissolved. To ensure complete solution of the test item in ethanol, the dose formulation of group 5 with a concentration of 160 mg/mL was slightly heated. Then, corn oil was added to give the appropriate final concentration of the test item in the solution. The mixtures were prepared using a magnetic stirrer.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The dose formulations were stored in glass beakers at room temperature (20 ± 5 °C).

DOSE VOLUME: 5 mL/kg body weight

DOSE CONCENTRATIONS:
Group 1: 0 mg/mL/day
Group 2: 0 mg/mL/day
Group 3: 16 mg/mL/day
Group 4: 48 mg/mL/day
Group 5: 160 mg/mL/day

VEHICLE
- Justification for use and choice of vehicle (if other than water): a variety of solvents was tested in a formulation trial for Harlan Laboratories Study C84885 (non-GLP) and was found to be unsuitable for p.o. application by gavage, as the test item was either insoluble in the vehicle or the resulting mixture solidified at room temperature: p. ex. aqua bi-dest., aqua bi-dest. + 20% dimethylsulfoxide, polyethylene glycol 300 (PEG 300), PEG 300 + 5% ethanol and corn oil.
Using a solution of 10% ethanol in corn oil, sufficiently high concentrations of test item in vehicle could be obtained; therefore 10% ethanol in corn oil was used as vehicle. Effects of ethanol administration to rats are well characterized, and 8 mL/kg of a 10% solution for 90 days are “well tolerated” (S.C. Gad et al., Int. J. Toxicol. 2006, 25, p. 499 – 521).

1) Corn oil
- Source: Carl Roth GmbH & Co. KG 76185 Karlsruhe / Germany
- Description: yellowish oily liquid
- Batch no.: 489110589
- Expiry date: 31-Mar-2011 (as handled by Harlan Laboratories Ltd.)
- Storage conditions: at room temperature (range of 20 ± 5 °C), light protected
2) Ethanol
- Source: Sigma Aldrich Chemie GmbH 9471 Buchs / Switzerland
- Description: colourless liquid
- Batch no.: 1297869
- Purity: ≥ 99.8% (v/v)
- Expiry date: 28-Jul-2011
- Storage conditions: at room temperature (range of 20 ± 5 °C), light protected
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using a GC-FID method provided by the sponsor.
After treatment start and during weeks 6 and 13, the following samples of approximately 2 g were drawn in duplicate prior to dosing for analysis of homogeneity and concentration: one sample of the control group (group 1) as well as three samples (top, middle and bottom) of the vehicle group (group 2) and of each test item-treated group (groups 3, 4 and 5).
Duplicate samples of about 2 g of each dose formulation of groups 2 to 5 were taken after treatment start to confirm stability (4 hours and 8 days at room temperature, 20 ± 5 °C).
The samples were stored at -20 ± 5 °C until analysis.
The test item was used as analytical standard.
Acceptance criteria for dose formulation concentration, homogeneity and stability determinations were ±20% of nominal content.

Results:
The formulations investigated during the study were found to comprise BAS 102 in the range of 97.9% to 109.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of BAS 102 in the preparations was approved because single results did not deviate more than 3.1% (<20%) from the corresponding mean.
In addition, the test item was found to be stable in formulations when kept eight days at room temperature due to recoveries which met the variation limit of 20% from the time-zero (homogeneity) mean.
The results indicate the accurate use of the test item BAS 102 and 10% ethanol in corn oil as vehicle during this study. Formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

For the results of the analysis please see also Section 8 Analytical methods (Entry: s_Sieber_2011)
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (groups 1 &2 ), 80 (group 3), 240 (group 4) and 800 mg/kg bw/day (group 5)
Basis:
other: actual dose received
No. of animals per sex per dose:
Main study and recovery groups:
Group 1: 10 males / 10 females (plus 5 males / 5 females as recovery group)
Group 2: 10 males / 10 females (plus 5 males / 5 females as recovery group)
Group 3: 10 males / 10 females
Group 4: 10 males / 10 females
Group 5: 10 males / 10 females (plus 5 males / 5 females as recovery group)

Control animals:
other: Group 1: control animals were treated with corn oil only... (see attached file)
Details on study design:
Dose selection rationale: the dose levels were selected based on a previous 14-day dose range-finding toxicity study (Harlan Laboratories study C84885 (non-GLP)). In this 14-day range-finding study, BAS 102 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose level of 100, 300 and 1000 mg/kg body weight/day for a period of 14 days. A control group was treated with the vehicle only, 10% ethanol in corn oil.
The groups comprised 5 animals per sex which were sacrificed after 14 days of treatment. Mortality, clinical signs, food consumption and body weights were recorded periodically during pretest and treatment periods.
At the end of the treatment period, all animals were killed, necropsied and examined post mortem. Organ weights were recorded.

Results:
- the ethanol-containing vehicle was well tolerated
- no test item-related effects on mortality
- no clinical signs were noted in any animal, except for slightly decreased activity and slight salivation in one male treated with 1000 mg/kg
- no test item-related effect on food consumption
- no differences in mean absolute body weight were observed
- weekly body weight gain was statistically significantly reduced (p<0.05, Dunnett's test) in males treated with 1000 mg/kg
- in both sexes, liver and kidney weights were increased in animals treated with 1000 mg/kg (kidneys in females not statistically significant)
- in females, increased liver weight was also observed in animals treated with 300 mg/kg
- increased adrenal weight was observed in females treated with 1000 mg/kg
- incidental findings: one control male, one control female and one female treated with 100 mg/kg were noted with dark red discolouration of the thymus

Based on the results of this dose range-finding study, doses of 80, 240 and 800 mg/kg/day were selected by the Sponsor for the subsequent 90-days oral (gavage) toxicity study.

- Post-exposure recovery period in satellite groups: 28 days ; after 91 days of treatment with ethanol-containing vehicle, rats may become addicted. To prevent the occurrence of ethanol withdrawal symptoms, recovery animals were treated with 10% ethanol in corn oil (groups 2 and 5), and corn oil only (group 1).
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS (in the result section mentioned as "Daily clinical signs"): Yes
- Time schedule: observations for viability / mortality were recorded twice daily. The animals were observed for clinical signs once before commencement of administration as well as twice daily during days 1 - 3 and daily on days 1 - 91/92 of the treatment period and once daily during days 1 - 28 of the recovery period.

DETAILED CLINICAL OBSERVATIONS (in the result section mentioned as "Weekly behavioural observations") : Yes
- Time schedule: the animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 13) thereafter.
- Cage side observations checked: appearance (piloerection, salivation, hunched posture), motor (ataxia, tremor/twitching, prostration, circling, spasm), behaviour (hyperactivity, somnolence, increased exploration, reduced grooming, vocalization), respiration (dyspnea, tachypnea, bradypnea), reflexes (blink, pinna, iridic light reflex, push-off (hind leg), pain response, startle/hearing, righting reflex) and miscellaneous (lacrimation, limbs cyanotic, mydriasis, miosis, exophthalmos, reduced muscle tone)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to a computer.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, main daily diet consumption calculated as g food/kg body weight / day was determined.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
- Time schedule for examinations: once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to a computer.

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: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: once on all animals during acclimatization and on groups 1, 2 and 5 during week 13.
- The ophthalmoscopic examinations of both eyes of the animals were performed after the application of a mydriatic solution (Ciba Vision AG, 3172 Niederwangen / Switzerland) using a direct ophthalmoscope. A description of any abnormality was recorded.

HAEMATOLOGY: Yes
- Time schedule for collection of blood and how many animals: after 13 weeks (all males and females of the main study and all recovery animals; blood samples of the males of the main study were taken one day after the blood samples of the females of the main study and recovery animals were taken) and after 17 weeks (only recovery animals); samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes; blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia by using a micro-hematocrit glass capillary tube.
- Animals fasted: Yes; the animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence), leukocyte count (total), differential leukocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes and large unstained cells), platelet count, methaemoglobin, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood and how many animals: after 13 weeks (all males and females of the main study and all recovery animals; blood samples of the males of the main study were taken one day after the blood samples of the females of the main study and recovery animals were taken) and after 17 weeks (only recovery animals); samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: Yes; the animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, bile acids, gamma-glutamyl-transferase, creatine kinase, sodium, potassium, chloride, calcium, phosphorus, protein (total), albumin, globulin and albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: after 13 weeks (all males and females of the main study and all recovery animals; blood samples of the males of the main study were taken one day after the blood samples of the females of the main study and recovery animals were taken) and after 17 weeks (only recovery animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; urine was collected during the 18 hours fasting period
- Parameters examined: urine volume (18 hour), specific gravity (relative density), colour, appearance, pH value, nitrite, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes and leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13 of the treatment period, relevant parameters (presented above under the heading "Detailed clinical observations") from a modified Irwin screen test were evaluated.
- Dose groups that were examined: all animals
- Battery of functions tested:
1) grip strength: each measurement was repeated three times; the means were calculated and recorded.
2) motor activity: 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 the measuring period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Sacrifice:
- after 13 weeks: all males and all females of the main study (males were killed one day later than the females)
- after 17 weeks: all recovery animals

All main study and recovery animals were weighed and necropsied. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. Descriptions of all macroscopical abnormalities were recorded. A necropsy including macroscopic examination was performed also on all animals which were found dead.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise: adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain - including section of medulla/pons, cerebral and cerebellar cortex, 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 including auricles, 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 and mandibular, mammary gland area, nasal cavity, ovaries, pancreas, pharynx, pituitary gland, prostate gland incl. coagulating glands, 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), tongue, trachea, ureter, urinary bladder (filled w/formalin at necropsy), uterus (w/ cervix, vagina and oviducts) and all gross lesions

ORGAN WEIGHTS:
The following organs from main study and recovery animals were weighed before fixation and the weights were recorded on the scheduled dates of necropsy: adrenal glands, brain - including section of medulla/pons, cerebral and cerebellar cortex, epididymides (fixed in Bouin's solution), heart including auricles, kidneys, liver, ovaries, spleen, testes (fixed in Bouin's solution), thymus and uterus (w/ cervix, vagina and oviducts). Relative organ weights were calculated on the basis of the body weight and brain weight. The terminal body weight was recorded immediately prior to necropsy.

HISTOPATHOLOGY: Yes
All organ and tissue samples, as stated for gross pathology above, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.
Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control (group 1 and 2) and high-dose groups (group 5) and all gross lesions from all animals were examined. Organ and tissue samples taken from animals which died spontaneously or which were killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group. As histomorphologic changes were observed in the liver, kidneys, stomach, prostate, nasal cavities and pharynx of high dose animals, examination of these organs was extended to the assigned animals of the mid- and low-dose groups.
Attempts were made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyse body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
- the Dunnett-test [see C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J.Amer. Stat. Assoc. 50, 1096-1121 (1955)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- the Steel-test [see R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981)] (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test [see R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).]

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1) Mortality:
- no premature deaths occurred during the acclimatization period and during the recovery period;
- treatment period: one female treated with 240 mg/kg/day and one female treated with 800 mg/kg/day were found dead during week 6. One male treated with 800 mg/kg/day spontaneously died during week 11 and one male treated with vehicle, 10% ethanol in corn oil, spontaneously died after treatment during week 13;
- the death of female treated with 240 mg/kg/day was caused by renal failure; the cause of death of the other decedents could not be established.

2) Daily clinical signs.
- no clinical signs were recorded in any animal during the acclimatization period and during the recovery period;
- treatment period: no clinical signs were recorded in animals treated with corn oil (group 1) and with vehicle (10% ethanol in corn oil, group 2) as well as in females treated with 80 and 240 mg/kg/day;
- one male treated with 80 mg/kg/day was noted with moderate salivation on a single day during week 13, this was probably caused by accidental regurgitation of the dosing solution and is thus related to the treatment procedure; no test item-related clinical signs were noted in males treated with 80 mg/kg/day;
- one male treated with 240 mg/kg/day was noted with slight dyspnea and slight to moderate salivation on two consecutive days during week 8; no other test item-related clinical signs were noted in males treated with 240 mg/kg/day;
- in males and females treated with 800 mg/kg/day, slight to moderate dyspnea and slight to moderated salivation were observed in single animals throughout the treatment period; these clinical signs were noted in various individuals and were mostly observed for a short period of several days only, never affecting more than 30% of the group. These clinical signs were considered test item-related;
- any other findings recorded in single animals such as fissures, scabs and wounds were considered incidental.

3) Weekly behavioural observations:
- no test item-related findings were recorded during the acclimatization, treatment and recovery period;
- slightly decreased activity was recorded in one male treated with corn oil and one male treated with 240 mg/kg/day during week 13 as well as in two females treated with 240 mg/kg/day during weeks 1 and 5, respectively; these findings were considered incidental.

BODY WEIGHT AND WEIGHT GAIN
- compared to vehicle-treated animals, absolute body weight and body weight gain were statistically significantly reduced in males treated with 800 mg/kg/day from the second week and first week onwards, respectively, until the end of the treatment period;
- in females treated with 800 mg/kg/day, statistically significantly lower absolute body weight and body weight gain was observed during weeks 1-4, 6 and 8 of the treatment period compared to vehicle-treated animals;
- the largest difference in absolute body weight between the high-dose group treated with 800 mg/kg/day and the vehicle group was -9.5% for males and -5.0% for females;
- in females treated with 240 mg/kg/day, body weight gain was slightly reduced during the first half of the treatment period compared to vehicle-treated animals, with the difference attaining statistical significance during week 4; however, the animals recovered towards the end of the treatment period.
During the 4-week recovery period, no statistically significant differences in absolute body weight between animals treated with corn oil, vehicle and 800 mg/kg/day were observed. Body weight gain of females treated with 800 mg/kg/day was increased in week 4 of the recovery period and body weight gain of males treated with 800 mg/kg/day was increased in weeks 2 to 4 of the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE
- food consumption was not affected by treatment with BAS 102.

OPHTHALMOSCOPIC EXAMINATION
- no test item-related changes were observed.

HAEMATOLOGY (changes in haematology parameters observed in animals treated with BAS 102 compared to animals treated with vehicle only)
After 13 weeks of treatment:
- increased prothrombine time and decreased partial thromboplasiten time were observed in animals treated with 800 mg/kg bw/d;
- a shift in reticulocyte caounts animals at 800 mg/kg bw/d (mainly females).
After the 4-week recovery period: no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.

CLINICAL CHEMISTRY
After 13 weeks of treatment: the following changes in clinical biochemistry parameters observed in animals treated with BAS 102 compared to animals treated with vehicle only were considered test item-related:
- decreased glucose levels at 800 mg/kg bw/day (-33% and -28% (p<0.01) for males and females, respectively);
- increased urea levels in males treated with 800 mg/kg/day (+38%, p<0.01) and females treated with 240 and 800 mg/kg/day (+20% (p<0.05) and +35% (p<0.01), respectively);
- increased creatinine levels in animals at 800 mg/kg/day (+35% (p<0.01) and +11% (p<0.05) in males and females, respectively);
- increased cholesterol levels in animals treated with 800 mg/kg/day (+29% and +50% (p<0.01) in males and females, respectively);
- increased triglyceride levels in animals at 800 mg/kg/day (+151% and +115% (p<0.01), in males and females, respectively);
- increased phospholipid levels in males and females treated with 800 mg/kg/day (+33% and +40% (p<0.01), respectively);
- increased lactate dehydrogenase levels (LDH) in males treated with 800 mg/kg/day (+44%, p<0.01);
- increased alkaline phosphatase levels (ALP) in females treated with 800 mg/kg/day (+25%, p<0.05);
- increased potassium levels in females treated with 240 and 800 mg/kg/day (+11% and +10% (p<0.01), respectively);
- increased calcium levels in males treated with 800 mg/kg/day (+3%, p<0.05) and in females treated with 80 and 800 mg/kg/day (+3% (p<0.05) and +5% (p<0.01), respectively);
- increased phosphorus levels in males treated with 800 mg/kg/day (+16%, p<0.01) and in females treated with 80, 240 and 800 mg/kg/day (+21%, +19% and +27% (p<0.01), respectively);
- increased protein levels in males treated with 800 mg/kg/day (+5%, p<0.01);
- increased globulin concentration and decreased albumin/globulin ratio (A/G ratio) in males and females treated with 800 mg/kg/day (+16% and -13% for males and +17% and -21% for females (p<0.01), respectively).
All values were within the range of historical control data, with the exception of increased cholesterol levels of females, increased phospholipid values of males and globulin concentration of males treated with 800 mg/kg/day.
After the 4-week recovery period: no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed, except reduced potassium levels in vehicle-treated males compared to corn oil-treated males or males treated with 800 mg/kg/day. As no changes in potassium levels were observed after 13 weeks of treatment, this finding was considered incidental.

URINALYSIS
After 13 weeks:
- increased urinary volume in males treated with 800 mg/kg/day (+131%, p<0.01);
- increased protein in males and females treated with 800 mg/kg/day (p<0.05 and, p<0.01, respectively);
All values were within the range of historical control data.
After the 4-week recovery period: no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.

NEUROBEHAVIOUR
1) Functional observational battery: no test item-related findings were recorded.
2) Grip strength: grip strength was not affected by treatment with BAS 102.
3) Locomotor activity: total locomotor activity was not affected by treatment with BAS 102.

ORGAN WEIGHTS
After 13 weeks:
- increased liver and kidneys weights in males (please also refer to "Any other information on results incl. tables" below; Table 1).
After the 4-week recovery period, the organ/body weight ratio of kidneys of males treated with 800 mg/kg/day was still statistically significantly increased (+15%, p < 0.05) compared to males treated with vehicle only.

GROSS PATHOLOGY
After 13 weeks:
- in the liver of two males treated with 240 mg/kg/day and of three males treated with 800 mg/kg/day, accentuated lobular pattern was noted; this finding correlated with hepatocellular hypertrophy;
- discoloration of the kidneys was observed in one male treated with 240 mg/kg/day and in two males treated with 800 mg/kg/day; these findings were considered test item-related.
After the 4-week recovery period, no test item-related findings were recorded in any animal.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following findings were observed in the target organs of toxicity and are presented in detail in "Any other information on results incl. tables" below (Table 2, 3 and 4).
Kidneys:
- in general, lesions recorded in the kidneys affected more males than females. They consisted of increased incidence or incidence and severity of hyaline droplets, tubular basophilia and mononuclear cell foci along with granular casts and tubular dilation in males treated with 80, 240 and 800 mg/kg/day. In addition, hyaline casts were increased in incidence in animals treated with 240 and 800 mg/kg/day, and tubular cysts were recorded in animals treated with 800 mg/kg/day;
- in females treated with 800 mg/kg/day, an increase in incidence or incidence and severity of tubular basophilia along with minimal granular casts, tubular dilation and tubular cysts was observed.
After the recovery period, increased incidences and severities of tubular basophilia, granular casts, mononuclear cell foci and tubular dilation along with increased incidence of hyaline casts were still recorded in males treated with 800 mg/kg/day.

Stomach:
- treatment-related lesions consisted of minor incidences and/or severities of epithelial hyperplasia, hyperkeratosis, focal glandular erosion and forestomach ulceration in animals treated with 240 and 800 mg/kg/day. In males treated with 800 mg/kg/day, the hyperkeratosis sometimes occurred along with minimal parakeratosis.
The findings resolved after the recovery period.

Liver (regarded as metabolic adaptation):
- minimal hepatocellular hypertrophy, mainly centrilobular, was recorded in few males treated with 240 and 800 mg/kg/day as well as in females treated with 800 mg/kg/day.

A number of findings observed in nasal cavity, nasopharyngeal duct and pharynx were related to regurgitation of the test compound and its irritant potential (not presented in detail):
- main findings recorded in the nasal cavity at different levels, in nasopharyngeal duct and pharynx consisted of necrosis and regenerative epithelium
- further findings in the nasal cavity consisted of olfactory epithelial disorganization/degeneration along with inflammatory changes (inflammatory exudate, inflammatory cell infiltrates and submucosal inflammation) as well as decreased eosinophilic inclusions.
After the recovery period, the following findings were still present in the nasal cavity but generally at reduced incidences and/or severities: necrosis and regenerative epithelium at level III as well as decreased eosinophilic inclusions in levels III and IV.

Other findings:
- minimal degrees of concretions along with few reactive granulocytes were recorded in the prostate gland of three males treated with 800 mg/kg/day. Minimal concretions were observed also in one male treated with 240 mg/kg/day, but without inflammatory cell reaction;
- a marked pyelonephritis was recorded in the examined kidney of one female (group 4: 240 mg/kg bw/day), which was found dead; this finding was along with chronic urinary bladder inflammation, marked lymphoid atrophy of the thymus, minimal lymphoid atrophy and increased tingible body macrophages in the mandibular lymph node, minimal erosion, ulceration and hyperkeratosis in the stomach as well as slightly increased granulopoiesis in the bone marrow.

Effect levels

Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: In females, no relevant effects were observed at this dose level. In males, beside signs of alpha2µ-globulin nephropathy (consedered not relevant for humans), no additional adverse effcets were seen.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Organ weights

 

Males

Females

80 mg/kg

240 mg/kg

800 mg/kg

80 mg/kg

240 mg/kg

800 mg/kg

Liver

Weight

 

+17% (**)

+12% (*)

 

 

 

 

Organ/

body weight ratio

 

+12% (*)

+31% (**)

 

 

+11% (*)

 

Organ/

brain weight ratio

 

+16% (**)

+10% (-)

 

 

 

Kidney

Weight

+16% (*)

+27% (**)

+28% (**)

 

 

 

 

Organ/

body ratio

+18% (*)

+22% (**)

+51% (**)

 

 

 

 

Organ/

brain weight ratio

+18% (*)

+26% (**)

+26% (**)

 

 

 

*/**/- : Dunnett-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Table 2: Microscopic findings (liver)

Findings

Group 1

Group 2

Group 3

Group 4

Group 5

Main test animals

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Hepatocellular hypertrophyIncidence/mean severity

0

0

0

0

0

0

1/1.0

0

2/1.0

4/.10

Table 3: Microscopic findings (kidney)

Findings

Group 1

Group 2

Group 3

Group 4

Group 5

Main test animals

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Hyaline droplets

Incidence/Mean Severity

2/1.0

0

4/1.0

0

 

10/2.3

0

10/2.2

0

10/2.7

0

Tubular basophilia

Incidence/Mean Severity

5/1.0

1/1.0

3/1.0

1/1.0

9/1.3

 

0

10/2.0

1/1.0

9/1.9

3/1.3

Hyaline cast(s)

Incidence/Mean Severity

0

0

1/1.0

0

1/1.0

0

3/1.0

0

7/1.0

1/1.0

Granular cast(s)

Incidence/Mean Severity

0

0

0

0

7/2.0

0

9/2.1

0

9/1.8

1/1.0

Mononuclear foci

Incidence/Mean Severity

1/1.0

1/1.0

1/1.0

0

3/1.0

1/1.0

5/1.2

1/1.0

3/1.0

1/1.0

Tubular dilation

Incidence/Mean Severity

0

0

0

0

1/1.0

0

7/1.0

0

10/1.8

8/1.0

Tubular cysts

Incidence

0

0

0

0

0

0

0

0

2

1

Recovery Animals

5 M

5 F

5 M

5 F

 

5 M

5 F

Hyaline droplets

Incidence/Mean Severity

2/1.0

0

2/1.0

0

1/1.0

0

Tubular basophilia

Incidence/Mean Severity

4/1.0

0

3/1.0

0

5/1.6

0

Hyaline cast(s)

Incidence/Mean Severity

0

0

1/1.0

0

3/1.0

0

Granular cast(s)

Incidence/Mean Severity

0

0

0

0

2/2.0

0

Mononuclear foci

Incidence/Mean Severity

2/1.0

0

1/1.0

0

4/1.3

1/1.0

Tubular dilation

Incidence/Mean Severity

0

0

0

0

3/1.0

0

Table 4: Microscopic findings (stomach)

Findings

Group 1

Group 2

Group 3

Group 4

Group 5

Main Test Animals

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

10 M

10 F

Epithelial hyperplasia

Incidence/Mean Severity

0

0

0

0

0

0

0

1/2.0

4/1.0

3/1.7

Hyperkeratosis

Incidence/Mean Severity

0

0

0

0

0

0

1/1.0

1/1.0

9/1.0

5/1.0

Parakeratosis

Incidence/Mean Severity

0

0

0

0

0

0

0

0

3/1.0

0

Erosion, glandular stomach

Incidence/Mean Severity

0

0

0

0

1/1.0

0

0

1/1.0

2/1.5

0

Ulceration, forestomach

Incidence/Mean Severity

0

0

0

0

0

0

0

1/1.0

1/1.0

0

Applicant's summary and conclusion

Conclusions:
NOAEL(male/female rats, oral): 240 mg/kg bw/day (actual dose received)
The primary target organ for the test item was considered to be the liver (kidney effects were not considered relevant for humans). Also, the stomach may also be regarded as primary target organ. The findings in the liver were deemed to be by metabolic adaptation.

In conclusion, based on the histopathological findings observed in kidneys, stomach, and liver, as well as findings related to accidental aspiration/regurgitation of the vehicle/test item, the histopathological NOEL for females could be established at 80 mg/kg/day. However, a histopathological NOEL for males could not be established.
The only test item-related findings recorded in males of the low dose group (80 mg/kg bw/day) were hyaline droplets, tubular basophilia, casts and tubular dilation. These findings, although elevated above the level of historical controls for animals of this strain and age, are common findings in untreated control animals.
Hyaline droplets, tubular basophilia and casts are typical signs of α2u-globulin nephropathy. This condition is specific to the male rat and is caused by the accumulation of α2u-globulin-komplexes in renal tubular cells. Hydrophilic compound bind to α2u-globulin, thus inhibiting its degradation in the tubular cells and leading to the accumulation in vesicles which are visible histologically as hyaline droplets (Peter Greaves: Histopathology of Preclinical Toxicity Studies, Third edition, 2007, p. 600-605, Academic Press, Elsevier.).
This condition in the male rat has been described for a wide variety of chemicals. Female rats and higher species, including humans, are not α2u-globulin excreters and are therefore not susceptible. (R. H. Alison: Neoplastic Lesions of Questionable Significance to Humans, Toxicol. Pathol. 22, 2, p. 179-186 (1994).).
Taking into account that the test item BAS 102 is trans-4-tert-butyl-cyclohexanol and therefore chemically very similar to other compounds known to bind to α2u-globulin (Borghoff et al. 1991), the cause of hyaline droplets, tubular basophilia, and casts observed in males treated with 80 mg/kg/day is most likely caused by 2u-globulin nephropathy and is therefore not relevant for humans.
Based on these considerations, a NOAEL for effects relevant to human risk assessment may be established at 240 mg/kg/day for females under the conditions of this study. This conclusion is supported by the observations in males because besides the signs of α2u-globulin nephropathy, no additional adverse effects occurred at 240 mg/kg/day.