Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented 52-week gavage study in rats. Appears to substanitally conform to modern standards of study conduct. However, there is no separate QA or GLP statement. The study report does not purport that the study conforms to either a national/international protocol nor national/international GLP standards. Though the study report includes a statement of chemical purity (98.8%), there is no separate C of A. As well, the study report asserts that a separate assessment of compound stability was conducted, showing that compound purity was stable, however, there is no separate report of this incorporated into this study report. The study report states that study substance was stable in the vehicle (corn oil) over the study period, however,there is no separate report of this incorporated into this study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Principles of method if other than guideline:

Graded doses administered daily via gavage over 13-weeks accompanied with body wt, clinical, and gross & histopathological observations.

GLP compliance:

not specified

Remarks:

no data The study report does not represent that the study conforms to either a national/international protocol or national/international GLP standards.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6731-36-8 (98.8%)

IUPAC Name:

6731-36-8 (98.8%)

Constituent 2

Reference substance name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

EC Number:

229-782-3

EC Name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Cas Number:

6731-36-8

Molecular formula:

C17H34O4

IUPAC Name:

1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane

Details on test material:

1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane (Lot No.:_______, CAS No.: 6731-36-8) was supplied by _______. This test substance is a colorless, transparent liquid of 98.8% purity. The stability of the test substance was validated by analyses performed by the supplier during the study period. Corn oil was utilized as the medium (Lot Nos. V2B6838, V3G3918, V3K4725, V3T0445 and V4A1005, supplied by Nakarai K.K.). The original materials of the test substance and medium were stored at room temperature in the test substance storage room (actual temperature:
16-25°C).

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

85 female and 85 male Crj:CD (SD) rats (Japan Charles River Company, Atsugi Breeding Center) were procured, and the males were quarantined and acclimatized for 7 days and the females were quarantined and acclimatized for 8 days. During these periods, the general condition was observed and the body weight was measured for all animals to insure no abnormality. Subsequently, 80 animals of each sex were selected and the study was conducted with 6-week-old animals. The body weights were 193.8-231.9 g for the males and 138.4-173.4 g for the females at the start of the study. The animals were raised in stainless steel hanger cages (w 260 x H 200 x D 380 mm), 2-3 per cage during acclimatization and 1 per cage during the administration period, in the No. 87 breeding room in C Ward, with a barrier system with the temperature set at 24°C (range: 21-27°C), humidity at 55% (range 35-75%), 12 h illumination (7 a.m.-7 p.m.) and a ventilation rate of 13-15 times/h. The cage stands were rotated to the right in the breeding room once a week during the administration period. In this regard, the highest temperature measured was 26°C and the lowest was 22°C, and the highest humidity measured was 61% and lowest was 49% during the study period. The feed was a solid feed (CRF-1, Oriental Yeast Industry K.K.) sterilized with high-pressure steam and the water was well water supplied from an automatic water supply device (water bottles were utilized during urine tests) after treating with sodium chlorite (about 2 ppm), both given freely. The diet was analyzed by the Japan Food Analysis Center, a foundation, and the water was analyzed by the Nankyu Science Research Center of Tsurushiro K.K., and both were validated to be within specifications. The feeding equipment was sterilized with high-pressure steam. The cage stands were exchanged once during grouping and 1-2 times every 4 weeks afterward, the cages were exchanged once during grouping and once every 2 weeks afterward, and the pans were exchanged 2-3 times weekly, and the breeding room was cleaned and mopped with disinfectant daily. The disinfectants were sodium chlorite and invert soap (2 kinds), used weekly in alternation.

The study groups consisted of 4 groups, including the3 dose groups (4, 20, and 100 mg/kg bw, plus a control group. The number of animals for necropsy was 10 each per dose group.

Stratified, continuous random grouping was carried out based on the body weights of the males and females one day before starting administration. An ear tag labeled with animal number was attached to each animal after grouping, and a label showing the study No., animal number, dose and sex was attached to the front of each cage.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Route of administration, administration method, administration and recovery period
Oral administration was determined as the route of administration in accordance with the OECD toxicity study guidelines for route of administration
as well as from the exposure route expected in humans. The administration was performed with a gastric tube, once a day, 7 days a week, for 52 weeks. The volume administered was 5 mL/kg and the control was administered the same volume of the medium. The amount of liquid administered was calculated based on the most recent body weight. In this regard, the day administration began was given as administration day 1 and the week administration began was given as administration week 1.

Method for preparing test substance and medium mixtures and frequency of preparation
The required amount of the test substance for each concentration was weighed and dissolved in corn oil to prepare 0.8, 4 and 20 mg/mL solutions. The solutions were prepared once a week and the prepared mixtures were stored in a shaded area in a low-temperature room or in a refrigerator (actual measured temperature: 1-8°C) in the specimen storage room in the animal breeding area. In this case, the 0.8 and 20 mg/mL solutions of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane dissolved in corn oil were verified to be stable for 8 weeks in a dark cold place (Attached data 2- not included). Also, analysis of all mixtures prepared for the first administration (administration week 1), during the midadministration period (administration week 26) and for the final administration (administration week 52) were performed, and the concentrations were verified to be within ±10% of the stipulated values.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no details are provided in the translated study report.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily, seven days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 4, 20, 100 mg/kg
Basis:
other: gavage

No. of animals per sex per dose:

20 males & 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

The study groups consisted of 4 groups, including 3 dose groups and a control group. The number of animals for necropsy was 10 each for the (13-week and) 52-week administration groups.

Stratified, continuous random grouping was carried out based on the body weights of the males and females one day before starting administration. An ear tag labeled with animal number was attached to each animal after grouping, and a label showing the study No., animal number, dose and sex was attached to the front of each cage.

Route of administration, administration method, administration
Oral administration was determined as the route of administration in accordance with the OECD toxicity study guidelines for route of administration as well as from the exposure route expected in humans. The administration was performed with a gastric tube, once a day, 7 days a week, for 13 weeks.The volume administered was 5 mL/kg and the control was administered the same volume of the medium. The amount of liquid administered was calculated based on the most recent body weight. In this regard, the day administration began was given as administration day 1 and the week administration began was given as administration week 1.

Method for preparing test substance and medium mixtures and frequency of preparation
The required amount of the test substance for each concentration was weighed and dissolved in corn oil to prepare 0.8, 4 and 20 mg/mL solutions. The solutions were prepared once a week and the prepared mixtures were stored in a shaded area in a low-temperature room or in a refrigerator (actual measured temperature: 1-8°C) in the specimen storage room in the animal breeding area. In this case, the 0.8 and 20 mg/mL solutions of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane dissolved in corn oil were verified to be stable for 8 weeks in a dark cold place. Also, analysis of all mixtures prepared for the first administration (administration week 1), during the midadministration period (administration week 26) and for the final administration (administration week 52) were performed, and the concentrations were verified to be within ±10% of the stipulated values.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Observations of general condition
The presence or absence of symptoms and mortality were observed twice daily, once prior to administration and once about 1-3 h after administration.

Body weight measurement
Body weight was measured once a week through administration week 13.

Measurement of food consumption
Food consumption was measured once a week through administration week 13. The feeder was filled with feed at 1-5 p.m, weighed and placed in the cage, and the feeder was taken out of the cage and the remaining food was weighed about 24 h later on the next day. The difference was given as the food consumption per day. In this regard, the day of the food consumption was given as the day the remaining food was measured.

Urine test
A urine test was performed for each animal at administration weeks 13. Fresh urine was sampled during the time frame of 8-12 a.m. (prior to administration) using a metabolic cage and the urine accumulated for the subsequent 24 h was also sampled. In this regard, feeding was started after fresh urine was sampled on the urine sampling day, but water was supplied as it was normally. The test items included: Amount of urine, Color, Osmotic pressure, Specific gravity, Sodium, Potassium, Chloride, Protein, Glucose, Ketone bodies, Bilirubin, Occult blood, Urobilinogen, Urine sediment(fresh centrifuged urine was examined for - Epithelial cells, Erythrocytes, Leukocytes, Casts, Acellular sediment)

Hematological examinations
Hematological examinations were performed on animals for necropsy at the end of administration weeks 13 and 52. 2-2.5 mL blood were sampled from the major posterior abdominal vein after intraperitoneal injection of 30 mg/kg pentobarbital sodium. The plasma utilized for blood coagulation testing was obtained by adding 0.9 mL blood to a test tube containing 0.1 mL of 3.8% sodium citrate, followed by centrifugation for 15 min at 1870 G (about 4°C). the sample utilized for other examinations was obtained by adding the remaining blood to a blood-sampling bottle (SB-41; Cysmex K.K.) containing 2 mg EDTA•2K. The animals were fasted for at least 18 h before blood sampling. In this case, the same operation was performed for the examinations on the midterm necropsy case. The test items: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Platelet count, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Leukocytes morphology Reticulocyte count, Prothrombin time, Activated partial thromboplastin time.

Blood biochemistry tests
The test was performed on animals for necropsy at the end of administration weeks 13 and 52. After blood was sampled for the hematological examinations, 3-5 mL blood was sampled from the major posterior abdominal vein under anesthesia, followed by allowing it to stand for about 60 min at room temperature, and then centrifugation for 10 min at 1870 G (about 4°C), and the serum obtained was utilized. In this case, the same operation was conducted in the examination for the midterm necropsy cases.The test items included: Total protein, Total bilirubin, Alkaline phosphatase, Total cholesterol, Triglycerides., Phospholipids, Glucose, Urea nitrogen, Creatinine, Inorganic phosphorus, Calcium, Serum protein fractionation, A/G Ratio, Sodium, Potassium, Chloride

Organ weight measurements
The weights (absolute weight) of the organs listed were measured after necropsy. Furthermore, the relative organ weights (relative weights) were calculated based on the body weights on the day the necropsy was performed. The same measurements were conducted on mortalities and the midterm necropsy cases. Brain, Pituitary, Thyroid (including parathyroid), Heart, Thymus, Lungs (including bronchi), Liver, Spleen, Kidneys, Adrenals, Testes, Epididymis, Ovaries, Uterus

Sacrifice and pathology:

Necropsies were performed for animals slated for sacrifice at the end of administration weeks 13 and 52. After blood was sampled, the animal was
exsanguinated and the necropsy was promptly performed, and all organs and tissues were examined for the presence or absence of abnormalities. In this case, the same examinations were conducted on mortalities and the midterm necropsy cases.

Histopathological examination
The listed organs/tissues were fixed and preserved in 10% neutral-buffered formalin solution (except that the eyes, optic nerves and Harderian glands were prefixed in 2.5% glutaraldehyde, and that the testes and epididymis were prefixed with Bouin's fluid). Microscopic examination was performed on specimens from the control group and the high-dose group after paraffin sectioning and staining with hematoxylin/eosin (H.E.). The result showed that test substance-related changes were observed in the livers, kidneys and mesenteric lymph nodes for males and females in the 100 mg/kg group in the examinations at the end of administration week 13, thus the same examinations were performed on the mesenteric lymph nodes for males and females in the 20 mg/kg group and on the livers and kidneys for females and males in the 4 and 20 mg/kg groups. Also, the same examinations were performed on cases for which tumors or "corns" (see NB below) were observed during necropsy.

Cerebrum, Tongue, Seminal vesicle, Cerebellum, Thymus, Prostate, Medulla oblongata, Liver Epididymis, Pituitary, Pancreas, Testes,Spinal cord (thoracic and lumbar), Spleen, Ovaries, Eyes, Kidneys, Uterus, Optic nerves, Adrenals, Vagina, Harderian glands, Esophagus, Femur (including marrow), Submaxillary lymph nodes, Stomach, Sternum (including marrow), Submaxillary glands, Duodenum, Mammary glands, Sublingual glands, Jejunum, Skin (lower abdomen),Subaural glands, Ileum, Aorta (thoracic),Thyroid, Cecum, Sciatic nerve, Parathyroid, Colon, m. biceps femoris, Heart, Rectum, Hind legs (right or left), Lungs (including bronchi), Mesenteric lymph nodes, Tumors, Trachea, Bladder

NB- Although the "original" data tables available in English on the internet include the word "corns", and the certified translations of this study report also originally had selected this term for the translation, when reviewed in context, the term is abiguous at best. Further review by other translators indicate that the report author might have been trying to convey the idea of thickened skin, callouses, or keratosis. In any event, as the effect was seen in both treated and untreated animals and does not appear to be dose related, neither is it likely to be treatment related. One plausible explanation is that the rats had developed toughened areas on their feet from housing in wire cages.

Statistics:

Means and standard deviations were calculated for body weight, food consumption, urine test (quantitative), hematological tests, blood biochemistry tests, organ weights and organ weight-body weight ratios for each group, and the homogeneity of the variances was tested by Bartlett's method. Comparison to the control group was performed with Dunnett's multiple comparison test for homogeneous variances and with Steel's multiple comparison test for heterogeneous variances. Steel's multiple comparison test was performed with the control group after the grades were converted to numbers with respect to the color and urine sediment results in the urine test by the test paper method. Also, Fisher's correct probability test was performed with respect to the necropsy results and Mann-Whitney's U-test was performed with respect to the results of histopathological examinations (excluding the results of immunological staining using anti-α2u-globulin antibody), in comparison to the control group. Two-sided tests were performed in both cases with 1 and 5% significance levels.

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 specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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:

no effects observed

Details on results:

General condition
Changes related to the administration of the test substance were not observed in any case throughout the administration period.

Body weight
Body weights showed almost the same time course of increase for each administration group and the control group throughout the administration period.

Food consumption
Changes related to the administration of the test substance were not observed in any administration group throughout the administration period.

Urine tests
Changes related to the administration of the test substance were not observed in any administration group in tests at week 52.

Hematological tests
In tests at the end of administration week 52, high leukocytes, neutrophils, lymphocytes, basophils, monocytes and unstained large cells in leukocytes morphology, low MCV and MCH, and prolonged PT and APTT were observed in males in the 100 mg/kg group. Also, low hemoglobin in females in the 20 and 100 mg/kg groups and low hematocrit in females in the 100 mg/kg group were observed.

Blood biochemistry tests
In tests at the end of administration week 52, low A/G ratio and albumin ratio [sic] and high α2-globulin ratio, β-globulin ratio and ALT were observed in males in the 100 mg/kg group. Also, low A/G ratio, albumin and sodium and total proteins, α1-globulin ratio, β-globulin ratio, total cholesterol and phospholipids were observed in females in the same group.

Necropsy
Hepatic hypertrophy was observed in 1 male in the 20 mg/kg group and in 4 males and 3 females in the 100 mg/kg group, and splenic hypertrophy was observed in 1 male in the 100 mg/kg group in examinations at the end of administration week 52.

Organ weights
High relative liver weights were observed in females in the 4 mg/kg group and high absolute and relative liver weights were observed in females in the 20 mg/kg group and in males and females in the 100 mg/kg group in examinations at the end of administration week 52. Also, high absolute and relative thyroid weights were observed in males in the 100 mg/kg and high absolute kidney weights were observed in females in the 100 mg/kg group.

Histopathological examinations
In examinations at the end of administration week 52, changes related to the test substance were observed in the spleens, duodenums, jejunums and ileums, in addition to the livers, kidneys and mesenteric lymph nodes. In the livers, mild to moderate centrilobular hepatocytic hypertrophy was again observed in 4 females in the 20 mg/kg group and in 2 males and all females in the 100 mg/kg group. In addition, mild to moderate fatty degeneration of the perilobular hepatocytes was observed in 6 males and 3 females in the control group, 1 female and 1 male in the 4 mg/kg group, 4 males and 2 females in the 20 mg/kg group, and in 5 males and 7 females in the 100 mg/kg group, with a significant increase in the females in the 100 mg/kg group compared to the control group. Also, biliary duct hyperplasia was observed in 1 female and 1 male in the control group, 2 females and 2 males in the 20 mg/kg group, and in 7 males and 1 female in the 100 mg/kg group, with a significant increase in males in the 100 mg/kg group compared to the control group. Furthermore, mild to moderate accumulation of foam cells in the sinusoids was observed in 2 males and 1 female in the 20 mg/kg group and in all males and 9 females in the 100 mg/kg group, while lymphocytic infiltration was observed in the surroundings. In the kidneys, mild to moderate basophilia of the tubules was observed in 5 cases in the control group, in 5 males each and 3 females each in the 4 and 20 mg/kg groups, and in 9 males and 5 females in the 100 mg/kg group, with a significant increase in females in the 100 mg/kg group compared to the control group. In the mesenteric lymph nodes, mild to marked accumulation of foam cells was again observed in 5 males and 3 females in the 20 mg/kg group and in all males and females in the 100 mg/kg group. In the spleens, mild to moderate accumulation of foam cells was observed in the red and white pulp in 1 male in the 20 mg/kg group and in 4 females and 4 males in the 100 mg/kg group. Mild accumulation of foam cells in the lamina propria was observed in the duodenum in 1 female and 1 male in the 100 mg/kg group and in the jejunums in 8 males and 6 females in the 100 mg/kg group. In the ileums, mild accumulation of foam cells in the lamina propria was observed in 6 males and 1 female in the 100 mg/kg group, and mild accumulation of foam cells in Peyer's patches was observed in 3 males and 1 female in the 100 mg/kg group.

Effect levels

Dose descriptor:

NOEL

Effect level:

< 4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical chemistry

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The 52 -week no observable effect level (NOEL) of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane appears to be 4 mg/kg/day for males and less than 4 mg/kg/day for females under the conditions of the present study.

Executive summary:

Multiple oral administrations of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane in female and male rats were conducted for 13 and 52 weeks at doses of 0 (control group), 4, 20, and 100 mg/kg. No changes related to the administration of the test substance were observed in the general condition, body weight or food consumption throughout the administration period. Changes related to the administration of the test substance were observed in the livers, kidneys and mesenteric lymph nodes at the end of administration week 13, and in addition to the livers, kidneys and mesenteric lymph nodes, also in the spleens, duodenums, jejunum and ileums at the end of administration week 52. In the livers, centrilobular hepatocytic hypertrophy was observed in histopathological examinations in females and males administered doses of 20 mg/kg or more at the end of administration weeks 13 and 52, with enhanced changes in females in the 100 mg/kg group at the end of administration week 52 compared to administration week 13.The hepatocytic hypertrophy observed in the present study seemed to suggest an increase of the smooth endoplasmic reticulum, specifically, drug metabolic enzyme induction, because of the presence of weakly eosinophilic cytoplasm. Furthermore, biliary duct hyperplasia in males and perilobular hepatocyte fatty degeneration in females in the 100 mg/kg group significantly increased at the end of administration week 52, compared to the control group. In addition to these findings, hepatic hypertrophy was observed at necropsy at the end of administration week 52 in males in groups administered 20 mg/kg or more and in females administered 100 mg/kg, and, for the organ weights, high liver weights in males in groups administered 100 mg/kg and in females administered 20 mg/kg or more at the end of administration week 13, high relative liver weights in females in the 4 mg/kg group, and high absolute and relative liver weights in females in the 20 mg/kg group and in females and males in the 100 mg/kg group were observed at the end of administration week 52. These changes suggest an influence of the test substance on hepatic function due to long-term administration. Furthermore, they are considered to be changes accompanying drug metabolic enzyme induction because drug metabolic enzyme induction in the liver was suggested as previously described even though no changes in the histopathological examinations were observed for the high absolute and relative thyroid weights in males in the 100 mg/kg group. Also, the change in the blood coagulation system in males in the 100 mg/kg group observed in the hematological examinations and the changes in the proteinaceous system and fatty parameters in females in the 20 mg/kg group and females and males in the 100 mg/kg group observed in the blood biochemistry tests to be described later are considered to be changes that accompany the changes observed in the livers. In the kidneys, hyaline droplets in the proximal tubule epithelium were observed in histopathological examinations at the end of administration week 13 in males in the groups administered 20 mg/kg or more and in males in the 100 mg/kg group at the end of administration week 52 (Photo 5), and the presence of α_2u-globulin at the locations where hyaline droplets were observed was validated. Said accumulation of α_2u-globulin is a change specific to male rats which has not been observed in humans, so the hyaline droplets observed in the proximal tubules were judged to be unrelated to the administration of the test substance. Also, basophilic tubules significantly increased in females in the 100 mg/kg group at the end of administration week 52, though no difference was observed between the control and 100 mg/kg groups at the end of administration week 13. This finding suggests that the influence on the kidney was through a pathway not mediated by α_2u-globulin, because increased basophilic tubules were only observed in females in the present study, but the mechanism could not be established because no precursor was identified. However, the change suggests that the influence on the kidney was related to the long-term administration of the test substance. In addition to the above findings, for the organ weights at the end of administration weeks 13 and 52, high absolute and relative kidney weights were observed in females in the 100 mg/kg group, and the change was considered to be related to the long-term administration of the test substance. In the mesenteric lymph nodes, foam cell accumulation was observed in the histopathological examinations at the end of administration week 13 in females and males in the 100 mg/kg group and at the end of administration week 52 in females and males in groups administered 20 mg/kg or more, and a significant increase in the incidence and worsening of the condition were observed at the end of administration week 52 compared to administration week 13. Also, the same foam cell accumulation was observed at the end of administration week 52 in the hepatic sinusoids and in the spleen red and white pulp in groups administered 20 mg/kg or more, and in the lamina propria mucosa in the small intestine (duodenum, jejunum and ileum) and in Peyer's patches in females and males in the 100 mg/kg group. Furthermore, splenic hypertrophy was observed at necropsy for a male in the 100 mg/kg group with moderate foam cell accumulation observed in the spleen red and white pulp. The series of foam cell accumulations observed suggest that the test substance was absorbed in the intestine, followed by lymph-borne or blood-borne transportation to the organs, where it was processed in the cells. The foam cell accumulation increased at the end of administration week 52 compared to administration week 13. In this regard, the adverse implications of the lymphocytic infiltration around the foam cells accumulated in the liver are unclear. In addition, in the hematological examinations, high leukocytes, neutrophils, lymphocytes, basophils, monocytes and unstained large cell in leukocytes morphological examinations, low MCV, MCH, hemoglobin and hematocrit, and prolonged PT and APTT were observed in males in the 100 mg/kg group at the end of administration week 52. Also, in the blood biochemistry tests, high β-globulin ratios and low chloride in males in the 100 mg/kg group and low A/G ratio and albumin ratio and high β-globulin ratio and total cholesterol in females in the same group, and high total cholesterol in females in the 20 mg/kg group were observed at the end of administration week 13. Furthermore, low A/G ratio and albumin ratio in males in the 100 mg/kg group, high α₂-globulin, β-globulin and ALT in males in the 100 mg/kg groupand low A/G ratio, albumin ratio and sodium, and high total protein, α₁-globulin ratio, β-globulin ratio, total cholesterol and phospholipids in females in the same group were observed at the end of administration week 52. In the urine test, no changes related to the administration of the test substance were observed at the end of administration week 13 or 52. From the above results, the 52 -week no observable effect level (NOEL) of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane appears to be 4 mg/kg/day for males and less than 4 mg/kg/day for females under the conditions of the present study.