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

Endpoint summary

Description of key information

  13-Week feeding of 0.5-4.0% resulted in dose-related reduced wt gain, increased relative liver wt, decrease in relative spleen wt. Mortality ranged from 20-80% in male & female mice. Hematopoesis suppressed in mice fed 2% and 4%,  leading to anemia. Dietary MTD estimated at <=0.5%.

   Female & male rats dosed via gavage for 13 weeks at doses of 0, 4, 20, and 100 mg/kg. Test substance-related changes were observed in the livers, and mesenteric lymph nodes: hepatocytic hypertrophy; foam cell accumulation. Hyaline droplet formation with alpha 2u globulin accumulation in male kidneys not compound related. a broad variety of blood marker changes were noted. 13-week NOEL of 4 mg/kg/day.

  Female & male rats dosed for 52 weeks at 0, 4, 20, and 100 mg/kg. Effects were seen on liver, mesenteric lymph nodes, kidneys, spleens, duodenums, jejunum and ileums at the end of administration week 52. Hepatocytic hypertrophy w/weakly eosinophilic cytoplasm suggests drug metabolic enzyme induction. Hepatic hypertrophy; Biliary duct hyperplasia; perilobular hepatocyte fatty degeneration (100 mg/kg) significantly increased at the end of week 52. High relative liver weights in females (4 mg/kg), and high absolute & relative liver weights in females (20 mg/kg) and in females and males (100 mg/kg) were observed at the end of administration week 52. Blood coagulation system changes were observed in males (100 mg/kg). Changes in protein and fats in females (20 mg/kg) and females and males (100 mg/kg). Observed kidney toxicity related to the long-term administration of the test substance, viz. week 52, high absolute and relative kidney weights in females at 100 mg/kg. Foam cell accumulation, males & females >=20mg/kg: hepatic sinusoids; spleen red and white pulp in groups administered 20 mg/kg or more; also, at 100mg/kg, in the lamina propria mucosa in the small intestine and in Peyer's patches in females and males. NOEL: 4 mg/kg/day for males and < 4 mg/kg/day for females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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

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.

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

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

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.

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.

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

Critical effects observed:

not specified

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.

 

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-day administration; 14-day recovery

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented study. This test was conducted in conformity with "Chemical Examination Law Toxicity Test Methods: Screening Toxicity Tests: 28-Day Repeat oral Toxicity Tests Using Mammals" (December 5 1986, Kanpogyo No. 700, Yakuhatsu No. 1039, 61-Kikyoku No. 1014, and "Chemical Substance GLP" (March 31, 1964, Kanpogyo No. 39, Yakuhatsu No. 229, 59-Kikyoku No. 85; revised November 18, 1988, Kankiken No. 233, Eisei No. 38, 63-Kikyoku No. 823). The study report makes reference to a C of A for test article, an analytical verification of test substance concentration, and stability of the test substance in study media. The study report does not include a separate statement of QA & GLP compliance.

Qualifier:

according to guideline

Guideline:

other: "Chemical Examination Law Toxicity Test Methods: Screening Toxicity Tests: 28-Day Repeat oral Toxicity Tests Using Mammals" (December 5 1986, Kanpogyo No. 700, Yakuhatsu No. 1039, 61-Kikyoku No. 1014,

GLP compliance:

yes

Remarks:

"Chemical Substance GLP" (March 31, 1964, Kanpogyo No. 39, Yakuhatsu No. 229, 59-Kikyoku No. 85; revised November 18, 1988, Kankiken No. 233, Eisei No. 38, 63-Kikyoku No. 823)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals and Housing Method
Sprague-Dawley rats (Crj:CD(SD)IGS, SPF, Charles River Japan K.K., Tsukuba Breeding Center) purchased 4 weeks after birth (Note 1) were acclimatize, with quarantine and habituation occurring over a 9-day period. Then 30 each females and males for whom no abnormalities of their general conditiohhad been observed were supplied for the trial (Note 2). During the entire housing period, the animals were kept in an animal housing room with standard temperature at 24 ± 1°C, standard humidity of 50-65%, air replacement rate of approximately 15 times/h, and 12 h of illumination (lights on from 7:00 a.m. to 7:00 p.m.). Animals were kept in metal floor cages (220 w x 270 d x 190 h mm), 1 animal per cage, and allowed free access to solid animal feed (CE-2, Clea Japan, Inc.) and tap water (Hatano Municipal Water Department water). During the housing period, the actual measured values of housing room temperature and humidity were within the standard levels, with 1 exception of the humidity dropping below the standard level for approxmately 1 h and 45 min due to periodic maintenance work on the air conditioner (Note 3). The food and water supplied did not contain any contaminant that could possibly interfere with the test.

Each animal was labeled on the ear with a series of animal numbers by ear punching. Animal cards, with a different color for each group, and on whichwere recorded the trial protocol number, sex, group (dosage level), and animal number, were attached to the animal cages to assist in the identification of each individual.

(Note 1): Date of animal purchase: January 13, 1999; Number received: 34 males, 34 females; Delivery weight: males: 75.6-88.2 g (average 83.2 g); females: 71.9-81.4 g (average 77.3 g)
(Note 2): Day of first administration: January 22, 1999; Body weight at first administration: males: 168.8-195.7 g (average 182.7 g); females: 138.9-164.0 g (average 152.3 g)
(Note 3): The actual measured temperature and humidity during the animal housing period were: temperature 23.5-24.5°C, humidity 35.5-64%.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The dosage levels for the present study were determined on the basis of results from a preliminary dose-finding study (Trial protocol No.: C-98-016;doses: 0, 500, and 1000 mg/kg). More specifically, when BPTC was repeatedly administered to Sprague-Dawley rats over a 7-day period, no changes were observed that could be clearly attributed to administration of the study substance with the exceptions of the transient hypersalivation immediately following administration to both sexes of groups receiving doses of 500 mg/kg or greater, and slight suppression of weight gain in females of the 1000 mg/kg group during the initial administration period. Based on these facts, 1000 mg/kg was determined to be a tolerable dose for 28 day repeat administration, so the doses established for the present study were a high dose of 1000 mg/kg for both sexes, and intermediate and lower doses at 300 and 100 mg/kg, with 3 as the approximate common divisor. A control group was also established in which both males and females were administered corn oil, which was the vehicle.

For the repeat administration test, 10 animals of each sex were assigned to the control and 1000 mg/kg groups, and 5 animals of each sex to the other groups.

Group assignment was conducted by measuring body weights after the quarantine period and then randomly assigning individuals according to weight class.

Beginning from the lowest animal numbers, 5 individuals of each sex were used for the recovery test in the control and 1000 mg/kg groups.

Administration method
The route of administration was oral, conforming to the guidelines of the Chemical Examination Law. The test article was administered using a stomach tube for rats, once daily between 9:00 a.m. and 3:00 p.m. for a 28-day period. The volume administered was 5 mL/kg, which was calculated for each individual based on the most recent body weight at the time of administration.

The system for recording the number of days and weeks during the administration period was to count the first day of administration as Administration Day 1 and the first week as Week 1, respectively. This method was followed for the system of recording the number of days and weeks during the recovery period, Recovery Day 1 and Recovery Week 1.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test articles were prepared by measuring the study substance for each dose, then adding this at a predetermined concentration and dissolving it
in corn oil (English name: corn oil, Japanese product name: corn oil, Lot No.: V8P7069, Nakaraitesuku Co., Inc.)

The test articles' content was measured at the Hatano Research Institute when they were prepared for the first time, and the average study substance content in solution was found to be 96.9-101% of the predetermined concentration (Appendix A - not available). A stability test conducted in advance of the animal trial confirmed that a 2 or a 20% w/v solution of the study substance was stable for 12 days after preparation when refrigerated and protected from light (Appendix B-not available). Therefore, the test articles were prepared at a rate of 1 batch weekly and stored in refrigerated, light-protected conditions until used. Because the test articles were solutions, no uniformity test was conducted. The study substance concentrationsof the test articles were measured by gas chromatography (Appendix C-not available).

Duration of treatment / exposure:

28-days administration; 14-days recovery

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
other: amount administered by gavage

No. of animals per sex per dose:

10/sex 1000mg/kg & Control
5/sex 100 & 300mg/kg

Control animals:

yes, concurrent vehicle

Positive control:

None

Observations and examinations performed and frequency:

General tests
The general condition of each individual was observed daily (during the administration period, before and after administration). The body weight and food consumption of each individual were measured at the frequency shown below. Body weight was measured during Administration Week 1 before and after administration on Day 4, and then again on Administration Day 4, and then twice weekly during Administration Week 2, through the rest of the administration period, and during the recovery period. It was also measured at the end of the administration period, at the end of the recovery period, on the day of necropsy, and at the time of death. Food consumption was measured as follows: consumption for 1 day was measured on Administration Days 1 and 2 during Administration Week 1, and it was then measured at a frequency of once weekly to the end of the recovery period.

Urinalysis
Each survivor of all the groups was placed in a metabolic cage during Administration Week 4 and at the end of the recovery period, 4-h urine or fresh urine was tested for the following parameters. Color and turbidity, pH, occult blood, protein, glucose, ketones,Urobilinogen, bilirubin


Hematology tests
In all cases animals were fasted for 18-24 h prior to blood sampling (prior to sacrifice and necropsy), when necropsies were performed at the end of
the administration period and at the end of the recovery period. The animals were anesthetized with pentobarbital sodium, and blood was sampled
from the abdominal caudal vena cava, using EDTA•2K as the anticoagulant. The parameters listed below were investigated. Blood used for measurement of prothrombin time and activated partial thromboplastin time was sampled using sodium citrate as the anticoagulant. A myelogram study was conducted for the control and high dosage groups on individuals sacrificed at the completion of the administration period, with smear prepared from thefemoral marrow of all animals subjected to periodic necropsy. This test determined that changes in bone marrow function did not occur, and this test was not performed for other groups.
Red blood cell count (RBC)
White blood cell count (WBC)
Hemoglobin (Hb)
Mean corpuscular volume (MCV)
Platelet count
Hematocrit (Ht)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Leukocyte classification
Myelogram test
Reticulocyte ratio
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)


Biochemical tests
As follow-up on the blood sampling described above, blood tests covering the following parameters were performed on blood sampled using heparinas anticoagulant, on all cases except mortalities, under similar anesthesia and sampling site conditions.
Albumin concentration
Total cholesterol concentration
Glucose concentration
Urea nitrogen concentration (BUN)
Creatinine concentration
ALP activity
GOT activity
GPT activity
gamma-GTP activity
Triglyceride concentration
Inorganic phosphorus concentration (inorg. phos.)
Calcium concentration
A/G ratio
Sodium concentration
Potassium concentration
Chloride concentration

Sacrifice and pathology:

Pathology investigations
After blood sampling, all individuals with the exception of mortalities were sacrificed by exsanguination by severance of the axillary artery. Organs and tissues were then visually examined, and the weights of the organs listed below were measured for each animal. The relative weight of each organ was calculated by subtraction from the body weight of the individual on the day of the necropsy.

Organs weighed: Brain, heart, liver, kidneys, thymus, spleen, adrenals, testes, epididymides, ovaries

After visual inspection, the following organs or tissues were extirpated and preserved. The testes and epididymides were fixed with Bouin's fluid (for long-term storage, 0.1M phosphate buffer 10% formalin solution was used), the other organs and tissues were fixed in 0.1M phosphate buffer 10% formalin solution. Samples were prepared from organs and tissues to which an asterisk (*) has been attached for all groups after completion of the administration period by paraffin-embedding, sectioning, and hematoxylin-eosin staining. A histological study was then made of the control and high dosage groups using the optical microscope. PAS-stained samples were prepared from the kidneys of all males in the control and high dosage groups. Samples were also prepared of organs and tissues in all cases where abnormalities had been visually observed, and these were histologically examined.

In the histological examination of specimens from the control and high dosage groups after completion of the administration period, changes were observed in the liver of both sexes, male kidneys, and female adrenals that were possibly related to the study substance, so histological examinations of the other groups were also conducted. The organ weight of the mortality was not measured; visual inspection and histological examination were conducted similar to that for cases of necropsy after completion of the administration period.

Fixed/Preserved Organs/Tissues:
Brain, pituitary, spinal cord, eyes, thyroid, parathyroid, *heart, trachea, bronchus, lung, *liver, *kidney, thymus, *spleen, *stomach, duodenum, jejunum, ileum, appendix, colon, rectum, *testes, *epididymides, prostate, seminal vesicle, *ovaries, uterus, vagina, mammary gland, bladder, mandibular lymph nodes, mesenteric lymph nodes, skeletal muscle (crural), sciatic nerve, femoral bone marrow, pancreas, mandibular gland, sublingual gland, tongue, esophagus, Harderian gland, skin, and lesions.

Statistics:

Data analysis
The average values and standard deviations were determined for each group for body weight and food consumption, as well as for hematological parameters, biochemical parameters, and organ weights of the individuals that were periodically sacrificed. In cases where there were 3 or more groups, including the test and control group structure, the uniformity of the distribution was determined using Bartlett's method (level of significance: 5%). When the distribution was uniform, one-way layout distribution analysis was conducted, and when intergroup significance (level of significance: 5%) was determined, multiple comparison was conducted using Dunnett's test. When the distribution was not uniform, the Kruskal-Wallis rank test was performed, and when intergroup significance (level of significance: 5%) was determined, multiple comparison was conducted using Dunnett's test. However, in cases where the distribution for any group is 0, the Kruskal-Wallis rank test was performed without conducting Bartlett's test, and if intergroup significance was determined, then multiple comparison was conducted using Dunnett's test. On the other hand, when there were 2 groups, a test group including a control group, then the determination of the difference in average values of the control and study substance groups was performed by first determining the average value and standard deviation for each group, then conducting the F-test (level of significance: 5%), and in homoscedastic cases Student's t-test was performed, but in heteroscedastic cases Aspin-Welch's t-test was used to determine significant difference. However, if the distribution for any group was 0, a t-test was not performed. For graded data from histopathological studies, the determination of significant difference (level of significance: 5%) between control and study substance groups was conducted by Mann-Whitney's U-test (two-sided test), and positive grade sums were subjected to Fisher's exact test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis 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:

no effects observed

Details on results:

Although no mortalities were observed during the administration period, hindlimb paralysis was observed in 1 male of the 1000 mg/kg group on Recovery Day 2; this progressed to abasia, the perirhinal fur became soiled, reddish urine thought to be hematuria was excreted, and piloerection was observed; on Recovery Day 3 he died. The other changes in general condition to be observed were transient hypersalivation occurring on Administration Day 2 and subsequently in males of groups receiving doses of 300 mg/kg or more, and in females of groups receiving doses of 100 mg/kg or more, which disappeared within 1 h after administration. During Administration Weeks 1-2, a significant reduction of food consumption occurred in both males and females of the 1000 mg/kg group in comparison to the control group, but otherwise no differences were observed for body weight or foodintake.

In hematology tests, the prothrombin time was significantly longer or tended to be longer for both sexes in the 1000 mg/kg group, and the activated partial thromboplastin time was significantly longer. There was also a significant decrease in hemoglobin levels of females in the 1000 mg/kg group, but no pronounced difference was observed in the myelograms between the control and test groups of either sex. In tests performed after completion of the recovery period, a significant reduction in hemoglobin levels was observed in females of the test groups, a significant reduction in hematocritlevels for both sexes of the test groups, and a significant increase in the mean corpuscular hemoglobin concentration in males of the test groups. Onthe other hand, the prolongation of blood clotting time seen at the end of the administration period was not observed for either sex.

In biochemical tests, a significant decrease in glucose concentration was observed in males of the 1000 mg/kg group, a significant increase in GPT
activity in both sexes of the same group, and a significant increase in γ-GTP activity in females of that group, respectively. Among females, a significant increase was observed in total protein concentration in the 1000 mg/kg group, and a significant decrease in albumin concentration in the 100 mg/kg group, respectively; there was also a significant decrease in the A/G ratio for all test groups. In groups receiving doses of 300 mg/kg or more, a significant increase in total cholesterol concentration was observed for females, and a significant decrease in triglycerides for males. In tests performed after the recovery period had been completed, a significant decrease was shown in A/G ratios for males of the test groups, and there was also a tendency for this to be lower for females in comparison with the control group, but other changes that had been seen after the administration
period were not observed.

In organ weight measurements, the weights of the liver and kidneys increased dose-dependently in both sexes. A significant increase in the absolute adrenal weight was seen in females in all of the groups receiving the study substance, and either significant increases or a tendency to increase was also observed for relative organ weights in these groups. Even after the recovery period had been completed, a significant increase was still observed in the relative liver and kidney weights of both sexes in the groups receiving the study substance.

The main macroscopic findings observed at the end of the administration period were darkening or enlargement of the liver, or the accentuation of the lobular pattern of the liver in some members of the 1000 mg/kg group. In histological studies, centrilobular hepatocytic hypertrophy was observed in males at a dosage of 300 mg/kg or greater, and in females at a dosage of 100 mg/kg or greater, and the degree of this change was greater according to the dose. Periportal fatty change increased dose-dependently in the livers of females. With regards to the kidneys, in all males of the groups receiving the study substance, eosinophilic bodies and eosinophilic intracytoplasmic inclusion bodies were observed in the proximal renal tubules, and their extent increased dose-dependently. As for the adrenals, hypertrophy of the zona fasciculata cytoplasm was observed in many females of the groups receiving the study substance. After the recovery period had been completed, there was a strong increase in basophilic tubules in the kidneys of males, but other changes seen at the end of the administration period either tended to decrease or disappear. The main finding for the mortality on Recovery Day 3 was an increase in findings similar to that at the end of the administration period: hemorrhaging was observed of brain and spinal cordsubdural space, bladder, thymus, mesenteric lymph nodes, and mandibular lymph nodes, and interstitial hemorrhage was observed for the testes, epididymides, and prostate gland.

In this way, for repeat administration of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane, there was not only centrilobular hepatocytic hypertrophy in females at a dosage of 100 mg/kg or greater and in males at a dosage of 300 mg/kg or greater, and periportal fatty changes which increased dose-dependently in the livers of females receiving doses of 100 mg/kg or greater, but a decrease of the A/G ratio was also observed. Additionally, as findings pertaining to the liver, a prolongation of blood clotting time and increased GPT activity, for example, were observed for both sexes of the 1000 mg/kg group, which suggests a hepatopathic effect. Other than the liver, eosinophilic intracytoplasmic inclusion bodies were observed in the proximal renal tubules of males receiving doses of 100 mg/kg or greater, which suggests nephrotoxicity, and hypertrophy of zona fasciculata cytoplasm was observed in many females receiving doses of 100 mg/kg or greater. Based upon these facts, it is thought that the no observable effect level of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane under the present test conditions is less than 100 mg/kg.

Dose descriptor:

NOEL

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: nephropathy

Critical effects observed:

not specified

Conclusions:

The no observable effect level (NOEL) of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane under the described test conditions is less than 100 mg/kg in both male and female rats, following 28-days of daily gavage administration and a 14-day period of follow-up observation.

Executive summary:

Repeat administration of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane, caused centrilobular hepatocytic hypertrophy in females at a dosage of 100 mg/kg or greater and in males at a dosage of 300 mg/kg or greater, and periportal fatty changes which increased dose-dependently in the livers of females receiving doses of 100 mg/kg or greater, but a decrease of the A/G ratio was also observed. Additionally, as findings pertaining to the liver, a prolongation of blood clotting time and increased GPT activity, for example, were observed for both sexes of the 1000 mg/kg group, which suggests a hepatopathic effect. Other than the liver, eosinophilic intracytoplasmic inclusion bodies were observed in the proximal renal tubules of males receiving doses of 100 mg/kg or greater, which suggests nephrotoxicity, and hypertrophy of zona fasciculata cytoplasm was observed in many females receiving doses of 100 mg/kg or greater. Based upon these facts, it is thought that the no observable effect level (NOEL) of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane under the described test conditions is less than 100 mg/kg.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 weeks

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented 13-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.

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:

The study report does not represent that the study conforms to either a national/international protocol or national/international GLP standards.

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

This 13-week subacute study was conducted as an interim/sub study to the overall 52-week study. accordingly, this description applies to all animals used in both studies:

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 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:

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 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 (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:

13 weeks

Frequency of treatment:

daily, 7-days/week

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

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 week 13. 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 Tumors
nodes
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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Blood biochemistry tests
In tests at the end of administration week 13, high β-globulin ratio and low chloride were observed in males in the 100 mg/kg group, and low A/G ratio and albumin and high β-globulin ratio and total cholesterol were observed in females of the same group. Furthermore, high total cholesterol was observed in females in the 20 mg/kg group.

Organ weights
High relative liver weights in females in the 20 mg/kg group and high absolute and relative liver weights in females in the 100 mg/kg group were observed in examinations at the end of administration week 13. Also, high relative kidney weights were observed in females in the 100 mg/kg group.

Histopathological examinations
In examinations at the end of administration week 13, changes related to the test substance were observed in the livers and the mesenteric lymph nodes. In the livers, mild to moderate centrilobular hepatocytic hypertrophy was observed in 2 females in the 20 mg/kg group and in 2 males and 9 females in the 100 mg/kg group, and the hypertrophied hepatocytes showed weak acidophilic, homogeneous cytoplasm. Mild accumulation of foam cells was observed in the mesenteric lymph nodes in 2 males and 1 female in the 100 mg/kg group.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: clinical chemistry, esp cholesterol.

Critical effects observed:

not specified

Conclusions:

A 13-week NOEL of 4 mg/kg/day was demonstrated for 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane in both males and female rats via gavage in corn oil.

Executive summary:

Multiple oral administrations of 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane in female and male rats were conducted for 13 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. 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. 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. In addition to these findings, hepatic hypertrophy was observed at necropsy in males in groups administered 100 mg/kg and in females administered 20 mg/kg or more at the end of administration week 13. 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. 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. 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. 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. In the urine test, no changes related to the administration of the test substance were observed at the end of administration week 13. According to these test results, it appears that a 13-week NOEL of 4 mg/kg/day was demonstrated for 1,1-bis[tert-butylperoxy]-3,3,5-trimethylcyclohexane in both males and female rats via gavage in corn oil.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The study is reliable with minor restrictions.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

After evaluating the data in terms of biological significance, degree and severity of the effect as well as considering the data as a whole for a specific parameter, the effects observed at 4 and 20 mg/kg/day were considered to be non-adverse in this 52-week study. These effects, as described below, are reduced hemoglobin level, hepatic hypertrophy, and presence of foam cells in liver.

The lower hemoglobin level reported for females at 20 mg/kg/day was not considered adverse. The marginal change in the hemoglobin level (8.6% reduction) was not accompanied by other typical changes expected in different types of anemia (e.g., erythrocyte count, reticulocyte count or total bilirubin level). There was no dose response for this effect either. This low level of change, without other serious health effects resulting from the marginal hemoglobin reduction, does not meet the criteria for classification (>20% reduction inhemaoglobinlevel).

Effects observed in the liver (i.e. hypertrophy, increased relative and/or absolute liver weight, fatty degeneration, biliary duct hyperplasia and foam cell accumulation), particularly at 4 and 20 mg/kg/day, were either observed in the controls, were mild to moderate in severity, occurred with low incidence, or only reached statistical significance in the 100 mg/kg/day group.

For hepatic effects in general, the authors made the following conclusion:

“Hepatocytic hypertrophy was reported in a 28-day multiple administration toxicity study in rats and a 13-week multiple administration toxicity study in mice previously conducted for this substance, with suggestions of drug metabolic enzyme induction. 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.”

 

“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.”

 

An accumulation of foam cells of the liver sinusoids was also reported. Foam cell accumulation occurred with low incidence in the 20 mg/kg/day group (2 males and 1 female) but was not accompanied by mononuclear cell infiltration in a dose-response manner. Therefore, this effect does not indicate an inflammatory process and the significance of this effect, in one or two animals, is questionable. 

Foam cell accumulation observed in the other organs was likewise not accompanied by inflammation of surrounding tissue and was attributed to absorption and metabolism of the test system.

 “The test substance was estimated to be absorbed and metabolized by the same

pathway as food lipids; specifically, it is absorbed in the small intestine and enters the veins

through lymphatic vessels and is transported to the tissues and organs. 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 theGuidance on information requirements and chemical safety assessment(R.8.2), the NOAEL is defined as “the highest dose level or concentration of the substance used in that test at which no statistically significant adverse effects were observed.” 

Based on the re-analysis of the data, we have concluded that the NOAEL for this study can be considered 20 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Well documented 52-week gavage study in rats.

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: blood coagulation; cardiovascular / hematological: hematopoiesis; digestive: liver

Justification for classification or non-classification

Regarding classification and labelling: for a substance to be classified as a STOT-RE, the cut off concentrations, for 90-day exposure, from the CLP guidance, are as follows for significant toxic effects:

Cat 1: C<10 mg/kg/day

Cat 2: 10 , C<100 mg/kg/day

Using Harber’s law (in accordance with the guidance) this equates into the following for a 52-week study:

Cat 1: C<2.5 mg/kg/day

Cat 2: 2.5 < C<25 mg/kg/day

If the NOAEL is below the guidance value (GV) the effective dose level (ED), i.e. the lowest dose inducing significant/severe target organ toxicity as defined in Section 3.9.2.2, should be determined based on the criteria described above. 

In this case the ED would be 100 mg/kg/day and would not meet the criteria for classification as a STOT-RE in accordance with CLP (v3).