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OECD 408 study

The oral toxicity of Tert-butyl-a,a-dimethylbenzyl peroxide in rats following daily oral administration for 13 consecutive weeks and recovery from any treatment-related effect during a period of 6 weeks, were investigated in this study (Lombogardi, 2018). Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 50, 100 and 400mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (corn oil) and acted as a control. Five additional animals for each sexwere included in the high dose and control groups for recovery assessment. The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, oestrus cycle, clinical pathology investigations (including bone marrow smears), terminal body weight, organ weights, macroscopic observations, histopathological examination and spermanalysis.

One male and one female from the control group were found dead on Days 21 and 83 of treatment period, respectively. One male animal dosed at 100mg/kg/day was found dead on Day 43 while one female rat dosed at 400mg/kg/day, was found dead on Day 23 of the treatment period.

No clinical signs were observed in the 2 control group animals prior to death. Matted fur, salivation and/or hunched posturewere seen in the males receiving 100mg/kg/day and in the females receiving 400mg/kg/day during the third week of treatment period. In addition, piloerection was seen in these 2 animals up to the day before death. Post mortem examination showed multiple, dark areas in the lungs with red colour in the cervical lymph nodes and thymus in the control male; red, fluid contents in the thoracic cavity and red colour in the lungs in the control female; pale/creamy contents in the thoracic cavity and adhesions between heart and all pulmonary lobes in the female dosed at 400mg/kg/day. Histopathological evaluation identified the factor contributory to the death of these unscheduled animals in a misdosing procedure.

Macroscopically, the male dosed at 100mg/kg/day showed enlarged and swollen liver; the histopathological evaluation revealed minimal, multifocal centrilobular hepatocytic hypertrophy in the liver and mild bilateral nephropathy, associated with hyaline droplet accumulation (a2µ-globulin) in the renal tubules. The pathological picture did not clearly establish the cause of death.

Salivation was observed in all male and female treated groups, and in one control female animal. This sign was evident starting fromWeek 1 in animals dosed at 400mg/kg/day, while it gradually appeared during the treatment period in those treated at 50 and 100mg/kg/day. Hunched posture and/or matted fur were also observed in male and female animals from all treated groups, with a dose-related severity and incidence. In addition, piloerection was seen in animals from all groups (including controls) with increasing severity and incidence, from Week 3 of the study up to the end of treatment period. These signs were no longer observed during recovery period. Skin fur staining and/or hairloss were also observed in a number of animals from all groups. A palpable mass was seen in a single female animal dosed at 100mg/kg/day starting from Day 90 of the treatment period.

Weekly detailed clinical signs (removal from cage and open field measurements)

No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity. No differences between treated and control groups were evident at the functional tests and at the motor activity measurements at the end of treatment and recovery periods.

Slight statistically significant decreases in body weight and body weight gain were observed in the males dosed at 400mg/kg/day when compared to controls during treatment period. Very slight reductions were also seen in the females dosed at 400mg/kg/day. These reductions gradually decreased during the recovery period.

Slight but statistically significant decreases in food consumption were occasionally observed in the animals receiving 400mg/kg/day during the treatment period. These decreases were no longer observed during the recovery period.

No significant findings were detected at the ophthalmoscopic examinations performed during the study.

No treatment-related anomalies were noted in the oestrous cycle when compared to controls.

A decrease of erythrocytes, haemoglobin and haematocrit was recorded in some females dosed at 400mg/kg/day. Changes were insufficient in magnitude to represent an adverse anaemia, even though they could represent an effect of the test item. No other changes of toxicological relevance were observed. The findings recorded at the Dosing Phase were no longer observed at the end of recovery.

No changes of coagulation of toxicological significance were observed.

Fluctuations of some biochemical parameters, were recorded in treated animals. Due to the slight severity and/or absence of dose-relation, theywere not considered to be of toxicological significance. Most of the findings recorded at the Dosing Phase were no longer observed at the end of recovery.

High proteinuria, ketonuria and increased urobilinogen were observed in few males dosed at 400mg/kg/day. Ketonuria was also recorded in one male receiving 100mg/kg/day. No findings were recorded at the end of recovery, confirming reversibility.

Two males and two females dosed at 400mg/kg/day showed a slight increase of the erythroid mature cells in bone marrow in week 13, leading to a decrease of theM/E ratio. Male no. A2196082 also showed slight decrease of segmented neutrophils. No other relevant changes in the number and/or morphology of cells were recorded. Due to the absence of peripheral erythrocytes changes, the above findings were considered of no toxicological relevance.

Terminal body weights showed statistically significant decreases in animals dosed at 400mg/kg/day when compared to controls at the end of treatment period. Partial or complete recovery was observed at the end of recovery period. Increases in absolute and/or relative kidney and liver weights were observed in animals from all treated groups at the end of treatment. No significant differences were observed at the end of recovery in either sexes.

The relative weight of the spleen was increased in the males receiving 400mg/kg/day, while the relative weight of the thyroid showed increases in the females dosed at 100 and 400mg/kg/day. Only a slight increase of the thyroid relative weight in the females was seen at the end of recovery.

In addition, increases in the relative weight of epididymides and testes were seen in the high dose males at the end of treatment and recovery. Without significant clear correlation with histopathology, the increases of the relative weights of the spleen, thyroid, testes and epididymides were considered to be of no toxicological relevance.

At final post mortem examination, a treatment-related change was noted in males receiving 400mg/kg/day (high dose), represented by an enlargement and/or swollen consistency and/or pale colour of the kidneys and liver. Enlarged and pale kidneys were again observed in 2 out of 5 recovery males dosed at 400mg/kg/day, as well as pale colour of the liver.

Treatment-related changes were seen in the kidney of males receiving Tert-butyla,a-dimethylbenzyl peroxide at >= 50mg/kg/day and in the liver of males and females dosed at 400mg/kg/day. The renal findings observed were represented by an increase of hyalin droplets or a2µ-globulin, in the proximal tubule in the cortex or cortico-medullary junction, confirmed immunohistochemically by a minimal to moderate positivity with a dose-related trend. Nephropathy was also seen associated with an increased dose-dependent severity mainly at >=100mg/kg/day. Minimal, multifocal centrilobular hepatocellular hypertrophy was only observed in the liver of male and female rats dosed at 400mg/kg/day. Hepatocyte hypertrophy could be considered an adaptative change. After the recovery period, the treatment-related changes in the liver were seen reversible in males and females. However, two males still showed a minimal increase of a2u-globulin, in the proximal tubule in the cortex, although not confirmed immunohistochemically. Mild to marked nephropathy was only noted in 2 out of 4 males involved. The renal pathology could be considered still undergoing reversal at the end of the treatment-free recovery period.

No differences were observed in spermmotility, morphology and concentration in treated males compared to controls at the end of treatment. Since no treatment-related effects were seen between control treated males no assessment was performed in males of the recovery group.

In conclusion, signs of effects related to treatment with Tert-butyla,a-dimethylbenzyl peroxide were observed with increasing incidence and severity in animals from all treated groups when administered by oral gavage for 13 consecutive weeks at the dosages of 50, 100 and 400mg/kg/day.

Most of these effects (clinical signs, slight decreases in body weight and food consumption, slight changes in haematological, biochemical and urinalysis parameters, slight increases in liver and/or kidney weights), observed at >= 50mg/kg/day, were not considered to be adverse, due to the low magnitude and/or complete reversibility. These findings were associated to a minimal centrilobular hepatocellular hypertrophy in male and female animal at 400mg/kg/day and a nephropathy in the male animals dosed at >= 100mg/kg/day. The centrilobular hepatocellular hypertrophy in the liver is often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes. Among these changes, adverse test item related effects (nephropathy) were observed in the kidneys of male rats receiving >= 100mg/kg/day. The hyaline droplets in renal tubular epithelium were associated with a2µ-globulin accumulation as confirmed by immunohistochemistry. Because humans do not have a protein that behaves in a manner comparable to a2u-globulin, on a qualitative basis, there is not concordance for this particular key event between male rats and humans and therefore this mode of action is qualitatively not relevant to humans (Hard et al., 2013)(1).

Therefore, it can be concluded that the high dose of 400mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for this study, excluding the male rat-specific a2u-globulin nephropathy.

OECD 422 study

The toxic effects on rats of both sexes after repeated dosing with Luperox 801 (97% tert-butyl cumyl peroxide) by oral route was evaluated in an OECD 422 study (Liberati, 2013). The test item, suspended in corn oil, was administered by oral gavage to 3 groups of 10 males and 10 females each as indicated below at the doses of 75, 150 and 600 mg/kg bw/d. A similar constituted control group (Group 1) received the vehicle alone during the treatment period.Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy. Females were treated for 2 weeks before pairing, thereafter during pairing, post coitum and lactation periods until Day 3 post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology, clinical chemistry and urinalysis), macroscopic observations, organ weights and histopathological examination.

Tremors, piloerection and salivation were the most relevant clinical signs detected in high dose males and females during the whole treatment period, at the daily clinical examination performed at 0.5-1 hour after treatment. In addition ataxia was detected in 7 out 10 high dose females on Day 2 of the pre mating period. Although its transient appearance of one day, the sign could be considered related to treatment, whereas it was also seen in the preliminary study (RTC Study No. 88780EXT) at the dosage of 1000 mg/kg/day. Salivation was also detected in mid-dose animals during the whole treatment period. One control female judged to be in extremis was killed on post coitum Day 18. Body weight and body weight gain of both sexes were significantly lower at statistical analysis in the high dose group of both sexes compared to controls throughout the study. Food consumption was reduced only in high dose females starting from post coitum Day 14. There were no effect on motor activity, sensory reaction to stimuli, and no neurotoxicity. There were no effect on haematological parameters and on clinical chemistry, and no effect on spermatogenic cycle. Terminal body weight was significantly lower at the statistical analysis in the high dose group of both sexes compared to controls. Statistically significant higher liver and kidneys weight was observed in high dose males and females compared to controls. In addition, a significant increase was also detected in the kidney weight of both sexes in the mid-dose group. At final sacrifice, treatment related changes were seen in the liver and kidneys. Statistically significant treatment-related findings were noted in the liver of the high dosed group of both sexes and in mid-dose male group, consisting of centrilobular hepatocytic hypertrophy associated with cytoplasmic eosinophilia. The change was of mild degree in the high dosed animals, and of minimal degree in the intermediate dose group. As the hepatocytic hypertrophy was not associated with other changes in the hepatocytes (i.e., degeneration and/or necrosis), this change was not considered as adverse, but rather as potentially adaptive. In the kidneys of the males treated with the intermediate and high dose, a dose-related increased incidence of minimal cortical basophilic tubules was noted. As organ weight data indicated a statistically significant increase of the relative kidney weight in these groups, as well as macroscopically cases of enlarged kidneys were seen only in the males treated with the high dose, it is suggested that the dose-related increased incidence of minimal cortical basophilic tubules is potentially related to treatment.

On the basis of the results obtained in the study, the NOAEL for general toxicity is 150 mg/kg/day for males and females, based on clinical signs, reduced body weight and body weight gain at 600 mg/kg bw/day (compared to controls).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase (allocation of the animals): 12 October 2016 End of experimental phase (last day of necropsy at the end of recovery): 01March 2017 Study completion: 24-01-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 91-117 g.
- Fasting period before study: No
- Housing: The animals were housed up to 3 of one sex to a cage, in clear polysulfone solid bottomed cages
- Acclimation period: 7 days prior to the start of treatment

DETAILS OF FOOD AND WATER QUALITY:
- Diet (e.g. ad libitum): 4 RF 21, Mucedola S.r.l., ad libitum throughout the study, except at the end of Week 13 of treatment, prior to blood sampling and during urine collection
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles, except at the end of Week 13 of treatment, prior to blood sampling and during urine collection

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of Tert-butylá,á-dimethylbenzyl peroxide was suspended in the vehicle.
The formulation was prepared weekly (concentrations of 10, 20 and 80mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in RTC Study no. 88760 in the range from1 to 200mg/mL.
Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 90-110%; precision CV < 5%).
A 24-hour and 8-day stability at room temperature was verified in the range from 1 to 200mg/mL.
The proposed formulation procedure for the test item was checked in the range from 10 to 80mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV<10%).
Samples of the formulations prepared on Weeks 1, 6 and 13 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Remarks:
group 4
No. of animals per sex per dose:
Each main phase group comprised 10 male and 10 female rats. Control and high dose groups included 5 additional animals per sex to be sacrificed after 6 weeks of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
On the basis of the results obtained in an OECD 422 study. The NOAEL for general toxicity was 150 mg/kg/day for males and females, based on clinical signs, reduced body weight and body weight gain at 600 mg/kg bw/day (compared to controls).
- Post-exposure recovery period in satellite groups: 6 weeks
Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day. Four animals died during the study and a complete necropsy was performed.

Clinical signs and neurotoxicity assessment
All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (approximately 5 - 30 minutes and 1.5 - 2 hours after dosing). Once before commencement of treatment and at least once per week during the study from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. Once duringWeek 12 of treatment an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength was also performed.

Motor activity assessment (MA)
The motor activity (MA) of all animals was measured once duringWeek 12 of treatment by an automated activity recording. Measurements were performed using a computer generated random order.

Body weight
Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and on the day of necropsy (after food deprivation).

Food consumption
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

Ophthalmoscopy
Both eyes of all animals were examined once prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome, Italy). A second evaluation was carried out on some animals which showed incomplete maturation of the lens. Data relevant to this additional evaluation were not reported but are archived with other raw data. Animals with non-resolving lesions were discarded and replaced with spare animals showing no ocular abnormality, from the batch initially ordered for the study. The eyes of all animals from main phase groups were re-examined duringWeek 13 of treatment.

Vaginal smears and oestrous cycle
From the first day of Week 10 and up to the end of the treatment period, vaginal smears were taken daily in the morning from all main phase animals. The vaginal smear data were examined to determine potential anomalies of the oestrous cycle.

Clinical pathology investigations
At the end of Week 13 of treatment, prior to blood sampling and during urine collection, the animals were put into individual metabolism cages, deprived of food and water for an overnight period of at least 16 hours and received approximately 10 mL/kg of drinking water by gavage, in order to obtain overnight urine samples suitable for analysis. Just prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava of 10 male and 10 female animals from each group. During Week 6 of the recovery period, blood and urine samples were taken from all surviving animals under identical conditions in order to re-evaluate the clinical pathology parameters, which showed possible treatment-related changes at measurements performed during the treatment period.
Blood samples were collected and analysed in the same order. The blood samples collected were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests

Hormone determination
At the end ofWeek 13 of treatment, prior to necropsy, additional blood samples (as much as possible, taking into account the amount required for clinical pathology investigations ) were taken from the abdominal vena cava, under isofluorane anaesthesia, of all surviving male and female animals and from 5 male and 5 female spare untreated rats from the same batch. Blood samples were collected into tubes without anticoagulant, centrifuged and serum samples were frozen at approximately -80°C for possible future analysis for T3, T4 and TSH evaluation. As no change was observed in the thyroid of the treated animals, these samples were not analysed and they will be destroyed after finalisation of the report.
Sacrifice and pathology:
Euthanasia
Animals that had completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, were subjected to necropsy, supervised by a pathologist.

Necropsy
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Organ weights
From all animals completing the scheduled test period, the organs (Adrenal glands - Brain (cerebrum, cerebellum, medulla/pons) - Epididymides - Heart - Kidneys - Liver - Ovaries - Spleen - Testes - Thymus (where present) - Thyroid gland - Uterus – cervix) were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

Bone marrow
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur.
Smears prepared from these samples were air dried, fixed in methanol, stained using a May-Grunwald-Giemsa procedure and stored. In the first instance, the smears were examined for abnormalities and a differential count was performed including calculation of the myeloid/erythroid cell ratio from main phase control and high dose animals.

Tissues fixed and preserved
Samples of Abnormalities - Adrenal glands - Aorta - Bone marrow (from sternum) - Brain (cerebrum, cerebellum, medulla/pons) - Caecum - Colon - DuodenumEpididymides - Eyes - Femur with joint - Heart - Ileum - Jejunum (including Peyer’s patches) - Kidneys - Liver - Lungs (including mainstem bronchi) - Lymph nodes – cervical - Lymph nodes – mesenteric - Mammary area - Oesophagus - Ovaries - Oviducts - Pancreas - Parathyroid glands - Pituitary gland - Prostate gland - Rectum - Salivary glands - Sciatic nerve - Seminal vesicles - Skeletal muscle - Skin - Spinal column - Spinal cord - Spleen - Stomach - Testes - Thymus (where present) - Thyroid gland - Trachea - Urinary bladder - Uterus – cervix - Vagina were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed inModified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are:
Abnormalities
Adrenal glands
Aorta
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary area
Oesophagus
Ovaries
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord
Spleen
Stomach
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. Immunohistochemical determination was also performed in all main and recovery phase males for the detection of a2µ-globulin in additional 5 micrometer thickness sections of the kidneys. In addition, the testes and epididymides of main group animals were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment.


Other examinations:
Sperm analysis
Sperm analysis was performed in all males killed at the end of the treatment period during the necropsy, shortly after the sacrifice. Since no treatment-related effects were seen between control treated males no assessment was performed in males of the recovery group.

Sperm motility and morphology
After the epididymal weight, the left or right cauda was separated from the corpus using a scalpel and subsequently kept for a few minutes at 37°C in a Petry dish containingMedium M199 and 5% albumine bovine serum (Sigma Aldrich), in order to allow spermrelease from the cauda in the medium. A sperm sample was taken from Petry dish, put into a KOVA GLASSTIC SLIDE (Hycor) and immediately examined. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope. Results were expressed as percentage of motile spermatozoa. In addition, spermatozoa from the same preparation were observed and classified as Normal or Abnormal. A detailed description was recorded only in presence of abnormalities (ie: few headless sperm, moderate sperm with abnormal flagellum). No abnormal sperm was found.

Sperm concentration
After the assessment of motility and morphology, the cauda was minced and homogenized in 20 mL of medium as detailed above. Following approximately 15 minutes of incubation at 37-39°C, a 0.1 mL sample of the suspension was taken and added to 0.9 mL of medium (a dilution factor of 10x was applied). An aliquot of the diluted suspension was taken and put into a KOVA SLIDE. The number of spermatozoa present in 2 grids of the slide were counted. The concentration was expressed as millions of spermper gram of cauda.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a non-parametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 1 in the attached document.
Salivation was observed in all male and female treated groups, and in one control female rat. This sign was evident starting from Week 1 in male and female animals of Group 4 (400mg/kg/day), while it gradually appeared during the treatment period in animals treated at 100 and 50mg/kg/day. Hunched posture and/or matted fur were also observed in male and female animals from all treated groups, with a dose-related severity and incidence. In addition, piloerection was seen in animals from all groups (including controls) with increasing severity and incidence, from Week 3 of the study up to the end of treatment period. These signs were no longer observed during recovery period. Hair loss and/or scabs and/or skin/fur staining were occasionally observed in individual animals of both sexes from all groups during the treatment and recovery periods. A palpable mass was seen in a single female animal (no. A2196057) dosed at 100mg/kg/day starting from Day 90 of the treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (no. A2196018) and one female (no. A2196009) from the control group were found dead on Days 21 and 83 of treatment period, respectively. One male animal dosed at 100mg/kg/day (no. A2196056, Group 3) and one female animal dosed at 400mg/kg/day (no. A2196071, Group 4) were found dead on Days 43 and 23 of the treatment period, respectively. No clinical signs were observed in the 2 control group animals prior to death. Matted fur, salivation and/or hunched posturewere seen in the mid-dose male (no. A2196056) and in the high dose female (no. A2196071) during the third week of treatment period. In addition, piloerection was seen in these 2 animals fromWeek 1 or 2 up to the day before death.
The following findings were reported at post mortem macroscopic and microscopic examinations:
Group 1
– control male (no. A2196018): multiple, dark areas in the lungs with red colour in the cervical lymph nodes and thymus;
– control female (no. A2196009): red, fluid contents in the thoracic cavity and red colour in the lungs;
Group 4
– high dose female (no. A2196071): pale/creamy contents in the thoracic cavity and adhesions between heart and all pulmonary lobes.
Histopathological evaluation revealed moderate alveolar haemorrhage with mild macrophage aggregation and inflammatory reaction in the lungs in the control male; moderate chronic inflammatory reaction in the aorta area of the heart, associated with haemorrhage also observed in the surrounding connective tissue involving oesophagus and trachea in the control female; marked diffuse chronic/acute inflammation in the pericardium and in the surrounding connective tissue, as well as in the lungs parenchima and pleura with presence of foreign material in the high dose female. The factor contributory to the death of the above unscheduled animals was attributed to a misdosing procedure.
Group 3
– mid-dose male (no. A2196056): macroscopically, this male showed enlarged and swollen liver; the histopathological evaluation revealed minimal, multifocal centrilobular hepatocytic hypertrophy in the liver and mild bilateral nephropathy, associated with hyaline droplet accumulation (a2µ-globulin) in the renal tubules. The pathological picture did not clearly establish the cause of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 6 and 7 in the attached document.
A slight statistically significant reduction in body weight (ranging from -6% to -12%) was observed starting from Day 29 of the treatment period in the males receiving 400mg/kg/day when compared to control animals. In the females, only very slight reductions (from -3% to -7%) were seen from Day 64 of treatment period up to the end of the recovery period. Body weight gain was also reduced in the males dosed at 400mg/kg/day (ranging from -10% to -20%) from Day 8 up to the end of treatment period and in the females receiving 400mg/kg/day (ranging from -6% to -16%) from Day 64 to Day 92.
These decreases were gradually reduced during recovery period. Decreases measured in animals from all groups on Day 92 of treatment period and Day 43 of recovery period were due to the overnight food deprivation for clinical pathology evaluation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Table in the attached document8.
A very slight but statistically significant decrease in food consumption (-9%) was observed in male animals dosed at 400mg/kg/day on Day 8 of the treatment
period. Decreases in food consumption, in some cases statistically significant, were occasionally noted in the females receiving 400mg/kg/day
(from -7% to -22%) during the treatment period. These decreases were no longer observed during the recovery period.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 9 in the attached document.
Before the start of treatment, animals showing no ocular abnormality at the ophthalmoscopic examination were selected for the study. Both eyes of all animals from the main phase groups were re-examined duringWeek 13 of treatment. No treatment-related findings were observed; only a spontaneous lesion (Lens, Cataract) was detected in a control male (no. A2196010).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See Tables 10 and 11 in the attached document.
Dosing Phase
A decrease of erythrocytes, haemoglobin and haematocrit was recorded in some females dosed at 400mg/kg/day (approximately 7% below mean control data). Changes were insufficient in magnitude and amply within the historical control data to represent an adverse anaemia, even though they could represent an effect of the test item. In addition, mean corpuscular haemoglobin concentration was decreased in females receiving 50 and 400mg/kg/day. Due to the minimal severity (3% and 2%, respectively), this finding was considered of no toxicological relevance. Males dosed at 100mg/kg/day showed a statistically significant increase of erythrocytes (8%), haemoglobin (8%), haematocrit (7%), lymphocytes (34%) and basophils (61%). Haemoglobin and haematocrit were also increased in males receiving 50mg/kg/day. Due to the absence of dose-relation, these findings were considered unrelated to treatment.

Recovery Phase
Changes recorded at the dosing phase were no longer observed, confirming reversibility. The statistically significant differences of platelets between control and treated males (20% above controls) were not observed during the dosing phase, therefore they were considered unrelated to treatment.

Coagulation
Dosing Phase
A statistically significant increase of prothrombin time was recorded in males dosed at 400mg/kg/day (8% above controls). Due to the slight severity and being within the historical control data, this finding was considered of no toxicological significance.
Recovery Phase
No changes were recorded.

Bone marrow smear evaluation - Week 13
Two males and two females dosed at 400mg/kg/day showed a slight increase of the erythroid mature cells, leading to a decrease of theM/E ratio.
Male no. A2196082 also showed slight decrease of segmented neutrophils. No other relevant changes in the number and/or morphology of cells were recorded. Due to the absence of peripheral erythrocytes changes, the above findings were considered of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 12 in the attached document.
Fluctuations of some biochemical parameters were recorded in treated animals. The severity of the findings observed was not considered to be suggestive of tissue/organ injury. Furthermore, they were within the normal ranges (Historical control data) for this strain and age of animals.
Recovery Phase
The findings recorded at the Dosing Phase were no longer observed, confirming complete reversibility. In males, urea values were comparable with controls, even though their data were similar to those recorded at the Dosing Phase. The statistically significant decrease of triglycerides and increase of glucose recorded in treated males at the end of recovery were not recorded at the Dosing phase, therefore they were considered to be unrelated to treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 13 in the attached document.
Dosing Phase
High proteinuria, ketonuria and increased urobilinogen were observed in few males dosed at 400mg/kg/day. Ketonuria was also recorded in one male receiving 100mg/kg/day (no. A2196068).
Recovery phase
No findings were recorded, confirming reversibility.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Weekly detailed clinical signs (removal from cage and open field measurements). See Table 2 in the attached document.
No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity. Statistically significant decreases in rearing number were occasionally seen (Day 73 of treatment period) in female animals dosed at 100 and 400mg/kg/day (-25% and -35%, respectively). No toxicological significance was attributed to this finding, since it was occasional and the values obtained were comparable to the other values recorded during the study. It was therefore considered to be incidental.

Neurotoxicity assessment (Functional Tests and Motor activity). See Tables 3, 4 and 5 in the attached document.
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of treatment and recovery periods. Motor activity measurements performed at
the end of the treatment and recovery periods did not show any toxicologically significant differences between treated animals and controls.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Tables 14 and 15 in the attached document.
Terminal body weights showed statistically significant decreases in animals dosed at 400mg/kg/day, when compared to controls at the end of treatment period (- 16% and - 11% in males and females, respectively). Partial or complete recovery was observed at the end of recovery period. Increases in absolute and relative kidney weights were observed in male animals from all treated groups (+20%, +14% and +32%, absolute; +14%, +13% and +56%, relative), while only the relative weights were increased in the females dosed at 100 and 400mg/kg/day (+11% and +21%). A slight increase of the relative kidneys weight was still observed in the males at the end of recovery (+17%). The absolute and relative weight of the liver was also statistically significantly increased in the males and females from all treated groups (+20%, +16% and +30% absolute, +14%, +15% and +55% relative in the males; +13%, +19% and +43% absolute, +12%, +23% and +60% relative in the females) at the end of treatment period. No significant differences were observed at the end of recovery in either sexes. The relative weight of the spleen was increased in the males dosed at 400mg/kg/day (+25%), while the relative weight of the thyroid showed increases in the females dosed at 100 and 400mg/kg/day (+24%). In addition, a decrease (-9%) in the absolute heart weight at the end of treatment period and increases in the relative weight of epididymides and testes (+16% and +20%, respectively) at the end of treatment and recovery (+34% and +7%, respectively) were seen in the males dosed at 400mg/kg/day. In absence of correlation with the histopathological examinations, the decrease of the absolute heart weigh and the increases of the relative weight of spleen, thyroid, testes and epididymides were considered to be of no toxicological relevance.
No other changes of toxicological significance were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 16 in the attached document.
Final sacrifice
At post mortem examination, a treatment-related change was noted in males receiving 400mg/kg/day (high dose), represented by an enlargement and/or swollen consistency and/or pale colour of the kidneys and liver.
Recovery sacrifice
At post mortem examination, enlarged and pale kidneys were again observed in 2 out of 5 males dosed at 400mg/kg/day, as well as pale colour of the liver.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 17 in the attached document.
Final sacrifice
Treatment-related changes were seen in the kidney of males receiving Tert-butyla,a-dimethylbenzyl peroxide at >= 50mg/kg/day and in the liver of males and females dosed at 400mg/kg/day. The renal findings observed were represented by an increase of hyalin droplets or a2µ-globulin, in the proximal tubule in the cortex or corticomedullary junction, confirmed immunohistochemically by a minimal to moderate positivity with a dose-related trend. A spectrum of renal lesions, defined as nephropathy and including multifocal tubular degeneration, tubular protein casts, thickening of the tubular and glomerular basement membrane,interstitial fibrosis and chronic inflammatory cell infiltration was also seen associated with an increased dose-dependent severity, mainly at >= 100mg/kg/day. In addition, the droplets seen in the proximal tubule of high dose males were mostly large, compared with the crystal-like or fine granules observed in males dosed at 50 or 100mg/kg/day.
Together, these findings, which directly related to the test item, are part of the so-called a2u-globulin nephropathy. Because humans do not have a protein that behaves in a manner comparable to a2u-globulin, on a qualitative basis, there is not concordance for this particular key event between male rats and humans and therefore this mode of action is qualitatively not relevant to humans (Hard et al., 2013).
Minimal, multifocal centrilobular hepatocellular hypertrophy was only observed in the liver of male and female rats dosed at 400mg/kg/day. Hepatocyte hypertrophy could be associated with microsomal enzyme induction secondary to the exposure to the test item; hepatocellular cytoplasm showed a pale, ground glass appearance and could be considered an adaptative change.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
The remaining lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology and/or physiological oestrous cyclic changes, commonly seen in untreated Sprague Dawley SD rats of this species and age under our experimental conditions.

Recovery sacrifice
The treatment-related changes in the liver were seen reversible in males and females.However, two males (nos. A2196096 and A2196098) still showed a minimal increase of a2µ-globulin in the proximal tubule in the cortex, although not confirmed immunohistochemically, and an increased severity of nephropathy (mild to marked) in 2 out of 4 males involved. The nephropathy observed in the remaining high dose males with minimal severity degree was considered more likely a spontaneous disease, in particular the albino strains, such as Sprague Dawley, are more inclined to develop this renal disease which occurs in both sexes but with greater frequency and severity in males. The renal pathology could be considered still undergoing reversal at the end of the treatment-free recovery period.
Other effects:
no effects observed
Description (incidence and severity):
Oestrus cycle
No treatment-related anomalies were observed, fromWeek 10 of the treatment period, in the oestrous cycle of the treated females, when compared to controls.

Sperm analysis
See Table 18 in the attached document.
Samples for evaluation of spermmotility, morphology and concentration were obtained from the epididymal cauda at necropsy from all males killed at the end of the treatment period. No differences were seen in motility, morphology and concentration expressed as million sperm/gram caudal epididymal tissue Since no treatment-related effects were seen between control and treated males no assessment was performed in males of the recovery group.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
One male and one female from control group and one female dosed at 400mg/kg/day died during the treatment period. The histopathological examination established that the factors contributory to the death were not related to the toxicity of the test item but to misdosing procedure. In addition, one male animal dosed at 100mg/kg/day was found dead on Day 43. Although changes that could be considered treatment-related (hepatocytic hypertrophy in the liver and mild bilateral nephropathy associated with hyaline droplet accumulation in the renal tubules) were observed, the pathological picture did not clearly establish the cause of death.
Salivation, hunched posture, matted fur and piloerection were the major clinical signs observed in all male and female treated groups, with a dose-related severity and incidence. These signs were reversible at the end of the recovery period. Body weight and food consumption measured during treatment showed in the animals dosed at 400mg/kg/day very slight reductions, which were not considered to be adverse.
No other significant signs of toxic or neurotoxic effects were seen during the in vivo phase of the study. No relevant lesions were recorded at ophthalmological examination. No treatmentrelated anomalies were noted in the oestrus cycle when compared to controls.
A decrease of erythrocytes, haemoglobin and haematocrit was recorded in some females dosed at 400mg/kg/day. Bone marrow smears examination carried out at the end of treatment showed a slight increase of the erythroid mature cells, leading to a decrease of the M/E ratio in two males and two females dosed at 400mg/kg/day. However, the above bone marrow changes were not related with changes in the haematological parameters of these animals and were considered of no toxicological relevance. Fluctuations of some biochemical parameters were recorded in treated animals. Due to the slight severity and/or absence of dose-relation, they were not considered to be of toxicological significance. High proteinuria, ketonuria and increased urobilinogen were observed in few males dosed at 400mg/kg/day. Ketonuria was also recorded in one male receiving 100mg/kg/day. No findings were recorded at the end of recovery, confirming reversibility.
Post mortem observations revealed increases in absolute and/or relative kidney and liver weights in animals from all treated groups at the end of treatment period (except the relative kidney weight of low dose females). No significant differences were observed at the end of recovery in either sexes. Macroscopic examination reported an enlargement and/or swollen consistency and/or pale colour of the kidneys and liver in the animas dosed at 400mg/kg/day. These changes were still present in 2 out of 5 males dosed at 400mg/kg/day at the end of the recovery phase.
At the end of treatment, no differences were observed at sperm analysis including sperm motility, concentration and morphology between the control and the group dosed at 400mg/kg/day.
At microscopic examination, treatment-related changes were seen in the kidney of males receiving Tert-butyla,a-dimethylbenzyl peroxide at >= 50mg/kg/day and in the liver of males and females dosed at 400mg/kg/day. The renal findings observed were represented by an increase of hyalin droplets or a2u-globulin, in the proximal tubule in the cortex or cortico-medullary junction, confirmed immunohistochemically by a minimal to moderate positivity with a dose-related trend. Nephropathy was also seen associated with an increased dose-dependent severity mainly at >=100mg/kg/day.
Because humans do not have a protein that behaves in a manner comparable to a2µ-globulin, on a qualitative basis, there is not concordance for this particular key event between male rats and humans and therefore this mode of action is qualitatively not relevant to humans.
Minimal, multifocal centrilobular hepatocellular hypertrophy was only observed in the liver of male and female rats dosed at 400mg/kg/day. Centrilobular hepatocellular hypertrophy in the liver is often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes. The treatment-related changes in the liver were seen reversible in males and females. However, 2 out of 4 males still showed a minimal increase of a2u-globulin in the proximal tubule in the cortex and an increased severity of nephropathy (mild to marked) in 2 out of 4 males involved.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

In conclusion, signs of effects related to treatment with Tert-butyla,a-dimethylbenzyl peroxide were observed with increasing incidence and severity in animals from all treated groups when administered by oral gavage for 13 consecutive weeks at the dosages of 50, 100 and 400mg/kg/day.
Most of these effects (clinical signs, slight decreases in body weight and food consumption, slight changes in haematological, biochemical and urinalysis parameters, slight increases in liver and/or kidney weights), observed at >= 50mg/kg/day, were not considered to be adverse, due to the
low magnitude and/or complete reversibility. These findings were associated to a minimal centrilobular hepatocellular hypertrophy in male and female animal at 400mg/kg/day and a nephropathy in the male animals dosed at >= 100mg/kg/day. The centrilobular hepatocellular hypertrophy in the liver is
often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes.
Among these changes, adverse test item related effects (nephropathy) were observed in the kidneys of male rats receiving >= 100mg/kg/day. The hyaline droplets in renal tubular epithelium were associated with a2u-globulin accumulation as confirmed by immunohistochemistry.
Because humans do not have a protein that behaves in a manner comparable to a2u-globulin, on a qualitative basis, there is not concordance for this particular key event between male rats and humans and therefore this mode of action is qualitatively not relevant to humans (Hard et al., 2013)(1).
Therefore, it can be concluded that the high dose of 400mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for this study, excluding the male rat-specific a2u-globulin nephropathy.
Executive summary:

The oral toxicity of Tert-butyl-a,a-dimethylbenzyl peroxide in rats following daily oral administration for 13 consecutive weeks and recovery from any treatment-related effect during a period of 6 weeks, were investigated in this study. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 50, 100 and 400mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (corn oil) and acted as a control. Five additional animals for each sexwere included in the high dose and control groups for recovery assessment. The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, oestrus cycle, clinical pathology investigations (including bone marrow smears), terminal body weight, organ weights, macroscopic observations, histopathological examination and spermanalysis.

One male and one female from the control group were found dead on Days 21 and 83 of treatment period, respectively. One male animal dosed at 100mg/kg/day was found dead on Day 43 while one female rat dosed at 400mg/kg/day, was found dead on Day 23 of the treatment period.

No clinical signs were observed in the 2 control group animals prior to death. Matted fur, salivation and/or hunched posturewere seen in the males receiving 100mg/kg/day and in the females receiving 400mg/kg/day during the third week of treatment period. In addition, piloerection was seen in these 2 animals up to the day before death. Post mortem examination showed multiple, dark areas in the lungs with red colour in the cervical lymph nodes and thymus in the control male; red, fluid contents in the thoracic cavity and red colour in the lungs in the control female; pale/creamy contents in the thoracic cavity and adhesions between heart and all pulmonary lobes in the female dosed at 400mg/kg/day. Histopathological evaluation identified the factor contributory to the death of these unscheduled animals in a misdosing procedure.

Macroscopically, the male dosed at 100mg/kg/day showed enlarged and swollen liver; the histopathological evaluation revealed minimal, multifocal centrilobular hepatocytic hypertrophy in the liver and mild bilateral nephropathy, associated with hyaline droplet accumulation (a2µ-globulin) in the renal tubules. The pathological picture did not clearly establish the cause of death.

Salivation was observed in all male and female treated groups, and in one control female animal. This sign was evident starting fromWeek 1 in animals dosed at 400mg/kg/day, while it gradually appeared during the treatment period in those treated at 50 and 100mg/kg/day. Hunched posture and/or matted fur were also observed in male and female animals from all treated groups, with a dose-related severity and incidence. In addition, piloerection was seen in animals from all groups (including controls) with increasing severity and incidence, from Week 3 of the study up to the end of treatment period. These signs were no longer observed during recovery period. Skin fur staining and/or hairloss were also observed in a number of animals from all groups. A palpable mass was seen in a single female animal dosed at 100mg/kg/day starting from Day 90 of the treatment period.

Weekly detailed clinical signs (removal from cage and open field measurements)

No changes of toxicological significance were found at the weekly clinical examination during treatment and recovery periods, which included an evaluation of neurotoxicity. No differences between treated and control groups were evident at the functional tests and at the motor activity measurements at the end of treatment and recovery periods.

Slight statistically significant decreases in body weight and body weight gain were observed in the males dosed at 400mg/kg/day when compared to controls during treatment period. Very slight reductions were also seen in the females dosed at 400mg/kg/day. These reductions gradually decreased during the recovery period.

Slight but statistically significant decreases in food consumption were occasionally observed in the animals receiving 400mg/kg/day during the treatment period. These decreases were no longer observed during the recovery period.

No significant findings were detected at the ophthalmoscopic examinations performed during the study.

No treatment-related anomalies were noted in the oestrous cycle when compared to controls.

A decrease of erythrocytes, haemoglobin and haematocrit was recorded in some females dosed at 400mg/kg/day. Changes were insufficient in magnitude to represent an adverse anaemia, even though they could represent an effect of the test item. No other changes of toxicological relevance were observed. The findings recorded at the Dosing Phase were no longer observed at the end of recovery.

No changes of coagulation of toxicological significance were observed.

Fluctuations of some biochemical parameters, were recorded in treated animals. Due to the slight severity and/or absence of dose-relation, theywere not considered to be of toxicological significance. Most of the findings recorded at the Dosing Phase were no longer observed at the end of recovery.

High proteinuria, ketonuria and increased urobilinogen were observed in few males dosed at 400mg/kg/day. Ketonuria was also recorded in one male receiving 100mg/kg/day. No findings were recorded at the end of recovery, confirming reversibility.

Two males and two females dosed at 400mg/kg/day showed a slight increase of the erythroid mature cells in bone marrow in week 13, leading to a decrease of theM/E ratio. Male no. A2196082 also showed slight decrease of segmented neutrophils. No other relevant changes in the number and/or morphology of cells were recorded. Due to the absence of peripheral erythrocytes changes, the above findings were considered of no toxicological relevance.

Terminal body weights showed statistically significant decreases in animals dosed at 400mg/kg/day when compared to controls at the end of treatment period. Partial or complete recovery was observed at the end of recovery period. Increases in absolute and/or relative kidney and liver weights were observed in animals from all treated groups at the end of treatment. No significant differences were observed at the end of recovery in either sexes.

The relative weight of the spleen was increased in the males receiving 400mg/kg/day, while the relative weight of the thyroid showed increases in the females dosed at 100 and 400mg/kg/day. Only a slight increase of the thyroid relative weight in the females was seen at the end of recovery.

In addition, increases in the relative weight of epididymides and testes were seen in the high dose males at the end of treatment and recovery. Without significant clear correlation with histopathology, the increases of the relative weights of the spleen, thyroid, testes and epididymides were considered to be of no toxicological relevance.

At final post mortem examination, a treatment-related change was noted in males receiving 400mg/kg/day (high dose), represented by an enlargement and/or swollen consistency and/or pale colour of the kidneys and liver. Enlarged and pale kidneys were again observed in 2 out of 5 recovery males dosed at 400mg/kg/day, as well as pale colour of the liver.

Treatment-related changes were seen in the kidney of males receiving Tert-butyla,a-dimethylbenzyl peroxide at >= 50mg/kg/day and in the liver of males and females dosed at 400mg/kg/day. The renal findings observed were represented by an increase of hyalin droplets or a2µ-globulin, in the proximal tubule in the cortex or cortico-medullary junction, confirmed immunohistochemically by a minimal to moderate positivity with a dose-related trend. Nephropathy was also seen associated with an increased dose-dependent severity mainly at >=100mg/kg/day. Minimal, multifocal centrilobular hepatocellular hypertrophy was only observed in the liver of male and female rats dosed at 400mg/kg/day. Hepatocyte hypertrophy could be considered an adaptative change. After the recovery period, the treatment-related changes in the liver were seen reversible in males and females. However, two males still showed a minimal increase of a2u-globulin, in the proximal tubule in the cortex, although not confirmed immunohistochemically. Mild to marked nephropathy was only noted in 2 out of 4 males involved. The renal pathology could be considered still undergoing reversal at the end of the treatment-free recovery period.

No differences were observed in spermmotility, morphology and concentration in treated males compared to controls at the end of treatment. Since no treatment-related effects were seen between control treated males no assessment was performed in males of the recovery group.

In conclusion, signs of effects related to treatment with Tert-butyla,a-dimethylbenzyl peroxide were observed with increasing incidence and severity in animals from all treated groups when administered by oral gavage for 13 consecutive weeks at the dosages of 50, 100 and 400mg/kg/day.

Most of these effects (clinical signs, slight decreases in body weight and food consumption, slight changes in haematological, biochemical and urinalysis parameters, slight increases in liver and/or kidney weights), observed at >= 50mg/kg/day, were not considered to be adverse, due to the low magnitude and/or complete reversibility. These findings were associated to a minimal centrilobular hepatocellular hypertrophy in male and female animal at 400mg/kg/day and a nephropathy in the male animals dosed at >= 100mg/kg/day. The centrilobular hepatocellular hypertrophy in the liver is often due to induction of metabolic enzymes and as such it is a direct effect of the test item. It is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver degenerative changes. Among these changes, adverse test item related effects (nephropathy) were observed in the kidneys of male rats receiving >= 100mg/kg/day. The hyaline droplets in renal tubular epithelium were associated with a2µ-globulin accumulation as confirmed by immunohistochemistry. Because humans do not have a protein that behaves in a manner comparable to a2u-globulin, on a qualitative basis, there is not concordance for this particular key event between male rats and humans and therefore this mode of action is qualitatively not relevant to humans (Hard et al., 2013)(1).

Therefore, it can be concluded that the high dose of 400 mg/kg/day may be considered as the No Observed Adverse Effect Level (NOAEL) for this study, excluding the male rat-specific a2u-globulin nephropathy.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2012 to 25 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A total of 90 Sprague Dawley SD rats (45 males and 45 virgin females), 6 to 7 weeks old and with a weight range of approximately 188 to 214 g for males and 160 to 176 g for females, were received from Charles River Italy S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 13 days was allowed before the start of treatment, during which time the health status of the animals was assessed by
thorough observations.

Animal room controls were set to maintain temperature and relative humidity at 22°C +- 2°C and 55% +- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

In-life data from 26 April 2012 to 25 June 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.
The required amount of Ter-Butyl Cumyl Peroxide was suspended in the vehicle (corn oil).
The formulations were prepared daily (concentrations of 15, 30 and 120 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied.
The formulations were gently mixed using mechanic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 15 to 120 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).
In RTC Study no. 88760, stability after 24 hours at room temperature was verified in the range from 1 to 200 mg/mL. According to RTC’s SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (90%-110% for concentration and CV<10% for homogeneity).
Samples of the formulations prepared on Weeks 1 and 6 were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC’s SOPs for suspensions (90-110% for concentration and CV <10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 88760), in the range from 1 to 200 mg/mL.
Duration of treatment / exposure:
Males were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Females were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum.
Frequency of treatment:
once a day
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 groups of 10 males and 10 females each . A similar constituted control group received the vehicle alone during the treatment period.
Control animals:
yes
Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
One control animal (no. 91770009) judged to be in extremis was killed.

Clinical signs
All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions

Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer generated random order. For males the tests were performed the day before necropsy and for females on Day 3 post partum.

Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order. For males the tests were performed the day before necropsy and for females on Day 3 post partum

Body weight
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to pairing and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Clinical pathology investigations
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood samples are listed below:

Haematology
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count

Differential leucocyte count
- Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests
Prothrombin time

Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (Only males)
At the same time interval as the clinical pathology investigations, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components
Sacrifice and pathology:
Parental animals were killed by exsanguination under isofluorane anaesthesia.
Parental males:
Males were killed after the mating of all females, after 33 days of treatment.
Parental females:
The females with live pups were killed on Day 4 post partum.
One female with total litter loss on Day 0 post partum was killed on Day 3 post partum.
The females which did not give birth 25 days after positive identification of mating were sacrificed on Days 26 or 27 post coitum.

Necropsy
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding one animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.
Females:
All females were examined also for the following:
a) external and internal abnormalities;
b) number of visible implantation sites (pregnant animals);
c) number of corpora lutea (pregnant animals).

Organ weights
From all animals (with the exception of animal humane killed) the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in section 4.5.6 were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
a) Tissues specified in Annex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
b) Tissues specified in Annex 1 from all animals killed during the treatment period.
c) All females sacrificed because no delivery, to investigate possible causes.
d) All abnormalities in all groups

Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.
The criterion for statistical significance was p<0.05 and p<0.01.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Tremor, piloerection and salivation were the most relevant clinical signs detected in high dose males and females during the whole treatment period at the daily clinical examination performed at 0.5-1 hour after treatment.
Salivation was also detected in mid-dose animals during the whole period. A palpable mass was also observed in one control female (animal no. 91770009) and one low dose female (animal no. 91770031). Salivation could be due to the irritating properties of the test item.
In addition, ataxia was detected in 7 out of 10 high dose females on Day 2 of the pre-mating period. Although its transient appearance of one day, the sign could be considered related to treatment, whereas it was also seen in the preliminary study (RTC Study No. 88780EXT) at the dosage of 1000 mg/kg/day. Brown stain of the cage tray was also seen in high dose males on Day 20 of treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (animal no.: 91770009) judged to be in extremis was killed on Day 18 post coitum.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly reduced mean body weight and body weight gain were recorded in high dose animals of both sexes compared to controls throughout the study.
In males, the decrease in body weight was statistically significant at the end of the first and second week of treatment (up to approximately 7%) and at the end of the fourth and fifth week of treatment (up to approximately 10%). Although not statistically significant, a trend to decrease in body weight was also observed in Group 3 (up to 3.8%).
In females, before pairing there was no effect on body weight in females although a very slight decrease, not statistically significant, was observed in Group 4 ( up to 3.6%).
Concerning the body weight gain, in males statistically significant decrease in Group 4 was detected at the end of the first (up to approximately 49%) and the fourth week of treatment (up to approximately 29%).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly reduced food consumption was recorded in high dose pregnant females starting from Day 14 post coitum (approximately 18%) up to Day 4 post partum (approximately 35%). No significant effects on food consumption were observed in males
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant changes recorded in males (mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, large unstained cells) were of minimal magnitude and/or not dose-related, therefore considered unrelated to treatment.
Females treated with 600 mg/kg/day showed slight decrease of erythrocytes, haemoglobin and haematocrit. However, changes were of low severity (approximately 10%), therefore considered of no toxicological importance.

Coagulation
No changes of toxicological importance were recorded.
The statistically significant decrement of prothrombin time observed in females receiving 150 mg/kg/day (4% compared with controls) was considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
One animal/sex treated with 150 mg/kg/day and 600 mg/kg/day (animal nos. 91770056, 91770074, 91770049 and 91770067) showed moderate to severe increment of transaminase enzymes. Changes were 2 to 18-fold and were not dose-related.
Additional increment of g-glutamyl transferase was recorded in animal no. 91770074 and increment of cholesterol was observed in animal no. 91770056.
Due to the low incidence and the lack of a dose correlation with histopathology (hypertrophy as adaptive phenomenon not degenerative, with the exception of one animal), the above changes cannot be conclusively attributed to treatment.
The statistically significant changes recorded for phosphorus, chloride and sodium in males were of minimal magnitude (approximately 4% to 15%), therefore considered of no toxicological relevance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Increase of urobilinogen and ketonuria was recorded in treated males. Due to lack of dose-relation, these changes cannot be conclusively attributed to treatment.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weight was significantly lower at statistical analysis in the high dose group of both sexes compared to controls. Statistically significant differences were noted in the absolute and/or relative organ weight of both sexes in group 4.Terminal body weight was significantly lower at statistical analysis in the high dose group of both sexes compared to controls.
Some statistically significant differences were noted in the absolute and/or relative organ weight of both sexes in the high dose group.
Slight, statistically significant increase was also noted in the relative kidney weight in the mid-dose group of both sexes (respectively of approximately 15% in males and 12% in females).

In males:
- Higher absolute and relative kidneys weight (28 and 42%, respectively) and liver weight (42 and 57%, respectively).
- Higher relative testes weight (16%) and relative brain weight (12%).
- Lower absolute and relative thymus weight (27 and 19%, respectively).
In females:
- Higher absolute and relative liver weight (30 and 56%, respectively).
- Higher relative kidneys weight (20%) and relative brain weight (15%).
- Lower absolute heart weight (15%) and absolute thymus weight (38%).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death
A single control female animal (no. 91770009) died during the study.
At necropsy, the following changes were noted: enlarged adrenals; pale brain, pituitary, pancreas, salivary glands, liver and heart; multiple dark, firm and raised areas in the lungs; enlarged spleen; dark area in the glandular stomach; small thymus, dark and/or red harderian glands and mediastinal lymph nodes; and firm mass in the subcutis, involving the forelimb.

Final sacrifice
Treatment-related changes were seen in the liver of both sexes treated with the high dose and in the kidneys of the males treated with the high dose.
The incidence of swollen and/or enlarged liver was higher in the high dose group, compared to the concurrent control group and these changes correlated with histopathological findings. Cases of enlarged kidneys were seen only in the males treated with the high dose.
The incidence of small size of the thymus was higher in the female high dose group, compared to the concurrent control group. However, the histopathological evaluation did not confirm a treatment-related effect.
All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled death
At histopathology, the main finding and the cause of death of the control female animal (no. 91770009) which died during the study, were subcutaneous hemangiosarcoma, which metastasized to the lungs.

Final sacrifice
Treatment related changes were seen in the liver and kidneys.
Statistically significant treatment-related findings were noted in the liver of the high dose group of both sexes and in the mid-dose male group, consisting of centrilobular hepatocytic hypertrophy associated with cytoplasmic eosinophilia. The change was of mild degree in the high dosed animals and of minimal degree in the mid-dose group. As the hepatocytic hypertrophy was not associated with other changes in the hepatocytes (i.e., degeneration and/or necrosis), this change is not considered as adverse, but rather as potentially adaptive.
In the kidneys of the males treated with the intermediate and high dose, a dose-related increased incidence of minimal cortical basophilic tubules was noted. As organ weight data indicated a statistically significant increase of the relative kidney weight in these groups, as well as macroscopically, cases of enlarged kidneys were seen only in the males treated with the high dose, it is suggested that the dose-related increased incidence of minimal cortical basophilic tubules is potentially related to treatment.

All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions. In particular, a single case of mammary carcinoma was seen in a female rat treated with the low dose. Such a tumour is known to occur sporadically in young SD rats (Son and Gopinath, 2004). In a single female animal of the high dose group, focal minimal hepatocytic necrosis was noted. As such a lesion was the only single case seen and similar cases are sporadically seen in untreated animals of the same strain and age, this case is not considered as related to treatment (Thoolen B, et al., JWard JM (2010). IN HAND - Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System. Toxicologic Pathology 38: 5S-81S).
Minimal to mild renal tubular dilation was seen in the male groups with the following incidence: 3, 1, 2, 5, respectively in the control, low, intermediate and high dose. As the incidence did not suggest a clear dose relation, and as this change was seen in 3/10 control animals, it is not considered as related to treatment. In a single female animal treated with the high dose, moderate renal tubular vacuolation was noted. As this change was seen only in a single animal in the study, it is considered as unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Oestrus cycle
The number of oestrus cycles was decreased in high dose females with respect to the control females. A trend to decrease was also observed in Groups 2 and 3. However, these values were comparable with the control historical data, therefore they were not considered to be of toxicological relevance.

Spermatogenic cycle
A detailed qualitative evaluation of testes was performed on 5 randomly selected control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002.
The PAS-H stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

BODY WEIGHT

MALES

 

Week of treatment

Body weight(g)

Before dosing

1

2

4

5

Dose levels

(mg/kg)

Control

256.82

345.58

373.62

412.84

416.37

75

257.11(+0.11%)

346.39 (+0.2%)

377.59 (+1.1%)

417.82 (+1.2%)

418.52 (+0.5%)

150

257.47 (+0.25%)

339.86 (-1.7%)

368.30 (-1.4%)

403.18 (-2.3%)

400.37 (-3.8%)

600

256.79 (+0.01%)

321.21 (-7.1%)**

350.71 (-6.1%)*

383.84 (-7.0%)*

374.58 (-10.0%)*

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

FEMALES

 

Day of post coitum phase

Body weight(g)

7

14

20

Dose levels

(mg/kg)

Control

294.69

333.91

424.19

75

294.84 (+0.05%)

332.21 (-0.5%)

417.67 (-1.5%)

150

285.93 (-3.0%)

323.24 (-3.2%)

400.86 (-5.5%)

600

275.69 (-6.4%)*

303.34 (-9.2%)**

346.43 (-18.3%)**

 

Day of post partum phase

1

4

Dose levels

(mg/kg)

Control

319.12

315.56

75

317.85 (-0.4%)

318.44 (+0.9%)

150

306.85 (-3.8%)

293.81 (-6.9%)

600

272.97 (-14.5%)**

258.13 (-18.2%)**

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

BODY WEIGHT GAIN

MALES

Week of treatment

Body weightgain(g)per day

1

4

Dose levels

(mg/kg)

Control

5.150

3.117

75

4.712 (-8.5%)

3.011 (-3.4%)

150

4.797 (-6.9%)

3.286 (+5.4%)

600

2.646 (-48.6%)**

2.216 (-28.9%)*

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01


FEMALES

 

Day of post coitum phase

Body weightgain(g)per day

14

20

Dose levels

(mg/kg)

Control

5.602

14.761

75

5.339 (-4.7%)

14.244 (-3.5%)

150

5.329 (-4.9%)

12.938 (-12.4%)

600

3.951 (-29.5%)**

    7.181 (-51.4%)**

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

FOOD CONSUMPTION

FEMALES

 

Day of post coitum phase

Day of post partum phase

Food consumption

(g/animal/day)

14

20

4

Dose levels

(mg/kg)

Control

24.87

25.69

30.54

75

25.35 (+1.9%)

25.83 (+0.5%)

33.40 (+9.4%)

150

23.75 (-4.5%)

25.31 (-1.5%)

28.82 (-5.6%)

600

20.37 (-18.1%)**

21.10 (-17.9%)*

19.79 (-35.2%)*

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

OESTRUS CYCLE

 

Number of

oestrus cycles

 

Fertility index

(%)

Precoital interval

(days)

Dose levels

(mg/kg)

Control

3.6

100

2.7+1.3

75

3.4 (-5.6%)

100

2.6+1.4

150

3.3 (-8.3%)

80

2.9+2.1

600

2.7 (-25.0%)

70

1.6+0.5

ORGAN WEIGHT

MALES

Terminal

body weight

(g)

Kidney weights

Liver weights

Absolute

wt. (g)

Relative

wt. (%)

Absolute

wt. (g)

Relative

wt. (%)

Dose

levels

(mg/kg)

Control

413.44

2.796

0.675

12.504

3.011

75

416.99 (+0.9%)

2.963 (+6.0%)

0.711 (+5.3%)

13.172 (+5.3%)

3.153 (+4.7%)

150

399.11 (-3.5%)

3.104 (+11.0%)

0.779** (+15.4%)

13.749 (+10.0%)

3.440 (+14.2%)

600

374.08* (-9.5%)

3.584** (+28.2%)

0.957** (+41.8%)

17.791** (+42.3%)

4.717** (+56.7%)

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

 

FEMALES

Terminal body weight

(g)

Liver weights

Thymus weight

 Absolute wt. (g)

Relative wt.(%)

 Absolute wt. (g)

Dose levels

(mg/kg)

Control

307.63

10.387

3.352

0.3057

75

309.00 (+0.4%)

12.644 (+21.7%)

4.042 (+20.6%)

0.2879 (-5.8%)

150

282.65 (-8.1%)

11.226 (+8.1%)

3.970 (+18.4%)

0.2643 (-13.5%)

600

257.00* (-16.5%)

13.518* (+30.1%)

5.224** (+55.8%)

0.1900** (-37.8%)

*  group mean is significantly different from control at level p < 0.05

** group mean is significantly different from control at level p < 0.01

Conclusions:
On the basis of the results obtained in the study, the NOAEL for general toxicity is 150 mg/kg/day for males and females, based on clinical signs, reduced body weight and body weight gain at 600 mg/kg bw/day (compared to controls).
Executive summary:

The toxic effects on rats of both sexes after repeated dosing with Luperox 801 (97% tert-Butyl Cumyl Peroxide) by oral route was evaluated in an OECD 422 study. The test item, suspended in corn oil, was administered by oral gavage to 3 groups of 10 males and 10 females each as indicated below at the doses of 75, 150 and 600 mg/kg bw/d. A similar constituted control group (Group 1) received the vehicle alone during the treatment period. Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy. Females were treated for 2 weeks before pairing, thereafter during pairing, post coitum and lactation periods until Day 3 post partum. The following investigations were performed in all groups: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology, clinical chemistry and urinalysis), macroscopic observations, organ weights and histopathological examination.

Tremors, piloerection and salivation were the most relevant clinical signs detected in high dose males and females during the whole treatment period, at the daily clinical examination performed at 0.5-1 hour after treatment. In addition ataxia was detected in 7 out 10 high dose females on Day 2 of the pre mating period. Although its transient appearance of one day, the sign could be considered related to treatment, whereas it was also seen in the preliminary study (RTC Study No. 88780EXT) at the dosage of 1000 mg/kg/day. Salivation was also detected in mid-dose animals during the whole treatment period.

One control female judged to be in extremis was killed on post coitum Day 18.

Body weight and body weight gain of both sexes were significantly lower at statistical analysis in the high dose group of both sexes compared to controls throughout the study.Food consumption was reduced only in high dose females starting from post coitum Day 14.

There were no effect on motor activity, sensory reaction to stimuli, and no neurotoxicity. There were no effect on haematological parameters and on clinical chemistry, and no effect on spermatogenic cycle.

Terminal body weight was significantly lower at the statistical analysis in the high dose group of both sexes compared to controls. Statistically significant higher liver and kidneys weight was observed in high dose males and females compared to controls. In addition, a significant increase was also detected in the kidney weight of both sexes in the mid-dose group.

At final sacrifice, treatment related changes were seen in the liver and kidneys.

Statistically significant treatment-related findings were noted in the liver of the high dosed group of both sexes and in mid-dose male group, consisting of centrilobular hepatocytic hypertrophy associated with cytoplasmic eosinophilia. The change was of mild degree in the high dosed animals, and of minimal degree in the intermediate dose group. As the hepatocytic hypertrophy was not associated with other changes in the hepatocytes (i.e., degeneration and/or necrosis), this change was not considered as adverse, but rather as potentially adaptive.

In the kidneys of the males treated with the intermediate and high dose, a dose-related increased incidence of minimal cortical basophilic tubules was noted. As organ weight data indicated a statistically significant increase of the relative kidney weight in these groups, as well as macroscopically cases of enlarged kidneys were seen only in the males treated with the high dose, it is suggested that the dose-related increased incidence of minimal cortical basophilic tubules is potentially related to treatment.

On the basis of the results obtained in the study, the NOAEL for general toxicity is 150 mg/kg/day for males and females, based on clinical signs, reduced body weight and body weight gain at 600 mg/kg bw/day (compared to controls).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study, Klimisch 1.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Critical effects observed:
not specified
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

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), tert-butyl-a,a-dimethylbenzyl peroxide (TBCP) is not classified for repeated dose toxicity.