Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

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

Applicant's summary and conclusion

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.