Registration Dossier

Administrative data

Description of key information

The oral administration of Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide, CAS# 78-63-7 to rats by gavage, for 90 days, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant adverse effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 150 mg/kg bw/day.

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:
Experimental starting date: 17th July 2013. Experimental completion date: 2nd June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no/minor deviations from standard test guidelines and/or minor methodlogical deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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 (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 202 to 223g, the females weighed 137 to 170g, and were approximately six to eight weeks old.

The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Appendix 28. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in corn oil. This vehicle was choosen for consistency with previous toxicity studies performed using this test item. Initially the test item formulations were prepared on a daily basis for seven days due to suspected low stability of the dosing formulations and dosing was completed with four hours of dose preparation. Further stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services and results showed the formulations to be stable for up to eight days. Formulations were then prepared weekly and stored at approximately 4 °C in the dark.

Samples of each test item formulation were taken and analyzed for concentration of di-tert-butyl 1,1,4,4-tetramethyl tetramethylene diperoxide (CAS# 78-63-7) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 23 - please see attached. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatograohic profile consisting of a single peak.

Analytical Procedure
Preparation of standard solutions
Stock solutions of test item in methanol were prepared for external standard calibration. An aliquot, 0.1 mg of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. This was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol to achieve the working concentration.

Preparation of Accuracy Samples
Samples of Corn Oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. The samples were then prepared for analysis as per the test samples previously.

Preparation of Linearity Standards
A range of standard solutions were prepared in methanol from a stock solution of 1 mg/mL by serial dilution covering the concentration range 0 to 0.1649 mg/mL.

Instrumental Setup
GC system: Agilent Technologies 5890 or 6890, incorporating autosampler and workstation
Column: DB-1 (30 m x 0.25 mm id x 0.23 micro-netres film)
Oven temperature program: Oven: 100°C for 2 minutes with 10°C/minute to 250°C for 5 minutes
Injection temperature: 150°C
Flame ionisation detector temperature: 150°C
Injection volume: 1 micro-litre
Retention time: ~ 7 minutes

Duration of treatment / exposure:
90-days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
15 mg/kg bw/day (3.75 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day (12.5 mg/mL)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day (37.5 mg/mL)
Basis:
actual ingested
No. of animals per sex per dose:
10 males
10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages were selected, in collaboration with the sponsor based on available toxicity data.

The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of corn oil.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Estrous Cycle Assessment
The stage of estrus were classified according to the following criteria:

Di-estrus (D) Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P) Predominantly epithelial cells, usually in significant numbers.
Early Estrus (E1) Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Estrus (E2) Predominantly cornified cells usually seen as clumps of cells.
Metestrus (M) Large numbers of leukocytes with discrete clumps of cornified cells.

The Estrous cycles are classified according to the following criteria:
Normal estrus The pattern of daily stages of estrus show a four to five day cycle, which is generally repeated over 21 days.
Extended estrus The observation of a predominance of epithelial/cornified cells for more than two days for more than one estrous cycle.
Extended di-estrus The predominant cell type is the leukocyte for more than three consecutive days over more than one estrous cycle.
Irregular cycle An irregular length of estrous cycle is observed over the 21 day evaluation period.
Acyclic No evidence of an estrous cycle is observed over the 21 day evaluation period.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

Laboratory Investigations
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling. Urinalytical investigations were performed on all control and test group animals during the final week of dosing. Urine samples were collected by housing the animals overnight in metabolism cages under normal hydration but without access to food.

The methods used for hematological, blood chemical and urinalytical investigations are given in Appendix 29 and normal ranges for hematological and blood chemical investigations are shown in Appendix 30 - please see attached.

Hematology
Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices - mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count - neutrophils (Neut), - lymphocytes (Lymph), - monocytes (Mono), - eosinophils (Eos), - basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)

Urinalysis
The following parameters were measured on collected urine:
Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Blood, Glucose, Appearance
Sacrifice and pathology:
Pathology
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

A vaginal smear was prepared at termination for all females and the stage of estrus was recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:

Adrenals, Liver, Brain, Ovaries, Left Cauda, Spleen, Left Epididymis, Left Testis, Right Epididymis, Right Testis, Heart, Thymus, Kidneys, Uterus

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate,
Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles,
Epididymides, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue, Lungs (with bronchi), Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary glands, Uterus (with Cervix), Muscle (skeletal), Vagina

All tissues were dispatched to the Test Site (Propath UK Ltd, Rotherwas, Hereford, HR2 6JU) for processing. All tissues from control and 150 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Since there were indications of treatment-related changes for the thyroid in both sexes and the kidneys in males in the 150 mg/kg bw/day dose group, histopathological evaluation of these tissues was extended to include the thyroids in both sexes and the kidneys for males in the low and intermediate dose groups using standard Hematoxylin and Eosin staining.

Immunohistochemical staining of the kidneys for α-2-microglobulin was also performed for males as appropriate. Sections of kidney were incubated with a goat anti-α-2-microglobulin antibody and detected using an anti-goat multimer antibody conjugated to horseradish peroxidase, followed by diaminobenzedine (DAB) to produce brown staining at the site of antibody binding, if present. Full details of staining reagents and equipment used were included in the raw data/study records.

Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology finding was conducted by the Test Facility. A complete histopathology phase report is presented in Appendix 22 (see attached) and represents the consensus view of both pathologists.

Sperm Analysis
At necropsy of adult males the following evaluations were performed:

i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.

ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC.

iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility. Approximately 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).

iv) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20ºC. At an appropriate later date the tissue were thawed and homogenised in an appropriate saline/detergent to determine the numbers of homogenisation resistant spermatids.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.


3.4.11.1 Statistical Analysis
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Sperm Analysis (excluding morphology), Absolute and Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
There were no deaths on the study.

Clinical Observations
Neither the type, incidence nor distribution of clinical signs observed during the study indicated any adverse effect of treatment at dosages up to 150 mg/kg bw/day.

At 150 mg/kg bw/day all animals showed increased salivation post dosing during the study. Similar increased salivation post dosing was also observed at 50 mg/kg bw/day but the incidence and number of animals affected was much lower. Increased salivation is frequently observed when animals are dosed via the oral gavage route and this is generally considered to reflect distaste/irritancy of the test item formulation rather than any systemic effect of treatment.

One control female had generalised fur loss between Days 46 and 91. Observations of this nature are commonly observed in group housed animals and in the absence of treatment is incidental.

Functional Observations
Behavioral Assessments
Assessment of the animals in a standard arena did not reveal any effect of treatment at 15, 50 or 150 mg/kg bw/day.

Functional Performance Tests
Assessment of functional performance using grip strength and measurement of motor activity did not indicate any obvious effects of treatment at dosages of 15, 50 or 150 mg/kg bw/day.

Sensory Reactivity Assessments
Sensory reactivity to different stimuli (auditory, visual and proprioceptive) appeared unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

Body Weight
There were no adverse effects of treatment on body weight development for either sex throughout the study at dosage up to 150 mg/kg bw/day.

Females treated with 150 and 50 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment whilst males treated with 150 mg/kg bw/day showed a statistically significant reduction in body weight gain During Weeks 6 and 11. Overall body weight gain was unaffected in these animals therefore the intergroup differences were considered not to be of toxicological importance.

Food Consumption
No adverse effect in overall food consumption or food efficiency was detected in treated animals when compared to controls.

Estrous Cycle Assessment
There were no treatment-related effects on female estrous cycles at dosages up to 150 mg/kg bw/day.

Water Consumption
Visual assessment of water intake did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.

Ophthalmoscopic Examination
Ophthalmic examination during the last week of treatment did not indicate any effect of treatment for either sex at 150 mg/kg bw/day.

Laboratory Investigations
Hematology
There was no clear effect of treatment on hematology parameters at 15, 50 or 150 mg/kg bw/day.

At 150 mg/kg bw/day, animals of either sex showed a statistically significant increase in mean platelet count when compared with controls. The majority of individual values were within the background ranges for rats of the strain and age used and in the absence of any associated histopathology correlates, these intergroup differences were considered to be of no toxicological significance.

Females from all treatment groups showed a statistically significant increase in hemoglobin, erythrocytes and hematocrit. The majority of individual values for these parameters were within the normal ranges for rats of the strain and age used and, in the absence of any histopathological correlates, these differences were considered incidental and unrelated to treatment.

Blood Chemistry
There was no clear effect of treatment on blood chemical parameters at 15, 50 or 150 mg/kg bw/day.

Males treated with 150 and 50 mg/kg bw/day showed a statistically significant reduction in glucose and alanine aminotransferase. Males treated with 150 mg/kg bw/day also showed a statistically significant reduction in alkaline phosphatase and bile acids. All of the individual values were within normal background ranges for these parameters and, in the absence of any associated histopathological changes, the intergroup differences were considered not to be of toxicological importance. Males treated with 15 mg/kg bw/day showed a statistically significant reduction in phosphorus. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.

Females from all treatment groups showed a statistically significant increase in total protein and calcium concentration. Females treated with 150 and 50 mg/kg bw/day also showed a statistically significant increase in albumin and females treated with 150 mg/kg bw/day also showed a statistically significant reduction in albumin/globulin ratio. The majority of the individual values were within normal background ranges for these parameters and, in the absence of any associated histopathological changes, the intergroup differences were considered not to be of toxicological importance.

Urinalysis
Group mean values and standard deviations for test and control group animals are given in Table 13. Individual data are given in Appendix 15.

No treatment-related effects were detected in the urinalytical parameters measured.

Pathology
Necropsy
Neither the type, incidence nor distribution of macroscopic abnormalities detected at terminal necropsy indicated any adverse effect of treatment.

One high dose male had a fluid filled left kidney, an enlarged right kidney and malformed seminal vesicles. In isoloation, this findings was considered to be incidental and unrelated to treatment.

Organ Weights
Animals of either sex treated with 150 mg/kg bw/day showed a statistically significant increase in absolute and body weight-relative liver weight when compared to controls. The majority of individual body weight-relative values exceeded the historical control range.

Males treated with 150 mg/kg bw/day also showed a statistically significant increase in absolute and body weight-relative kidney weight. Body weight-relative values are probably the more accurate indicator of toxicological effect and at 150 mg/kg bw/day five of the ten individual values exceeded the historical control group.

No toxicologically significant effects were detected in animals of either sex treated with 50 or 15 mg/kg bw/day.

Absolute and body weight-relative ovary weight in all treatment groups were statistically significantly lower than control but values did not show any consistent dosage relationship. The differences in group mean values observed, in the absence of any histopathological change, were considered incidental and unrelated to treatment. Females treated with 150 mg/kg bw/day also showed a statistically significant increase in absolute and body weight-relative kidney weight. In the absence of any histopathological change in female kidneys, the intergroup difference was considered not to be of toxicological importance.

Absolute and body weight-relative spleen and left cauda weights in males from all treatment groups were statistically significantly higher than control but values did not show any consistent dosage relationship. Absolute brain weight in males from all treatment groups and body weight-relative brain weight in males treated with 150 and 15 mg/kg bw/day were statistically significantly higher than control whilst body weight-relative brain weight for males treated with 50 mg/kg bw/day was lower than control. All values did not show any consistent dosage relationship. The differences in group mean values observed, in the absence of any histopathological change, were considered incidental and unrelated to treatment.


Histopathology
The following treatment related microscopic abnormalities were detected:

Kidneys: An increase in the severity and incidence of intra-epithelial hyaline droplets was present in all treated male groups. In males given 150 mg/kg bw/day there was also an increase in the incidence and severity of basophilic tubules and, in eight out of the ten males, granular casts were observed at the corticomedullary junction. Furthermore in one male given 50 mg/kg bw/day and all males given 150 mg/kg bw/day there was minimal single cell necrosis of cortical tubules. These findings were consistent with an etiological diagnosis of alpha-2µ-microglobulin nephropathy using immunohistochemistry in which there was a clear increase in staining intensity at the highest dosage.

Thyroids: The incidence of minimal or mild diffuse hypertrophy of the follicular epithelium was greater in animals of either sex from all treated groups.

Sperm Analysis
No adverse effect of treatment on sperm concentration, motility, morphology or homogenisation resistant spermatid counts was apparent.

Males treated with 150 mg/kg bw/day showed a statistically significant reduction in epididymal spermatid count. In the absence of any associated histopathological correlates the intergroup difference was considered of no toxicological importance.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
The oral administration of Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide, CAS# 78-63-7 to rats by gavage, for 90 days, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant adverse effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 150 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

 

i) The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study (Adopted 21 September 1998).  

ii) This study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registrarion, Evaluation, Authorisation and Restriction of Chemicals (REACH)     

 

Methods…….

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 15, 50 and 150 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (corn oil).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Estrous cycle assessment was performed during the final three weeks of the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Urinalytical investigations were performed during the final week of the study. Ophthalmoscopic examination was also performed on control group and high dose animals prior to the start of treatment and during Week 12 of the study.

 

All animals were subjected to gross necropsy examination, which included male sperm assessments, and histopathological evaluation of selected tissues from high dose and control animals was performed. 

 

Results…….

Mortality

There were no unscheduled deaths on the study.

 

Clinical Observations

Neither the type, incidence nor distribution of clinical sign observed during the study indicated any effect of treatment at dosages up to 150 mg/kg bw/day.

 

Behavioral Assessment

There were no treatment-related changes in behavioral parameters measured.

 

Functional Performance Tests

There were no treatment-related changes in functional performance.

 

Sensory Reactivity Assessments

Sensory reactivity to different stimuli (auditory, visual and proprioceptive) appeared unaffected by treatment at 15, 50 or 150 mg/kg bw/day.

 

Body Weight

There were no adverse effects of treatment on body weight gain for either sex at 15, 50 or 150 mg/kg bw/day.

 

Food Consumption

There were no effects of treatment on food consumption or food conversion efficiency for either sex at 15, 50 or 150 mg/kg bw/day.

 

Estrous Cycle Assessment

There were no treatment-related effects on female estrous cycles.

 

Water Consumption

There were no treatment-related effects detected in water consumption.

 

Ophthalmoscopy

Ophthalmic examination of animals receiving 150 mg/kg bw/day at the end of the study did not indicate any effect of treatment.

 

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

 

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Urinalysis

Assessment of urine parameters at the end of the study did not indicate any obvious effect of treatment at 15, 50 or 150 mg/kg bw/day.

 

Necropsy

Neither the type, incidence nor distribution of macroscopic abnormalities detected at terminal necropsy indicated any adverse effect of treatment.

 

Organ Weights

Males treated with 150 mg/kg bw/day showed a statistically significant increase in absolute and body weight-relative kidney weight.

 

No toxicologically significant effects were detected in females treated with 150 mg/kg bw/day or animals of either sex treated with 50 or 15 mg/kg bw/day.

 

Sperm Analysis

There were no toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts.

 

Histopathology

The following treatment related microscopic abnormalities were detected:

 

Kidneys: increased incidence of intra-epithelial hyaline droplets in all treated male groups. In males given 150 mg/kg bw/day, associated granular cast formation, an increase in basophilic tubules and single cell necrosis of cortical tubules was also evident.

 

Thyroids: The incidence of minimal or mild diffuse hypertrophy of the follicular epithelium was increased in animals of either sex from all treatment groups.

 

Conclusion

The oral administration of Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide, CAS# 78-63-7 to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any toxicologically significant adverse effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore considered to be 150 mg/kg bw/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 2011- 06 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: • Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Animals:
Wistar Rat, RccHanTM: WIST(SPF)

- Source:
Harlan Laboratories B.V. / Kreuzelweg 53 / 5961 NM Horst / Netherlands

- Age at study initiation:
Seven to eight weeks

- Weight at study initiation:
Males weighed 190 to 214 g. Females weighed 142 to 162 g.

- Fasting period before study:
Animals were not fasted prior to dosing.

- Housing:
In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding and paper enrichment.

- Diet:
Pelleted standard Harlan Teklad 2914C (batch nos. 68/10 and 85/10) rat / mouse maintenance diet, available ad libitum.

- Water (e.g. ad libitum):
Community tap-water from Itingen was available ad libitum in water bottles

The diet and drinking water was tested to confirm absence of contaminants.

- Acclimation period:
Seven days.


ENVIRONMENTAL CONDITIONS

(temp. range: ; ). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

- Temperature (°C):
22 ± 3 °C

- Humidity (%):
5relative humidity range: 30 - 70%

- Air changes (per hr):
10 - 15 air changes per hour

- Photoperiod (hrs dark / hrs light):
The low intensity fluorescent lighting was controlled to give twelve hours continuous light (with music) and twelve hours darkness.

IN-LIFE DATES:
Delivery of Animals:
15-Feb-2011

Randomization:
16-Feb-2011

Acclimatization:
15 to 21-Feb-2011

Administration / Treatment:
22-Feb to 21-Mar-2011

Recovery:
22-Mar to 5-April 2011

Termination (Necropsy):
22-Mar-2011 (after fours weeks treatment)
06-Apr-2011 (after two weeks recovery)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Method of administration:
Gavage

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations
as a solution in corn oil.

VEHICLE:
Corn oil

- Justification for use and choice of vehicle(if other than water):
Corn oil provided better quality formulations.

- Concentration in vehicle:
Group 2: 4 mg/mL/day
Group 3: 12 mg/mL/day
Group 4: 40 mg/mL/day

- Amount of vehicle (if gavage):
5 ml/kg bodyweight

- Lot/batch no. (if required):
400159216

- Purity:
Fulfills Ph.Eur.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dose formulations was determined by gas chromatography. The dose formulations were sampled on the first day of treatment and during week 3. The analytical method has been shown to yield satifactory linearity, specificity and accuracy for the purposes of the study.
:
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Remarks:
Doses / Concentrations:
Dose levels of 20, 60 and 200 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Male and Female: 10 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 20 mg/kg/day
Male and Female: 5 animals per sex at 60 mg/kg/day
Male and Female: 10 animals per sex at 200 mg/kg/day
Control animals:
yes, concurrent vehicle
Details on study design:
EXAMPLE:
- Dose selection rationale:
Based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study D10333

- Rationale for animal assignment (if not random):
Random by body weight.

- Rationale for selecting satellite groups:
Required by Japanese guideline.

- Post-exposure recovery period in satellite groups:
Yes

- Section schedule rationale (if not random):
Not applicable
Positive control:
None
Observations and examinations performed and frequency:
VIABILITY / MORTALITY OBSERVATIONS: Yes
- Time schedule:
Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS: Yes
The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period and once daily during days 1 to 14 of the recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter.

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule:
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results are presented in the summary and
individual tables of the weekly detailed clinical observations under week 4.

Grip Strength: Yes
Forelimb and hind limb grip strength measurements were performed during the FOB at week 4

Locomotor Activity: Yes
Locomotor (decreased or increased) activity was measured quantitatively during the fourth treatment week for a 60-minute period and the total
activity of this time period was recorded.


BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy.

FOOD CONSUMPTION: Yes
The cage food consumption was recorded once during the acclimatization period and weekly thereafter.


OPHTHALMOSCOPIC EXAMINATION: No data:
Not applicable
- Time schedule for examinations:
Not applicable
- Dose groups that were examined:
Not applicable


HEMATOLOGY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (Day 29) and end of the recovery period (Day 42)
- Anaesthetic used for blood collection: Yes
Isoflurane
- Animals fasted:
Yes
- How many animals:
Day 29
Groups 1 and 4: 10 males and 10 females
Groups 2 and 3: 5 males and 5 females
Day 42
Groups 1 and 4: 5 males and 5 females

- Parameters checked in table [No.?] were examined.
Routine hematological parameters were examined.


CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (Day 29) and end of the recovery period (Day 42)
- Anaesthetic used for blood collection: Yes
Isoflurane
- Animals fasted: Yes
- How many animals:
Day 29
Groups 1 and 4: 10 males and 10 females
Groups 2 and 3: 5 males and 5 females
Day 42
Groups 1 and 4: 5 males and 5 females


URINALYSIS: Yes
- Time schedule for collection of urine:
Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage. Samples taken at the end of the treatment
period (Day 29) and end of the recovery period (Day 42)
- Anaesthetic used for blood collection: Yes
Isoflurane
- Animals fasted: Yes
- How many animals:
Day 29
Groups 1 and 4: 10 males and 10 females
Groups 2 and 3: 5 males and 5 females
Day 42
Groups 1 and 4: 5 males and 5 females



Sacrifice and pathology:
EXAMPLE:
GROSS PATHOLOGY: Yes (see tables)

All animals were subjected to a full external and internal macroscopic examination and any abnormalities were recorded.

HISTOPATHOLOGY: Yes (see tables)

All control and high dose animals were subjected to a full histological examination and any abnormalities were recorded.
Other examinations:
EXAMPLE:
MORTALITY DATA
ORGAN WEIGHTS
See Tables
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings: The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex, The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution, Fisher's exact-test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:

Observations
Viability / Mortality

All animals survived until their respective days of scheduled necropsy.

Daily Observations

There were no test item-related changes evident during the daily clinical observations. One male treated with 60 mg/kg/day had minor evidence of scabbing and hair loss in the dorsal area of the neck. This was considered to be a background finding of no toxicological relevance.

The females were without clinical signs at all dose levels.


Weekly Behavioral Observations

There were no findings at any dose level during the weekly behavioral observations (weeks 1-3).


Functional Observational Battery (Screen)
There were no findings at any dose level during the functional observation battery performed at week 4.
- Grip Strength
There were no test item-related changes in the mean fore- or hindlimb grip strength values at any dose level.
In males treated with 60 mg/kg/day, a statistically significant reduction in mean forelimb grip strength (p<0.05) was noted when compared with the controls. In the absence of similar changes at 200 mg/kg/day, this was considered to be unrelated to the test item.
- Locomotor Activity
At 200 mg/kg/day, significantly reduced locomotor activity was noted in males at measurement intervals of 0-10 minutes, 40-50 minutes and for the total measurement interval 0-60 minutes (all p<0.05). Significantly reduced locomotor activity was also seen in male treated with 60 mg/kg/day, during 20-30 minutes (p<0.05) , 40-50 minutes (p<0.01), 50-60 minutes (p<0.01) and for the overall measurement interval of 0-60 minutes (p<0.01). Males treated with 20 mg/kg/day also had intervals of significantly reduced locomotor activity: reductions were noted from 0-10 minutes (p<0.05) and 10-20 minutes (p<0.05) when compared with the controls. In contrast, females treated with 200 mg/kg/day had significantly increased locomotor activity from 0-10 minutes (p<0.05) when compared with the controls. No other differences were noted in females at any dose level.
Similar reductions of locomotor activity to those seen in the males were not reflected in the females, nor were dose-response relationships seen in the severity of the differences noted in the males. These differences were considered to be unrelated to the treatment with the test item.


Food Consumption
The mean daily food consumption of all test item-treated males very marginally exceeded that of the control males at all measurement intervals during the treatment and recovery periods. The difference was minimal and was considered to be unrelated to the test item. The relative food consumption largely reflected these minimal differences and there was no dose- response relationship.

The mean daily food consumption of the test item-treated females was generally similar to that of the control females during the treatment and recovery periods.


Body Weights
There were no test item-related differences in the mean body weights when compared with the respective controls, nor were late effects detected during the recovery period.

The mean body weight gain of the test item-treated males and females was not affected. In males treated with 200 mg/kg/day, the mean body weight gain was significantly elevated (p<0.05) on day 8 of the recovery period when compared with the controls. In the absence of changes of similar magnitude during the treatment period, this finding was considered not to be a late effect.


Clinical Laboratory Investigations
Hematology
Minor deviations of the hematology parameters seen in males treated with 200 mg/kg/day remained within the ranges of the historical control data and were considered to be unrelated to the treatment with the test item.
The mean red cell count and mean corpuscular volume were reduced (although only the latter parameter attained statistical significance (p<0.05)) when compared with the control. The significant reduction in white blood cells (p<0.05) was due almost exclusively to the reduced absolute lymphocyte count (p<0.05). The relative white blood cell populations largely reflected the pattern seen in the absolute count, although very slight increases in the mean eosinophil count and the mean basophil count attained statistical significance (p<0.05). The mean relative lymphocyte count was also reduced but the difference did not attain statistical significance when compared with the controls, nor did it exceed the historical control values.
No relevant differences in the hematology parameters were noted in the males after the recovery period.

Clinical Biochemistry
No test item-related changes in the parameters of clinical biochemistry were noted after four weeks of treatment, and no late effects were noted at the end of the recovery period. A small number of statistically significant differences to the control values were noted but in the absence of clear dose response-relationships, all were considered to be without toxicological relevance.
At 200 mg/kg/day, significantly elevated triglycerides were noted in males (p<0.01). Potassium levels were significantly elevated (p<0.01) in males but this difference was not clearly related to dose. Females at this dose level had significantly elevated cholesterol levels (p<0.05), sodium levels (p<0.01) and chloride levels (p<0.01) when compared to the controls. The latter two differences exceeded the upper range of the historical control data. The mean globulin level and the albumin/globulin ratio were significantly elevated in females (both p<0.01), but the difference in globulin was considered to be an artifact which resulted from a low control value.
After the recovery period, the mean triglyceride level in the males remained significantly higher (p<0.05) than that of the control males. In males and females, the aspartate aminotransferase activity was significantly lower (both p<0.05), and exceeded the lower limit of the historical control values in females only but is considered to be without toxicological relevance insofar as no differences were noted after the treatment period. The mean phosphorus levels were significantly lower in males (p<0.01) and mean chloride levels were significantly lower in females (p<0.05) but remained within range of their respective historical control values.
At 60 mg/kg/day, significantly elevated glucose and urea levels (both p<0.05) were noted in males, and the mean total bilirubin levels were significantly lower (p<0.05) in both genders. These differences were not seen at the highest dose level and were therefore considered to be unrelated to the test item. Alkaline phosphatase activity was increased in females and although the difference was statistically significant (p<0.01), these were largely due to a single outlier (female no 46) and was considered to be unrelated to the test item.
Potassium levels were significantly elevated (p<0.01) in males whereas females were unaffected. However, sodium and chloride levels (both p<0.05) were significantly elevated in females only but only the sodium level exceeded the upper range of the historical control data. Phosphorus was significantly reduced (p<0.05) in females and the value exceeded the lower limit of the historical control data with any relationship to dose. All differences in the electrolytes were considered to be unrelated to the treatment with the test item.
At 20 mg/kg/day, the clinical biochemistry parameters of the male and female rats were generally similar to those of the respective controls


Urinalysis
In males treated with 200 mg/kg/day for four weeks, the pH of the urine was slightly (but significantly, p<0.05) acidic when compared with the control males, and the number of leukocytes in the urine were also significantly higher (p<0.05) when compared with the control males.
The post-recovery urine parameters of the males were unremarkable.
The urinalysis parameters of females treated at all dose levels were unaffected when compared to the controls and no late effects were noted during the recovery period.


Pathology
Organ Weights
After 4 Weeks’ Treatment
After four weeks’ treatment, no differences of statistical significance were noted in the males at any dose level when compared with the control males. However, several organs of male no. 22 treated with 200 mg/kg/day showed marked differences in weight compared to the other males of that group and to the control males. Specifically, the liver was remarked macroscopically as reduced in size. When the absolute liver weight of male 22 is deleted from the calculation of the mean for that group, it increases from 9.63 g to 10.49 g, representing a 27% increase in the mean liver weight compared to the control males. When this deletion of male no. 22 is repeated for the mean liver-to-body weight and mean liver-to-brain ratios, the values increased from 3.27% to 3.59% and from 513.68% to 565.74%, respectively. Therefore, males treated with the test item at 200 mg/kg/day are considered to have elevated mean absolute and relative liver weights which are test item related.
Marginally elevated mean absolute liver weight (ca +9%) was also noted in the females treated with 200 mg/kg/day. This difference was also reflected in the liver-to-body weight ratio (p<0.01) and in the mean liver-to-brain weight ratio (not significant). The mean thyroid-to-body weight ratio was significantly elevated (p<0.05) when compared with the control females but was identical to the females treated with 20 mg/kg/day; a clear dose response relationship was not evident. All other organ weight compared favorably with those of the control females.

At 60 mg/kg/day, slightly but not significantly elevated mean absolute liver weights were noted in males when compared with controls. These differences are also reflected in the mean relative liver weights of these males and were considered to be test item-related differences.
No differences of toxicological relevance were noted in the females treated with 60 mg/kg/day.
At 20 mg/kg/day, no differences of statistical or toxicological relevance were noted in males or females.

After 2 Weeks’ Recovery
In the males sacrificed after the 2-week recovery period, the mean absolute brain weight and the mean brain-to-body weight ratio values were significantly lower (p<0.05) than those of the control males. The significantly elevated kidney-to-brain weight ratio (p<0.05) noted in the high-dose males was considered to be an artifact as the kidney-to-body weight ratio was not affected.
There were no changes in the females treated with the test item.


Macroscopic Findings
In the liver, accentuated lobular pattern in four males and reduction in size in one male were recorded at 200 mg/kg/day in main study. All other gross lesions recorded were considered to be within the range of normal background alterations.

Microscopic Findings
Kidney
In main study, increased incidence/ severity of the hyaline droplets and tubular basophilia was observed in males of at 60 mg/kg/day and 200 mg/kg/day. Minimal tubular necrosis was also observed in one male at 200 mg/kg/day.
In recovery study, increased incidence/ severity of hyaline droplets and tubular basophilia was observed in males at 200 mg/kg/day. Minimal to slight tubular necrosis was also observed in four males at 200 mg/kg/day.

Liver
Diffuse intracytoplasmic microvesicles were observed in three males at 200 mg/kg/day in main study. Although macroscopical finding “accentuated lobular pattern” and “reduced in size” were recorded in the liver, corresponding histological findings were not specified.

Other Findings
Myocardial hypertrophy in the heart was observed in one male at 200 mg/kg/day in main study. Although this change is uncommon in animals of this strain and age, similar or test-item related findings were not observed in the heart from the rest of the animals and the association with the test-item was unclear.
Tubular degeneration/atrophy in the testis was observed bilaterally in two males (no.22; mild, no.23; minimal) at 200 mg/kg/day in main study. Slight similar change was observed unilaterally in one male at 60 mg/kg/day and three males at 60 mg/kg/day. Since this change (unilateral/bilateral) can happen incidentally, and the difference in its incidence/ severity was unclear among groups, the association with the test-item was unclear.

Oligospermia and cellular debris in the epididymis and colloid reduction in the prostate gland, seminal vesicle and coagulating gland was observed in animal no.22. These findings were considered to be secondary changes related with the tubular degeneration/atrophy in the testis observed in the same animal.

As a result of thorough examination of the mesenteric lymph node of males, no significant difference was detected in its histology among groups.

The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOEL
Remarks:
EXAMPLE:
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Not applicable
Critical effects observed:
not specified

Test item-related findings were generally restricted to increased mean absolute and relative liver weights noted in both genders treated with 200 mg/kg/day and in males treated with 60 mg/kg/day. The increased liver weights were largely reversible after the recovery period, and were therefore considered to be adaptive changes related to the metabolism of the test item.

 

In the liver, accentuated lobular pattern in four males and reduction in size in one male were recorded at 200 mg/kg/day after four weeks. Although uncommon findings in animals of this strain and age, corresponding histological findings were not specified and therefore the significance of these findings was unclear.All other gross lesions recorded were considered to be within the range of normal background alterations.

 

Test-item related microscopic findings were observed in the kidney and the liver of males.

 

In the kidney, increased incidence/severity of hyaline droplets and tubular basophiliawas observed at 60 mg/kg/day and 200 mg/kg/day and tubular necrosis was observed at 200 mg/kg/day. These were considered to be test-item related and were still observed after the
2-week recovery period with an enhanced incidence/severity of the tubular basophilia and tubular necrosis.
 

 

In the liver, diffuse intracytoplasmic microvesicles (characterized by fine, vaguely-outlined vacuoles and morphologically different from fatty change, which is characterized by medium- to large-sized, well-defined vacuoles) was observed at 200 mg/kg/day after four weeks’ treatment and was considered to be test-item related. No test-item related findings were observed after the recovery period and the intracytoplasmic microvesicles regressed after recovery.

 

Although myocardial hypertrophy in the heart was observed in one male and bilateral tubular degeneration/atrophy in the testis was observed in two males at 200 mg/kg/day in main study, the association between these findings and the test-item treatment was unclear.

 

Based on the results of this study, 20 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) was established as the no-observed-effect-level (NOEL) and 200 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) as the no-observed-adverse-effect-level (NOAEL).

Conclusions:
Based on the results of this study, 20 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) was established as the no-observed-effect-level (NOEL) and 200 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) as the no-observed-adverse-effect-level (NOAEL).

Oral administration of the test material, test material, to rats for a period of up to twenty-eight consecutive days at dose levels of 1000/500, 150 and 15 mg/kg/day resulted in the premature deaths at 1000/500 mg/kg/day and minor histopathological changes at 150 mg/kg/day. No such effects were demonstrated at 15 mg/kg/day and the “No Observed Effect Level” (NOEL) was, therefore, considered to be 15 mg/kg/day.

The minor change observed at 150 mg/kg/day was not indicative of serious damage to health as defined in the EU labelling guide of Commission Directive 93/21/EEC. This dose level can therefore be regarded as a "No Observed Adverse Effect Level" (NOAEL).
EXAMPLE:
Executive summary:

General

In this subacute toxicity study, di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) was administered daily by oral gavage to-bred Wistar rats of both sexes at dose levels of 20, 60 and 200 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 200 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

 

Mortality / Viability

All animals survived until their respective days of scheduled necropsy.

Clinical Signs (Daily and Weekly)

There were no test item-related changes evident during the daily clinical observations or during the weekly behavioral observations (weeks 1 - 3).

  

Functional Observational

There were no findings at any dose level during the functional observation battery performed at week 4.

 

Grip Strength

There were no test item-related changes in the mean fore- or hindlimb grip strength values at any dose level.

 

Locomotor Activity

Similar intermittent reductions of locomotor activity to those seen in the males were not reflected in the females, nor were dose-response relationships seen in the severity of the differences noted in the males. These differences were considered to be unrelated to the treatment with the test item.

Food Consumption 

There were no test item-related differences in the mean daily food consumption of the males or females at any dose level, nor were differences noted during the recovery period.

Body Weights

There were no test item-related differences in the mean body weights when compared with the respective controls, nor were late effects detected during the recovery period. 

 

Clinical Laboratory Investigations

Hematology

Minor deviations of the hematology parameters seen in males treated with 200 mg/kg/day remained within the range of the historical control data and were considered to be unrelated to the treatment with the test item. No relevant differences in the hematology parameters were noted in the males after the recovery period.

 

Clinical Biochemistry

No test item-related changes in the parameters of clinical biochemistry were noted after four weeks of treatment, and no late effects were noted at the end of the recovery period. A small number of statistically significant differences to the control values were noted but in the absence of clear dose response-relationships, all were considered to be without toxicological relevance.

 

Urinalysis

There were no test item-related differences in the urinalysis parameters after four weeks’ treatment or after two weeks’ recovery.

 

Organ Weights

Increased mean absolute and relative liver weights noted in both genders treated with 200 mg/kg/day and in males treated with 60 mg/kg/day were considered to be related to the treatment with the test item. The increased liver weights were largely reversible after the recovery period, and were therefore considered to be adaptive changes related to the metabolism of the test item.

 

Macroscopic / Microscopic Findings

In the liver, accentuated lobular pattern in four males and reduction in size in one male were recorded macroscopically after fours weeks’ treatment with 200 mg/kg/day.Allothergross lesions recorded were considered to be within the range of normal background alterations.

Test-item related microscopic findings were observed in the kidney and the liver of males.

In the kidney, increased incidence/severity of the hyaline droplets and tubular basophiliawere observed at 60 mg/kg/day and 200 mg/kg/day and tubular necrosis was observed at 200 mg/kg/day; these were considered to be test-item related. These changes persisted after two weeks’ recovery with an enhanced incidence/severity of the tubular basophilia and tubular necrosis.

In the liver, diffuse intracytoplasmic microvesicles (characterized by fine, vaguely-outlined vacuoles and were morphologically different from fatty change which is characterized by medium- to large-sized, well-defined vacuoles) were observed at 200 mg/kg/day after four weeks’ treatment and were considered to be test-item related. No test-item related findings were observed after two weeks’ recovery and the intracytoplasmic microvesicles were considered to be reversible.

 

Conclusion

Based on the results of this study, 20 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) was established as the no-observed-effect-level (NOEL) and 200 mg/kg body weight/day of di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS #78-63-7) as the no-observed-adverse-effect-level (NOAEL).

Endpoint:
repeated dose toxicity: oral
Remarks:
other: Dose-range finding
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18-Nov-2010 to 30-Nov-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 7-Day Range-Finding Oral (Gavage) Toxicity Study was done to standard test method by a reliable GLP laboratory.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
7-Day Range-Finding Oral (Gavage) Toxicity Study in the Wistar Rat as part of OECD 407
Deviations:
not applicable
Remarks:
7-Day Range-Finding Oral (Gavage) Toxicity Study in the Wistar Rat
GLP compliance:
no
Remarks:
Not Required.
Species:
rat
Strain:
other: Rat, Ra,HanRcc:: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Animals:
Rat, Rat,HanRcc:: WIST(SPF)
Rationale:
Recognized by international guidelines as a recommended test system.
Breeder:
Hlarlan Nederland Kreuzelweg 53 5960 ad I lorsi / The Netherlands

Number of Animals:
Group 1: 3 males and 3 females
Group 2: 3 males and 3 females
Group 3: 3 males and 3 females
Group 4: 3 males and 3 females
Age (at Delivery):
Ca 7 weeks
Body Weight Range (at Acclimatization):
Males: ca. 190 g (±20%)
Females: Ca. 150 g (±20%)
Identification:
Cage card and tail mark
Randomization:
Computer-generated random algorithm.
Acclimatization:
Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour. continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There will be 12- hour fluorescent light/12-hour dark cycle with music during the light period.

Accommodation:
In groups of 3 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ Schill AG, 4132 Mutienz/Switzerland).

Diet:
Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi KIlba SA, 4303 Kaiseraugst / Switzerland) will be available Ad libitum.

Water:
Community tap-water from Itingen will be available Ad libitum in water bottles
Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Corn oil
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil provided the best quality application mixture.

- Concentration in vehicle: 200, 600 or 1000 mg/kg body weight for a period of seven days. A control group received a similar dose volume (5 mL/kg body weight) of the vehicle, corn oi.

- Amount of vehicle (if gavage): 200, 600 or 1000 mg/kg body weight for a period of seven days. A control group received a similar dose volume (5 mL/kg body weight) of the vehicle, corn oil.

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
7 days.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
200, 600 or 1000 mg/kg body weight
Basis:
other: nominal in corn oil
No. of animals per sex per dose:
Number of Animals:
Group 1: 3 males and 3 females
Group 2: 3 males and 3 females
Group 3: 3 males and 3 females
Group 4: 3 males and 3 females
Control animals:
yes, concurrent vehicle
Details on study design:
EXAMPLE:
Randomization: Computer-generated random algorithm.

Activities and Observations
The following observations were recorded:

Please see attachment "Tables and appendix 1" for full details..

Viability / Mortality: Twice daily
General Clinical Observations:

- Acclimatization Period Once

- Treatment Period Twice daily on day 1; once daily thereafter
Food Consumption:

- Acclimatization Period Day 1

- Treatment Period Days 1 to 3, 3 to 5, 5 to 7
Body Weights:
- Acclimatization Period Day 1

- Treatment Period On days 1, 3, 5 and 7

Treatment
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily
Daily Dose Levels.

Group 1:0 mg/kg/day
Group 2:200 mg/kg/day
Group 3:600 mg/kg/day
Group 4:1000 mg/kg/day

Rationale for Dose Level Selection: To evaluate appropriate doses for the subsequent 28-day study.
Dose Volume:10 ml/kg body

Dose Concentrations:

Group 1:0mg/ml/day
Group 2:20mg/ml/day
Group 3:60mg/ml/day
Group 4:100mg/ml/day

Duration of Acclimatization Period:5 days

Duration of Treatment Period:7 days
EXAMPLE:
Positive control:
Not recorded in study report.
Observations and examinations performed and frequency:
Activities and Observations
The following observations were recorded:
Viability / Mortality: Twice daily
General Clinical Observations:
- Acclimatization Period: Once
- Treatment Period: Twice daily on day 1; once daily thereafter :
Food Consumption:
- Acclimatization Period: Day 1
- Treatment Period: Days 1 to 3, 3 to 5, 5 to 7
Body Weights:
- Acclimatization Period: Day 1
- Treatment Period: On days 1, 3, 5 and 7
Sacrifice and pathology:
EXAMPLE:
Gross Pathology: Yes.
Organ Weights and ratios: Yes
Histopathology: Yes
Other examinations:
None
Statistics:
The following statistical methods will be used to analyze the body weight, organ weights and all ratios:

• The Dunnett-test (many to one t-test) based on a pooled variance estimate will be applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) will be applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.

Additional methods of statistical analysis will be used at the discretion of the statistician. The methods and the results will be described in the report.
Statistical Analysis
The following statistical methods will be used to analyze the body weight, organ weights and all ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate will be applied if the variables can be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) will be applied instead of the Dunnett-test when the data can not he assumed to follow a normal distribution.
Additional methods of statistical analysis will be used at the discretion of the statistician. The methods and the results will be described in the report.

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BWs slightly lower in males of group 3 and both sexes of group 4.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced in males; no changes in females
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
:
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality / Viability

All animals survived until scheduled necropsy.

Clinical Signs

No clinical symptoms were evident at any dose level.

Food Consumption

In males, the mean daily food consumption over the 7-day treatment period was less than the control males. The differences at recorded at 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day were -2.3%, -6.0% and -14.7%, respectively, when compared with the controls. Of these differences, only those seen at 600 mg/kg/day and 1000 mg/kg/day were considered to be possibly related to the treatment with the test item.

In females, the mean daily food consumption exceeded or compared favorably with that the control females.


Body Weights

After 7 days’ treatment with the test item, marginally lower mean body weights noted in males treated with 600 mg/kg/day and 1000 mg/kg/day, when compared with the control males, were considered to be related to the treatment with the test item. The mean body weight gain values over the 7-day treatment period were affected accordingly: +11.3 % in controls, +9.1% at 600 mg/kg/day and +7.3% at 1000 mg/kg/day, respectively.

In females, lower mean body weights were seen at 200 mg/kg/day and at 1000 mg/kg/day. The former difference was unrelated to dose (females treated with 600 mg/kg/day had higher body weights than those treated with 200 mg/kg/day) and were therefore considered to be related to the lower initial mean body weights. The mean body weight gain values largely reflected these differences: When compared with control females (+6.8%), the mean body weight gain values in females treated with 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day were +5.8%, +6.1% and +1.9%, respectively. The latter difference was considered to be related to the treatment with the test item.

Organ Weights

Test item-related changes in mean absolute and relative organ weights were noted in the liver and thymus.

In males treated with 1000 mg/kg/day, the mean absolute and mean liver-to-body weight liver weights were elevated by +29.5% and +34.2%, respectively, when compared with the controls. In females, these parameters were increased by +67.2% and +65.6%, respectively, when compared with the control females. In the thymus, contrary differences to the respective control weights were noted between genders: the mean absolute thymus weights and mean thymus-to-body weights were decreased in males (-22.7% and -20.5%, respectively) and increased in females (+16.9% and +15.2%, respectively).

At 600 mg/kg/day, males also had markedly increased mean absolute liver and liver-to-body weights which were +18% and +21.7% higher, respectively, than those of the control males. In females, these differences to the control values were +42.3% and +39.1% higher, respectively, than those of the controls. In males, the mean absolute thymus weights were reduced (-13%) and the mean thymus-to-body weight was reduced (-11.1%) when compared with the control value.

At 200 mg/kg/day, the mean absolute liver weights and liver-to-body weights were higher (+7.1% and +6.6%, respectively) than the control males. The differences to the control values were more clearly defined in females: the mean absolute liver weights and the mean liver-to-body weights were elevated by +14.6% and +16.3%, respectively.

Macroscopic Findings

No test item-related macroscopical changes were noted at any dose level.

At 200 mg/kg/day, unilateral renal pelvis dilation was noted in one male (no. 4).

At 600 mg/kg/day, there were no macroscopical findings noted in any animal.

At 1000 mg/kg/day, the left adrenal gland of one male (no. 10) was found to be slightly reduced in size, which corresponded with the slightly lower combined adrenal weight.

Microscopic Findings

Hepatocellular hypertrophy was recorded in all animals treated with 1000 mg/kg/day as treatment-related finding. Hypertrophic alteration was seen mainly in centrilobular region in all males and one females (expressed as grade 1, minimal severity), and seen in two females more diffusely (expressed as grade 2; slight severity).

Hepatocellular hypertrophy was recorded in all animals of the high dose group. Hypertrophic alteration was seen mainly in centrilobular region in all males and one female, and seen in two females more diffusely. Hepatocellular hypertrophy correlated with significantly increased absolute and relative organ weights of the liver. There were no further indicators of liver injury, and hence, this lesion was considered to be of metabolic nature and of adaptive character typical of enzyme induction.


Dose descriptor:
NOEL
Remarks:
EXAMPLE:
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
EXAMPLE:
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Mortality / Viability

All animals survived until scheduled necropsy.

 

Clinical Signs

 

No clinical symptoms were evident at any dose level.

Food Consumption

 

In males, the mean daily food consumption over the 7-day treatment period was less than the control males. The differences at recorded at 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day were -2.3%, -6.0% and -14.7%, respectively, when compared with the controls. Of these differences, only those seen at 600 mg/kg/day and 1000 mg/kg/day were considered to be possibly related to the treatment with the test item.

 

In females, the mean daily food consumption exceeded or compared favorably with that the control females.

 

 

Body Weights

 

After 7 days’ treatment with the test item, marginally lower mean body weights noted in males treated with 600 mg/kg/day and 1000 mg/kg/day, when compared with the control males, were considered to be related to the treatment with the test item. The mean body weight gain values over the 7-day treatment period were affected accordingly: +11.3 % in controls, +9.1% at 600 mg/kg/day and +7.3% at 1000 mg/kg/day, respectively.

 

In females, lower mean body weights were seen at 200 mg/kg/day and at 1000 mg/kg/day. The former difference was unrelated to dose (females treated with 600 mg/kg/day had higher body weights than those treated with 200 mg/kg/day) and were therefore considered to be related to the lower initial mean body weights. The mean body weight gain values largely reflected these differences: When compared with control females (+6.8%), the mean body weight gain values in females treated with 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day were +5.8%, +6.1% and +1.9%, respectively. The latter difference was considered to be related to the treatment with the test item.

 

 

Organ Weights

 

Test item-related changes in mean absolute and relative organ weights were noted in the liver and thymus.

 

In males treated with 1000 mg/kg/day, the mean absolute and mean liver-to-body weight liver weights were elevated by +29.5% and +34.2%, respectively, when compared with the controls. In females, these parameters were increased by +67.2% and +65.6%, respectively, when compared with the control females. In the thymus, contrary differences to the respective control weights were noted between genders: the mean absolute thymus weights and mean thymus-to-body weights were decreased in males (-22.7% and -20.5%, respectively) and increased in females (+16.9% and +15.2%, respectively).

 

At 600 mg/kg/day, males also had markedly increased mean absolute liver and liver-to-body weights which were +18% and +21.7% higher, respectively, than those of the control males. In females, these differences to the control values were +42.3% and +39.1% higher, respectively, than those of the controls. In males, the mean absolute thymus weights were reduced (-13%) and the mean thymus-to-body weight was reduced (-11.1%) when compared with the control value.

 

At 200 mg/kg/day, the mean absolute liver weights and liver-to-body weights were higher (+7.1% and +6.6%, respectively) than the control males. The differences to the control values were more clearly defined in females: the mean absolute liver weights and the mean liver-to-body weights were elevated by +14.6% and +16.3%, respectively.

 

 

Macroscopic Findings

 

No test item-related macroscopical changes were noted at any dose level.

 

At 200 mg/kg/day, unilateral renal pelvis dilation was noted in one male (no. 4).

 

At 600 mg/kg/day, there were no macroscopical findings noted in any animal.

 

At 1000 mg/kg/day, the left adrenal gland of one male (no. 10) was found to be slightly reduced in size, which corresponded with the slightly lower combined adrenal weight.

 

 

Microscopic Findings

Hepatocellular hypertrophy was recorded in all animals treated with 1000 mg/kg/day as treatment-related finding.Hypertrophic alteration was seen mainly in centrilobular region in all males and one females (expressed as grade 1, minimal severity), and seen in two females more diffusely (expressed as grade 2; slight severity).

 

Table1       Incidence and Mean Severity Grade of Main Findings inLiver

Finding
Incidence /
Mean Severity
Grade

Group 1

Group 2

Group 3

Group 4

Liver

3M

3F

3M

3F

3M

3F

3M

3F

Hepatocellular hypertrophy
Incidence/Mean Severity

0

0

NE

NE

NE

NE

3/1.0

3/1.7

NE, Not examined.

 

Hepatocellular hypertrophy was recorded in all animals of the high dose group. Hypertrophic alteration was seen mainly in centrilobular region in all males and one female, and seen in two females more diffusely. Hepatocellular hypertrophycorrelated with significantly increased absolute and relative organ weights of the liver. There were no further indicators of liver injury, and hence, this lesion was considered to be of metabolic nature and of adaptive character typical ofenzymeinduction.

 

 

Conclusions:
Based on the results of this seven-day dose range-finding study, test item-related reductions in mean food consumption were noted in males treated with 600 mg/kg/day and 1000 mg/kg/day (but not in females), reduced body weight gain in males at 600 mg/kg/day and in males and females at 1000 mg/kg/day.

Increased mean absolute and relative liver weights were noted at 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day in males and females. Although such changes are often primarily due to metabolic adaptation to the treatment with the test item and are tolerable for short periods, longer periods of administration at such dose levels may lead to impaired organ function and secondary effects. The organ weight changes in the thymus (decreased in males at 600 mg/kg/day and 1000 mg/kg/day and increased in females at 1000 mg/kg/day) were considered to be secondary stress-related reactions. Microscopically, hepatocellular hypertrophy was recorded in all animals treated with 1000 mg/kg/day. Hypertrophic alteration was seen mainly in centrilobular region in all males and one female, and more diffusely in two females. Hepatocellular hypertrophy correlated with significantly increased absolute and relative organ weights of the liver. There were no further indicators of liver injury, and hence, this lesion was considered to be of metabolic nature and of adaptive character typical of enzyme induction.

Therefore, dose levels of 20, 60 and 200 mg/kg body weight/day were selected for the subsequent 28-day study (Harlan Laboratories Study D10344) with di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS 78-63-7), with the expectation that hepatic hypertrophy is likely to be seen after 28 days’ repeated administration at 200 mg/kg/day.
Executive summary:

The purpose of this oral toxicity range-finding study will be to assess the toxicological protile of the test item when administered daily to rats by oral gavage for a period of 7 days. Based on the results of this seven-day dose range-finding study, test item-related reductions in mean food consumption were noted in males treated with 600 mg/kg/day and 1000 mg/kg/day (but not in females), reduced body weight gain in males at 600 mg/kg/day and in males and females at 1000 mg/kg/day.

Increased mean absolute and relative liver weights were noted at 200 mg/kg/day, 600 mg/kg/day and 1000 mg/kg/day in males and females. Although such changes are often primarily due to metabolic adaptation to the treatment with the test item and are tolerable for short periods, longer periods of administration at such dose levels may lead to impaired organ function and secondary effects. The organ weight changes in the thymus (decreased in males at 600 mg/kg/day and 1000 mg/kg/day and increased in females at 1000 mg/kg/day) were considered to be secondary stress-related reactions. Microscopically, hepatocellular hypertrophy was recorded in all animals treated with 1000 mg/kg/day. Hypertrophic alteration was seen mainly in centrilobular region in all males and one female, and more diffusely in two females. Hepatocellular hypertrophy correlated with significantly increased absolute and relative organ weights of the liver. There were no further indicators of liver injury, and hence, this lesion was considered to be of metabolic nature and of adaptive character typical of enzyme induction. Therefore, dose levels of 20, 60 and 200 mg/kg body weight/day were selected for the subsequent 28-day study (Harlan Laboratories Study D10344) with di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (CAS 78-63-7), with the expectation that hepatic hypertrophy is likely to be seen after 28 days’ repeated administration at 200 mg/kg/day.

Endpoint:
chronic toxicity: oral
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Apparently well conducted GLP study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
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

The oral administration of Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide, CAS# 78-63-7 to rats by gavage, at dose levels of 15, 50 and 150 mg/kg bw/day, did not result in any adverse effects of treatment.

 

Clinical observations were confined to increased salivation observed for both sexes at 50 and 150 mg/kg bw/day. At 150 mg/kg bw/day all animals were affected but at 50 mg/kg bw/day the overall number of animals and incidence tended to be lower. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be related to systemic toxicity. There were considered to be no effects of treatment on behavioral or functional assessments, body weight, food consumption or food conversion efficiency for either sex throughout the study. Hematological and blood chemistry investigations at 150 mg/kg bw/day did not reveal any adverse effect of treatment for either sex.

 

For both sexes at 150 mg/kg bw/day, there was an increase in absolute and body weight relative organ weights for the liver, suggestive of an adaptive change, although this was not accompanied by any evidence of microscopic change and was considered not to represent an adverse effect of treatment. In the thyroid glands for both sexes there was a greater incidence of minimal or mild diffuse hypertrophy of the follicular epithelium than in controls at all dosage levels. The underlying mechanism for this treatment-related change was not apparent but the most likely explanation for its development is a perturbation of thyroid hormone synthesis, transport or metabolism. Since the rat thyroid gland has been shown to be markedly more sensitive than humans in its response to xenobiotics it seems reasonable to conclude that the minor difference seen in this study would not carry significant risk to humans.

 

Microscopic examination of kidney sections revealed a dosage-related increase of intra-epithelial hyaline droplets in males that affected all the treated groups. In males given 150 mg/kg bw/day the presence of an increase in hyaline droplets was associated with granular cast formation, an increase in basophilic tubules and single cell necrosis of cortical tubules. These findings were consistent with an etiological diagnosis of alpha-2µ-microglobulin nephropathy using immunohistochemistry, in which there was a clear increase in staining intensity at the highest dosage. It was, therefore, concluded that all the treatment-related renal changes were a consequence of alpha-2µ-microglobulin nephropathy and, since this condition is species specific, of no relevance to human exposure. The increased levels of hyaline droplets and the other changes observed in males at the highest dosage were considered responsible for the increased absolute and body weight-relative kidney weights observed for these animals.

 

Overall it was considered that the No Observed Adverse Effect Level (NOAEL) for this study was 150 mg/kg bw/day.

Justification for classification or non-classification

The 90 -day NOAEL is 150 mg/kg bw/day, the highest dose tested. The increase in liver weight was not considered adverse and the effect on the kidney, observed in the males, is species specific. These types of effects and this dose level do not warrant classification for STOT-RE.