Registration Dossier

Administrative data

Description of key information

The oral administration of Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide (read-across substance), CAS 78-63-7 to rats by gavage, for 90 days, at dose levels of 15, 50 and 150 mg/kgbw/day, did not result in any toxicologically significant adverse effects. The ‘No Observed Adverse Effect Level’ (NOAEL) was, therefore, considered to be 150 mg/kgbw/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:
from 17th July 2013 to 2nd June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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 223 g, the females weighed 137 to 170 g, 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
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
15 mg/kg bw/day (3.75 mg/mL)
Basis:
actual ingested
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:
50 mg/kg bw/day (12.5 mg/mL)
Basis:
actual ingested
Dose / conc.:
150 mg/kg bw/day (actual dose received)
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.

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:
effects observed, treatment-related
Description (incidence and severity):
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.

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.
Mortality:
no mortality observed
Description (incidence):
There were no deaths in the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect in overall food consumption or food efficiency was detected in treated animals when compared to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual assessment of water intake did not indicate any effect of treatment at 15, 50 or 150 mg/kg bw/day.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination during the last week of treatment did not indicate any effect of treatment for either sex at 150 mg/kg bw/day.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 findings:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected in the urinalytical parameters measured.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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.

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.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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.
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.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Details on results:
Estrous Cycle Assessment
There were no treatment-related effects on female estrous cycles at dosages up to 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.

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.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
no

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.

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:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 17th July 2013 to 2nd June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to the attached read-across justification.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
no
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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key study (OECD 408, read-across)

A repeated-dose study via oral route is not available. Consequently, read-across was applied using study results obtained from Di-Tert-Butyl 1,1,4,4-Tetramethyl Tetramethylene Diperoxide (CAS 78 -63 -7), a structure analogue of the substance to be registered.

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.
Bases on the data available, the test substance was not classified and labelled according to Regulation (EC) No 1272/2008 (CLP).