Registration Dossier

Administrative data

Description of key information

A 90-day repeat dose study is available. The registered substance was administered to rats at 30, 150 and 450 mg/kg/day by gavage. Followed by a 28 day treatment free period in sub-groups of animals previously dosed at 450 mg/kg bw/day.

 

The NOEL from this study is 30 mg/kg bw/day. There were no effects of treatment for females that received 30 mg/kg bw/day (low dose group) and the findings observed for males at this dosage appeared to be associated with hydrocarbon nephropathy associated with α2-microglobulin. This condition, while adverse to the male rat, is deemed not to be a hazard to human health as this is principally a rodent only phenomenon, as described above.

 

Also in this study local effects on the stomach were seen at 150 and 450 mg/kg bw/day, as written abovethis finding may have limited relevance in the assessment of human health.

 

At 150 mg/kg bw/day, adaptive effects in the liver were seen and nephropathy as described above in males. Further, statistically significant reductions for hemoglobin, red blood cell count (RBC),

haematocrit and mean cell hemoglobin concentration were apparent for males but all values were within the historical control range. Females at this dosage showed statistically significant increase for mean cell volume and mean cell hemoglobin and also decreased higher mean cell hemoglobin concentration (MCHC) but the majority of individual values were within the historical control range. These findings were similar to anemia present for both sexes at 450 mg/kg bw/day but, at this lower dosage, there was no clear effect of spleen histopathology. In the absence of such effect, and with individual erythrocyte values being generally within the normal range, these findings were considered not to represent an adverse effect at this dosage.

 

At 450 mg/kg bw/day several significant treatment findings were apparent for both sexes during the study. Males showed lower body weight gain and marginally lower food intake. They also had a slightly lower food conversion. The overall activity in males was lower. In both sexesstatistically significant reductions for hemoglobin, red blood cell count (RBC) and hematocrit and increases in mean cell volume (MCV), mean cell hemoglobin (MCH) and a reduction in mean cell hemoglobin concentration (MCHC), significant increase in circulating neutrophils. In females higher platelets counts attained statistical significance at this dose level. Males showed slight but statistically significant increase in blood urea levels, increase in plasma glucose levels, protein and significant increase in inorganic phosphorus and lower sodium levels.

Both sexes statistically significant increases in absolute and body weight relative liver weights and centrilobular hypertrophy was seen. A significant increase in absolute and relative adrenal weights were identified in both sexes accompanied by zona fasciculata hypertrophy. Significant increases in absolute and relative spleen weights were identified in both sexes with increased hematopoiesis, increased hemosiderin. One male showed an enlarged spleen and one female showed discoloration of the spleen. In both sexes statistically significant increases in absolute and body weight relative kidney weights were observed. In males this was seen together with hyaline droplets with associated pathological changes). In the thymus atrophy was present males and females. Hypertrophy of the follicular cells in the thyroid was present in 5 of 10 females.

Although some of these findings were clearly adaptive in nature or of little relevance to man, the extent of findings for both sexes was considered to preclude this dosage from being considered as a NOAEL for the purposes of human risk assessment.

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: 06 October 2016 Experimental Completion Date: 04 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification : 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5)
Physical State/Appearance : Clear colorless liquid
Purity : 92.5%
Batch Number : 1504510829
Date Received : 21 June 2016
Storage Conditions : Approximately 4°C, in dark; used/formulated under
ambient conditions and light
Expiry Date : 01 June 2018
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to the appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 194 to 229g, the females weighed 143 to 188g, and were approximately six to eight weeks old.

Animal Care and Husbandry
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, Envigo RMS (UK) Limited., Oxon, UK) was used. 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 Envigo Research Limited, 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.

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
Details on route of administration:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
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 Arachis oil BP. Recovery group animals were maintained for a further twenty-eight days following termination of treatment.
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentrations in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepared working standard solutions in dilution solvent with a concentration of 0.1 mg/mL. The standard solutions contained the equivalent of vehicle to that of the relevant samples.

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

Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum.

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

Preparation of Accuracy and Precision Samples
The concentration of Test Item in the final solution was quantified by GC using FID detection

Instrumentation Parameters
GC System: Agilent Technologies 6890, incorporating autosampler and workstations
Column: ZB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature programme: Oven - 260°C for 5 minutes with 10°C/minute to 100°C
Injection temperature: 250°C
Flame ionisation detector temperature: 250°C
Injection volume: 1 µL
Retention timeL ~4.1 minutes

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms was measured.
Duration of treatment / exposure:
90 days treatment, followed by a 28-day recovery period for the highest dose group
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males, 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen following review of available toxicity data. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Positive control:
None
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
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. During the treatment-free period, animals were observed daily. All observations were recorded.

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.

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

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery 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 non-recovery 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. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. Motor activity was also conducted in recovery animals as a precautionary measure only; this data will be maintained with the study records but has not been included in the study report. 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 non-recovery 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

Ophthalmoscopic Examination
The eyes of all animals were examined pre-treatment and those of the non-recovery and high dose animals 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.

Estrous Cycle Assessment
Vaginal smears were taken daily for 21 days, on all non-recovery test and control group females, during the final three weeks of treatment. The stage of estrous was recorded for each day.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the treatment period (Day 90) and on all surviving recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 or 119. Animals were not fasted prior to sampling.

Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during Week 16. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
The methods used for hematological and blood chemical and urinalysis are given in Annex 6 and normal ranges are shown in Annex 7.

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
Sacrifice and pathology:
Terminal Investigations
Necropsy
On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all surviving 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.

Sperm Analysis
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.

For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.

For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. In the absence of any associated treatment-related finding in sperm concentration, motility or morphology no further investigations was performed on the testicular tissue.

Morphological assessment was performed on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

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
Cecum
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 gland
Uterus (with cervix)
Muscle (skeletal)
Vagina

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing. All tissues from non-recovery control and 450 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, examination was subsequently extended to include similarly prepared sections of adrenal glands (male), kidneys (male; including immuno-histochemical staining of the kidneys for α2-microglobulin), thyroid glands (female), duodenum (females), liver (male and female), spleen (male and female), stomach (male and female) and thymus (male and female) from animals in the low, intermediate and recovery dose groups.

Pathology
Microscopic examination was conducted by the Study Pathologist. A histopathology peer review was conducted by the Responsible Scientist at the Test Site (Envigo CRS Limited, Huntingdon).


Statistics:
Please see "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Surviving high dose (450 mg/kg bw/day) animals showed a high incidence of post-dose increased salivation over the course of the treatment period, being first apparent for males from Day 1 and females from Day 12. Similar post-dose increased salivation was also present, to a lesser extent, for either sex treated at 150 mg/kg bw/day. Such increased post-dosing salivation is frequently observed when administering test items via the oral gavage route and is considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment. Four control males also showed post-dose increased salivation but this was on one occasion only and was considered most likely to reflect difficulty in dosing these particular animals on that isolated occasion.

At 450 mg/kg bw/day, six males and two females showed sporadic episodes of body tremors, generally during the later stages of the study. This observation was usually observed post-dosing, although on occasions it persisted through to dosing the following day. Additionally hunched posture was also observed for six females (including one female during behavioural assessment arena observations) at this dosage. Isolated incidents of this clinical sign were observed for two animals early in the study (Day 20 and 28 respectively), for the remainder of the animals this clinical sign was apparent between Days 70 to 79 of the study.

Two males showed isolated instances of noisy respiration (as did one control male) but this probably reflected the increased salivation apparent at this dosage, which may have impacted upon respiration. Generalized fur loss was also observed in one male during the latter days of dosing but, in isolation, was considered to be incidental and of no toxicological significance.

No clinical signs were apparent for either sex receiving 30 mg/kg bw/day or for control females. Additionally, no clinical signs were apparent for animals previously treated at 450 mg/kg bw/day during the treatment-free recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Male No. 105, receiving 450 mg/kg bw/day was killed in extremis on Day 76 after showing increased salivation, pilo-erection, respiratory distress (gasping, laboured and decreased respiration), prostration, and lethargy. Macroscopic necropsy examination revealed a small pus and fibrous filled mass under the right forelimb and fluid in the thoracic cavity; collectively these findings indicate that the decline in the animal’s clinical condition was due to accidental trauma during the dosing procedure. Other necropsy findings included red contents in the urinary bladder, yellow stomach contents and raised limiting ridge and epithelial sloughing of the non-glandular region of the stomach.

Male No. 90 (Control) was killed in extremis on Day 80 after showing a slight nasal discharge, respiratory distress (noisy, gasping and decreased respiration), staining around the mouth/snout, hunched posture and lethargy. Macroscopic necropsy examination revealed a hole in the right axilla region of the thoracic cavity leading to a fluid filled mass. These findings indicate that the decline in the animal’s clinical condition was due to accidental trauma during the dosing procedure. Necropsy also revealed a mottled appearance to the liver.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the first week of dosing, males treated with 450 mg/kg bw/day showed a statistically significant reduction in body weight gain in relation to controls. Although subsequent improvement was evident, sporadic instances of slightly lower weekly body weight gains in these males were also noted during the remainder of the treatment period, resulting in an overall lower body weight gain than the controls at the end of dosing period.

There was no effect of treatment on bodyweight gain for both sexes at 30 or 150 mg/kg bw/day or females at 450 mg/kg bw/day. Occasional differences in body weight gain attained statistical significance when compared with control but, in the absence of any effect on overall body weight gain, these were considered to reflect normal biological variation and were unrelated to treatment.

During the treatment-free period, males previously receiving 450 mg/kg bw/day showed a clear body weight recovery such that overall body weight gain at the end of the treatment free period was comparable with controls. Body weight fluctuations were evident in females previously treated with 450 mg/kg bw/day, however females at this stage of the study are no longer on a growth curve and the differences from control observed were considered to reflect normal biological variation, rather than some underlying effect of prior exposure to the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At commencement of treatment, either sex treated with 450 mg/kg bw/day and females receiving 150 mg/kg bw/day showed marginally lower dietary intake than controls. Subsequent improvement was evident although sporadic instances of marginally lower food intake persisted in the 450 mg/kg bw/day males during the remainder of the treatment period.

There was no effect of treatment on food intake for either sex treated with 30 mg/kg bw/day or for males receiving 150 mg/kg bw/day.

During the treatment-free period, recovery 450 mg/kg bw/day males were seen to have a marginally higher group mean food consumption value in relation to that of the controls. This finding was consistent with the recovery of body weight gain observed for these animals during the recovery period. There was no effect on food intake for females previously treated with 450 mg/kg bw/day
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food conversion efficiency for males receiving 450 mg/kg bw/day was slightly lower than control during the first week of treatment. Thereafter, there were no consistent intergroup differences in food conversion efficiency that indicated any effect of treatment for either sex at 30, 150 and 450 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no overt intergroup differences.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Findings observed during ophthalmoscopic examination of the eyes of animals receiving 450 mg/kg bw/day during Week 12 of the study did not indicate any effect of treatment.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological determinations performed for either sex at 450 mg/kg bw/day at the end of the treatment period highlighted statistically significant reductions for hemoglobin, red blood cell count (RBC) and hematocrit compared to control. These were accompanied in both sexes by increases in mean cell volume (MCV), mean cell hemoglobin (MCH) and a reduction in mean cell hemoglobin concentration (MCHC) in relation to controls. For mean cell volume, all individual values for males and 7/10 individual values for females exceeded the historical control range; one individual control value also exceeded the historical control range. For red blood cell count, 3/10 individual values for males and 2/10 individual values for females were below the respective historical control range, however for the control group one individual male value exceeded and 2/10 individual female values were below the respective historical control range. Additionally for mean cell hemoglobin, 2/10 individual values for treated females were below the historical control range.

Similar statistically significant reductions for hemoglobin, red blood cell count (RBC) and hematocrit were observed for males treated at 150 mg/kg bw/day and these were accompanied by a statistically significant reduction in mean cell hemoglobin concentration, compared to control. For all parameters, the individual values for these treated males were all within the historical control range. Females from this test group showed statistically significant higher mean cell volume (MCV) and mean cell hemoglobin (MCH) and lower mean cell hemoglobin concentration (MCHC) in comparison to control. For mean cell volume, 3/10 individual values for these females exceeded the historical control range compared to one in the control group.

For recovery males previously treated at 450 mg/kg bw/day, red blood cell count at the end of treatment free period was statistically significantly lower than control, but all individual values for these treated males were within the historical control range. Hematocrit was statistically significantly higher than control, with four individual values for treated males exceeding the historical control range compared to only one for the control group. This increase in hematocrit was in contrast with the lower mean value, compared with control, observed during the treatment period, and probably indicates slight over compensation during recovery from the initial effects during treatment. Higher mean cell volume and mean cell hemoglobin remained statistically significant when compared to control; for these treated animals, 6/9 individual values for mean cell volume and 4/9 individual values for mean cell hemoglobin exceeded the historical control range. Lower mean cell hemoglobin concentration also attained statistical significance when compared with control, with 7/9 individual values for these treated males being lower than the historical control range compared to only 2/9 for the control group. For recovery females, there were no statistically significant differences from control for hematology parameters at the end of the treatment free period.

A statistically significant increase in circulating neutrophils was identified in either sex treated at 450 mg/kg bw/day and in females treated at 150 mg/kg bw/day, in comparison with controls. For males at 450 mg/kg bw/day, 9/10 individual values exceeded the historical control range compared to only one control value. For females at 150 and 450 mg/kg bw/day, whilst 8/10 and 9/10 individual values respectively exceeded the historical control so did 5/10 individual control values, therefore any association with treatment and this finding for females is considered to be more equivocal.
Males treated at 450 mg/kg bw/day also showed a statistically significant increase in plasma eosinophil levels. However, all individual values for these treated animals were within the historical control range whilst one control individual control value exceeded this historical range; this finding was therefore considered to be incidental and unrelated to treatment.

For males, a statistically significant decrease in activated partial thromboplastin time, compared to control, was detected in all dosages at the end of the treatment period, although no dosage relationship was apparent at 30 and 150 mg/kg bw/day. All individual values for treated males were within the historical control range and, whilst there may have been some influence of altered liver metabolism, as indicated by centrilobular hypertrophy of the hepatocytes, this finding was considered unlikely to be related to treatment and to be of no toxicological significance. For females, prothrombin times at all dosages were lower than control but mean values showed no dosage relationship. All individual values for treated animals were within the historical control range whilst two individual control values exceeded this historical range and this finding was considered to be incidental and unrelated to treatment.

For females at 450 mg/kg bw/day, higher platelets counts attained statistical significance when compared with control, with all individual values for these treated animals exceeded the background data range. Platelet count was also higher than control for males at this dosage but differences did not attain statistical significance. For treated males, 7/10 individual values exceeded the historical control range compared to only 2/10 for the control group. This finding may, at least in part, have been influenced by the anemia among non-recovery rats at this dosage. For males previously treated at 450 mg/kg bw/day, lower platelet count attained statistical significance at the end of the treatment free period, when compared with concurrent control. Only one individual value for the treated males was below the historical control range, and this finding was considered incidental and unrelated to previous exposure to the test item. There were no statistically significant differences from control for platelet count for females previously treated at 450 mg/kg bw/day at the end of the treatment free period.

There were no noteworthy findings in the hematological parameters examined in animals of either sex treated at 30 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, males treated with 450 mg/kg bw/day exhibited a slight but statistically significant increase in blood urea levels in relation to controls, with 9/10 individual values for these treated animals exceeding the historical control range compared to 3/10 for the control group. This may reflect altered liver metabolism, as indicated by centrilobular hypertrophy of the hepatocytes or a change to renal efficiency due to pathological kidney changes due to hyaline droplets.

For females receiving 150 mg/kg bw/day, blood urea levels were also statistically significantly higher than control, with 7/10 individual values for these treated females exceeding the historical control range compared to only 3/10 for the control group. However in the absence of any similar increase for females receiving 450 mg/kg bw/day, this finding was considered to be incidental and of no toxicological significance.

Males treated with 450 mg/kg bw/day also exhibited a statistically significant increase in plasma glucose levels, compared to control, at the end of the dosing period, with 8/10 individual values exceeding the historical control range. This finding may reflect altered liver metabolism, as indicated by centrilobular hypertrophy of the hepatocytes.

For males at 450 mg/kg bw/day, a slight but statistically significant decrease in total protein together with an increase in the albumin/globulin ratio was also evident at the end of the dosing period, compared to control. For these treated males, 4/10 individual values for albumin/globulin ratio exceeded the historical control range, but all individual values for total protein were within the respective historical control range. Whilst there may have been some influence of altered liver metabolism, as indicated by centrilobular hypertrophy of the hepatocytes, this finding at the level observed was considered to be no toxicological significance.

For males at 450 mg/kg bw/day, a statistically significant increase in inorganic phosphorus was apparent at the end of the treatment period. All individual values for these treated animals exceeded the historical control range compared to only one individual control value. This finding may reflect a change to renal efficiency due to pathological kidney changes due to hyaline droplets for animals at this dosage.

For males at 150 or 450 mg/kg bw/day, lower mean sodium levels also attained statistical significance when compared to control. At 150 mg/kg bw/day, all individual values were with the historical control and this finding was considered most likely to be incidental and unrelated to treatment. However, at 450 mg/kg bw/day, 3/10 values were below the historical control range and this finding may reflect a change to renal efficiency due to pathological kidney changes due to hyaline droplets for animals at this dosage.

For males at 450 mg/kg bw/day, there was a statistically significant decrease in mean cholesterol compared to control, but all individual values for these treated males were within the historical control range. Whilst there may have been some influence of altered liver metabolism, as indicated by centrilobular hypertrophy of the hepatocytes, this finding at the level observed was considered to be of no toxicological significance.

For recovery males previously given 450 mg/kg bw/day, there was a statistically significant increase in mean aspartate aminotransferase activity compared to control at the end of the treatment free period, with 6/9 individual values for treated males exceeding the historical control. No similar effect was apparent for males during the treatment period, therefore this finding, in isolation, was considered unlikely to be of any toxicological significance.

For recovery females previously given 450 mg/kg bw/day, there was a statistically significant increase blood urea levels at the end of the treatment free period, compared to control, with 7/10 individual values for the treated females exceeding the historical control, compared to only one in the control group. No similar effect on blood urea was apparent for females at this dosage during the treatment period, therefore this finding, in isolation, was considered unlikely to be of any toxicological significance. Additionally for recovery females, there was a statistically significant decrease in mean alkaline phosphatase activity compared to control at the end of the treatment free period, but all individual values for the treated animals were within the historical control range. As this reduced enzyme level was seen with no obvious pathological liver pathology this finding was considered to be incidental and unrelated to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related adverse effects identified at any dose level in animals of either sex on urinalysis parameters that were evaluated towards the end of the treatment or treatment-free periods.

At 450 mg/kg bw/day, collected urine volume was statistically significantly higher than control at the end of the treatment period, although there was no corresponding effect on specific gravity. Whilst there may have been some influence of pathological kidney changes due to hyaline droplets, this finding, which was not associated with any obvious increase in water consumption, probably reflected normal biological variation and, at the level observed was considered not to be adverse. Furthermore, no statistically significant differences in urine volume were apparent for males at the end of the four week treatment-free recovery period
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Behavioral Assessments
At 450 mg/kg bw/day, hunched posture and pilo-erection was observed for one female during the Week 4 observation and a further female during the Week 5 observations, with hunched posture also being observed for this latter animal during the Week 6 and 7 evaluations. Similar hunched posture was observed during routine post-dosing assessments of clinical signs at this dosage.

There were no clinical signs apparent during behavioural assessments for either sex at 30 or 150 mg/kg bw/day or for males at 450 mg/kg bw/day.

Functional Performance Tests
Motor activity assessment during Week 12 revealed a statistically significant reduction in overall activity in males treated with 450 mg/kg bw/day in relation to controls, although activity during the final 20% of the observation period was similar to control.

There were no statistically significant differences from control in motor activity of either sex at 30 or 150 mg/kg bw/day or for females at 450 mg/kg bw/day.

During Week 12 of the treatment period, males from the 450 mg/kg bw/day dose group showed a statistically significant increase in hind limb strength during Test 2 relative to controls (p<0.05) with females from this dose group showing a statistically significant increase in forelimb strength (p<0.05) during Test 3. These differences were evident in only one of three tests and as such were considered most likely to be due to biological variation.

Sensory Reactivity Assessments
All scores during sensory reactivity assessments were the same for treated and control animals of either sex.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, statistically significant increases in absolute and body weight relative liver weights were identified in both sexes that received test item at 450 mg/kg bw/day in comparison with controls. Increased liver weights were also identified in the males treated at 150 mg/kg bw/day. For this organ, body weight relative values are generally considered to be the best indicator of toxicological effect and all individual body weight relative values for both sexes at 450 mg/kg bw/day and 8/10 individual body weight relative values for males receiving 150 mg/kg bw/day exceeded the historical control range. However, it should be noted the historical control range was also exceeded by 7/10 control males and 7/10 control females at the end of the treatment period. These increases in liver weights were supported by histopathological change (centrilobular hypertrophy) observed for the liver at the end of the treatment period. Following the four week recovery period at 450 mg/kg bw/day, statistically significant increased liver weight were still apparent for females and was again supported by evidence of histopathological change for this sex, although at a lower incidence.

At the end of the treatment period statistically significant increases in absolute and relative adrenal weights were identified in both sexes that received test item at 450 mg/kg bw/day in comparison with controls. For these treated males, 9/10 individual absolute values and all individual body weight relative values exceeded the historical control range, however so did 4/10 absolute and 8/10 body weight relative control values. For the treated females, 7/10 individual absolute values and 9/10 individual body weight relative values exceeded the historical control range, however this historical range was also exceeded by 5/10 absolute and 5/10 body weight relative control values. The increase in adrenal weights for males was supported by evidence of histopathological change (zona fasciculata hypertrophy). Following the four week recovery period at 450 mg/kg bw/day, there were no statistically significant differences from control for absolute and body weight relative adrenal weights.

At the end of the treatment period statistically significant increases in absolute and relative spleen weights were identified in both sexes that received test item at 450 mg/kg bw/day in comparison with controls. For these treated males, 8/10 individual absolute values and all individual body weight relative values exceeded the historical control range, compared to only 2 /10 absolute and 2/10 body weight relative control values. For the treated females, all individual absolute and body weight relative values exceeded the historical control range, compared to 1 /10 absolute and 1/10 body weight relative control values. The increase in spleen weights for males was supported by evidence of histopathological change (increased hematopoiesis, increased hemosiderin). Following the four week recovery period at 450 mg/kg bw/day, there were no statistically significant differences from control for absolute and body weight relative spleen weights.

At the end of the treatment period, statistically significant increases in absolute and body weight relative kidney weights were identified in both sexes that received test item at 450 mg/kg bw/day in comparison with controls. Increased kidney weights were also identified in the males treated at 30 or 150 mg/kg bw/day. For this organ, body weight relative values are generally considered to be the best indicator of toxicological effect and all individual body weight relative values for both sexes at 450 mg/kg bw/day, all individual body weight relative values for males receiving 150 mg/kg bw/day and 9/10 individual body weight relative values for males receiving 30 mg/kg bw/day exceeded the historical control. However it should be noted the historical control range was also exceeded by 6/10 control males and 5/10 control females at the end of the treatment period. Although there was no evidence of histopathological changes for females at 450 mg/kg bw/day, for males at all dosages the increases in kidney weights were supported by histopathological change (hyaline droplets with associated pathological changes) for the kidneys at the end of the treatment period. Following the four week recovery period at 450 mg/kg bw/day, there were no statistically significant differences from control for absolute and body weight relative kidney weights.

At the end of the treatment period statistically significant lower absolute and relative brain weights were identified in males that received test item at 30, 150 or 450 mg/kg bw/day in comparison with controls. However, there was no consistent dosage relationship and only isolated individual for these treated groups (and control) exceeded the historical control range. In the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.

At the end of the treatment period statistically significant lower absolute and relative thymus weights were identified in males that received test item at 450 mg/kg bw/day in comparison with controls. However, only one isolated individual body weight relative value for these treated animals was below the historical control range while 4/10 individual absolute and 4/10 individual body weight relative values exceeded the historical control range. While the lower thymus weights was supported by a higher incidence of atrophy observed during microscopic evaluation of this organ, the differences in organ weight are considered to be primarily due to unusually high control values and, as such, are considered to be incidental and of no toxicological significance.

At the end of the treatment period, statistically significant higher absolute and relative ovary weights were identified in females that received test item at 450 mg/kg bw/day in comparison with controls. For these treated females, 4/10 individual absolute values and 4/10 individual body weight relative values exceeded the historical control range and one absolute value was below this historical control range. However for the control group, 3/10 individual absolute values and 2/10 individual body weight relative values were below the historical control range. In the absence of any supporting histopathological change this finding was considered to be incidental and of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy
At the end of the dosing period, animals of either sex treated with 450 mg/kg bw/day exhibited thickening and sloughing of the non-glandular region of the stomach. Other gastric changes at this dosage included raised limiting ridge (2 males and 2 females) pale areas on the glandular region (one male) and thinning of the glandular region (one male). At 150 mg/kg bw/day there was a lower incidence of similar stomach findings; two males and one female showed thickening and/or sloughing of the non-glandular region and two males showed pale areas on or thinning of the glandular region. At 450 mg/kg bw/day, at the end of the four week recovery period, stomach findings were restricted to raised limiting ridge in two males and three females.

At 450 mg/kg bw/day, four males, at the end of the treatment period, showed an enlarged spleen, with three of these spleens also showing discoloration. Two females at this dosage, also showed enlarged spleen, with one also showing discoloration, and a further three females showed discoloration of the spleen, at the end of the treatment period. At 150 mg/kg bw/day, one male showed an enlarged spleen and one female showed discoloration of the spleen. At 450 mg/kg bw/day, following the four week recovery period, enlarged spleen was only evident for one male animal.
A mottled appearance to one or both of the kidneys was observed for one, five and nine males at 30, 150 and 450 mg/kg bw/day respectively, at the end of the treatment period. While the precise aetiology of this condition is unknown it may reflect pathological kidney changes due to hyaline droplets. No similar findings were apparent in recovery test animals following the twenty-eight day treatment-free period.

Red patchy coloration of the lungs was observed for both sexes across non-recovery and recovery dose groups including controls. The incidence and distribution of this finding did not suggest any association with treatment and this finding is often observed within this laboratory and is consistent with the exsanguination procedure employed on this study.

One male at 450 mg/kg bw/day showed an enlarged heart at necropsy at the end of the treatment period. One female previously treated at 450 mg/kg bw/day also showed an enlarged heart at necropsy at the end of the treatment-free period. In the absence of any supporting effects on heart weights or supporting histopathological change these findings were considered to be incidental and unrelated to treatment.

Other incidental findings involved isolated instances of pale patches on the liver and kidneys with increased pelvic space (hydronephrosis) in one or both kidneys. Findings of this nature are consistent with normally expected low incidence findings in laboratory maintained rats within this laboratory and were therefore considered to be unrelated to exposure to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Administration of the test item at dose levels of 30, 150 and 450 mg/kg bw/day resulted in treatment-related changes in the following tissues:
Adrenals - hypertrophy (zona fasciculata) was present in 7 of 10 males treated at 450 mg/kg bw/day. Similar changes were not present in males treated at 30 or 150 mg/kg bw/day or in recovery males previously treated at 450 mg/kg bw/day.

Kidney - an increase in hyaline droplets was identified in all non-recovery males treated with the test item at 30, 150 and 450 mg/kg bw/day. This condition was associated with pathological changes, proteinaceous casts and basophilic tubules and probably accounted for the mottled appearance noted at necropsy. Immunohistochemical staining proved positive for α2-microglobulin in all males. After the recovery period there was no increase in hyaline droplets and the pathological changes had largely reversed with only a minor increase in the appearance of basophilic tubules at a minimal severity remaining.

Liver - centrilobular hypertrophy of hepatocytes was present in 3 of 10 males treated at 150 mg/kg bw/day and in 6 of 10 males and all females that received 450 mg/kg bw/day. Centrilobular hypertrophy persisted in 3 of 10 recovery females previously treated at 450 mg/kg bw/day.

Thyroid Gland - hypertrophy of the follicular cells was present in 5 of 10 non-recovery 450 mg/kg bw/day females and had fully reversed by the end of the recovery period.

Spleen - hematopoiesis was increased in all non-recovery animals treated at 450 mg/kg bw/day (mild or moderate) compared to 2 of 10 non-recovery control males (mild). At 150 mg/kg bw/day 4 of 10 females also showed a minor increase (one moderate and three mild) but in these animals this condition was considered to be of questionable significance. The incidence of hematopoiesis for other treated animals was considered to show no association with treatment. This condition was not present in recovery animals at 450 mg/kg bw/day at the end of the treatment-free recovery period.
Increased hemosiderin (mild) was identified at the end of the treatment period in the spleen of 7 of 10 non-recovery males and 8 of 10 non-recovery females that received 450 mg/kg bw/day. This change persisted in 7 of 9 recovery males (mild) and all recovery females (7 mild, 3 moderate) at 450 mg/kg bw/day with this finding only being present at a mild severity in one control recovery male and one control recovery female.

Stomach - hyperplasia of the non-glandular stomach, of mild or moderate severity, was noted in 9 of 10 males and all females treated at 450 mg/kg bw/day. Minimal or mild hyperplasia was present in 4 of 10 males and 3 of 10 females treated at 150 mg/kg bw/day.

Hyperplasia at minimal severities was observed in 3 of 10 recovery 450 mg/kg bw/day males indicating an almost complete recovery in the males and complete reversibility in females.

Duodenum - mild mucosal hypertrophy was present in one non-recovery control female and in 6 of 10 non-recovery 450 mg/kg bw/day females only and had completely reversed in animals examined at the end of the twenty-eight day recovery period.

Thymus - atrophy (minimal) was present in 6 of 10 non-recovery 450 mg/kg bw/day males and 5 of 10 females from this test group and in 2 of 10 control females. This change had completely reversed in the recovery animals.

No other findings were present at histopathology which could be attributed to treatment with the test item or correlated with in-life changes noted.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the nature of estrous cycle detected in females over the last three weeks of dosing.

There were no effects of treatment on sperm concentration or motility at the end of the dosing period for males receiving 30, 150 or 450 mg/kg bw/day.

There were no effects of treatment on sperm morphology at the end of the dosing period for males receiving 450 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Remarks:
Systemic effects
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Local effects
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic effects
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
duodenum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Oral administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 30, 150 and 450 mg/kg bw/day followed by a 28 day treatment free period in sub-groups of animals previously dosed at 450 mg/kg bw/day resulted in treatment related changes in all male dose groups and in females that received 450 or 150 mg/kg bw/day precluding identification of a No Observed Effect Level (NOEL) in animals at these dosages. However, under the conditions of this study the NOEL for females was considered to be 30 mg/kg bw/day. At 30 mg/kg bw/day, only the presence of hyaline droplets in the kidney precluded this dosage from being a NOEL for the male rat and this finding has no relevance in the assessment of potential hazard to human health. Excluding effects associated with hyaline droplets in the male kidney, the No Observed Adverse Effect Level (NOAEL) for local toxicity for the male and females rat within this study is considered to be 30 mg/kg bw/day, due to local irritant effects of the Test Item to the stomach, and the NOAEL for systemic toxicity for both sexes is 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)       OECD Guidelines for Testing of Chemicals No. 408 “Repeated Dose 90-Day Oral Toxicity Study in Rodents” (Adopted 21 September 1998)

ii)     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 Registration, 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 30, 150 and 450 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (450 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period. Urinalysis was performed for all non-recovery and surviving recovery animals towards the end of the treatment and treatment-free periods, respectively. Ophthalmoscopic examination was also performed on all animals prior to start of treatment and on non-recovery control and high dose animals at the end of the treatment period.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all non-recovery control and high dose animals as well as any gross lesions was performed in the first instance. As there were treatment-related findings identified in both sexes of non-recovery 450 mg/kg bw/day animals, examination of affected tissues was subsequently extended to include relevant animals from all the remaining dose groups on the study.

Results

Mortality

There were two unscheduled deaths during the course of the study. One male treated at 450 mg/kg bw/day was killedin extremison Day 76 and a control male was killedin extremison Day 80. The decline in clinical condition resulting in the early termination of both animals was considered to have resulted from accidental trauma during the dosing procedure and, as such, these deaths were clearly unrelated to treatment.

Clinical Observations

During the treatment phase, all animals treated at 450 mg/kg bw/day showed transient episodes of increased salivation. Similar post-dose increased salivation was also present, to a lesser extent, for both sexes at 150 mg/kg bw/day. Four control males also showed post-dose increased salivation but on only one single occasion. Two males at 450 mg/kg bw/day and one control male showed isolated instances of noisy respiration. 

At 450 mg/kg bw/day, six males and two females showed sporadic episodes of body tremors, generally during the later stages of the study. Hunched posture was also observed for six females during the study with the majority of these animals being affected between Days 70 to 79 of the study.    

No clinical signs were apparent for either sex receiving 30 mg/kg bw/day.

No clinical signs were apparent for animals previously treated at 450 mg/kg bw/day during the treatment-free recovery period.

Behavioral Assessment

At 450 mg/kg bw/day, one female showed hunched posture and pilo-erection during Week 4 assessments and a further female showed hunched posture and/or pilo-erection during assessments for Weeks 5, 6 and 7. 

There were no clinical signs apparent during behavioural assessments for either sex at 30 or 150 mg/kg bw/day or for males at 450 mg/kg bw/day. 

Functional Performance Tests

Motor activity assessment during Week 12 revealed a statistically significant reduction in overall activity in males treated with 450 mg/kg bw/day in relation to controls, although activity during the final 20% of the observation period was similar to control.

There were no statistically significant differences from control in motor activity of either sex at 30 or 150 mg/kg bw/day or for females at 450 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity assessments were unaffected by treatment at 450 mg/kg bw/day.

Body Weight

At 450 mg/kg bw/day, body weight gain of males was statistically significantly lower than control during the first week of treatment. Although subsequent improvement was evident, sporadic instances of slightly lower weekly body weight gains were observed during the study and resulted in an overall lower body weight gain than the controls by the end of dosing period.

There was no effect of treatment on bodyweight gain for both sexes at 30 or 150 mg/kg bw/day or females at 450 mg/kg bw/day.

Following the cessation of treatment, recovery phase 450 mg/kg bw/day males showed a clear body weight recovery such that overall body weight gain was similar to control at the end of the study. Fluctuations of body weight gain for recovery phase 450 mg/kg bw/day females during the treatment free period were considered to have been unaffected by previous exposure to the test item. 

Food Consumption

Both sexes treated at 450 mg/kg bw/day and females receiving 150 mg/kg bw/day showed marginally lower dietary intake than controls during the first week of the study. For males at 450 mg/kg bw/day, sporadic instances of marginally lower food intake persisted during the remainder of the treatment period. Food conversion efficiency for males receiving 450 mg/kg bw/day was also slightly lower than control during the first week of treatment.

There was no effect of treatment on food intake for either sex treated with 30 mg/kg bw/day or for males receiving 150 mg/kg bw/day.

During the treatment-free period, recovery 450 mg/kg bw/day males showed marginally higher food consumption compared to control consistent with their higher body weight gain during this period. There was no effect on food intake for females previously treated with 450 mg/kg bw/day.

Water Consumption

Daily visual inspection of water bottles revealed no overt intergroup differences.

Ophthalmoscopy

Ophthalmoscopic examination of the eyes of animals receiving 450 mg/kg bw/day during Week 12 of the study did not indicate any effect of treatment.

Estrous Cycling

There was no effect of treatment on estrous cycle for females receiving 30, 150 or 450 mg/kg bw/day of the test item.

Hematology

At 450 mg/kg bw/day, statistically significant lower hemoglobin, red blood cell count, hematocrit and mean cell hemoglobin concentration and statistically significant higher mean cell volume and mean cell hemoglobin was apparent for both sexes, compared to control. Statistically significant lower hemoglobin, red blood cell count and hematocrit were observed for males receiving 150 mg/kg bw/day, while females at this dosage showed statistically significant higher mean cell volume and mean cell hemoglobin and statistically significant lower mean cell hemoglobin concentration.

At the end of the treatment free period, males previously treated at 450 mg/kg bw/day showed statistically significantly lower red blood cell count and mean cell hemoglobin concentration and statistically significant higher mean cell volume and mean cell hemoglobin. Hematocrit was statistically significantly higher than control, in contrast to the reduction observed during the treatment period. For recovery females previously treated at 450 mg/kg bw/day, there were no statistically significant differences from control for hematology parameters at the end of the treatment free period.

For females at 150 mg/kg bw/day and both sexes at 450 mg/kg bw/day there was a statistically significant increase in circulating neutrophils at the end of the treatment period.

At 450 mg/kg bw/day, higher platelet counts were apparent for both sexes but differences from control only attained statistical significance for females. This finding may, at least in part, have been influenced by the anemia among non-recovery rats at this dosage.

There were no noteworthy findings in the hematological parameters examined in animals of either sex treated at 30 mg/kg bw/day.

Blood Chemistry

At 450 mg/kg bw/day, statistically significant higher blood urea, plasma glucose and inorganic phosphorus levels and statistically significant lower mean sodium levels, compared to control, were apparent for males at the end of treatment period. 

Urinalysis

There were no treatment-related effects detected in the urinalytical parameters.

Necropsy

At 450 mg/kg bw/day, both sexes exhibited thickening and sloughing of the non-glandular region of the stomach at the end of the treatment period. Other gastric changes at this dosage included raised limiting ridge, pale areas on the glandular region and thinning of the glandular region. At 150 mg/kg bw/day there was a lower incidence of similar stomach findings. At the end of the four week recovery period, stomach findings for animals previously treated at 450 mg/kg bw/day were restricted to raised limiting ridge in two males and three females.   

At 450 mg/kg bw/day, four males and five females showed enlarged and/or discoloured spleen at the end of the treatment period. At 150 mg/kg bw/day, one male showed an enlarged spleen and one female showed discoloration of the spleen. At 450 mg/kg bw/day, following the four week recovery period, enlarged spleen was only evident for one male animal.

A mottled appearance to one or both of the kidneys was observed for one, five and nine males at 30, 150 and 450 mg/kg bw/day respectively, at the end of the treatment period. No similar findings were apparent in males animals previously treated at 450 mg/kg bw/day at the end of the treatment-free recovery period.  

Sperm Analysis

There were no effects of treatment on sperm concentration or motility at the end of the dosing period for males receiving 30, 150 or 450 mg/kg bw/day.

There were no effects of treatment on sperm morphology at the end of the dosing period for males receiving 450 mg/kg bw/day.

Organ Weights

At 450 mg/kg bw/day, statistically significant higher absolute and body weight relative liver, kidney, spleen and adrenal weights, compared to control, were apparent for both sexes at the end of the treatment period.

At 150 mg/kg bw/day, statistically significant higher absolute and body weight relative liver and kidney weights, compared to control, were apparent for males at the end of the treatment period.

At 30 mg/kg bw/day, statistically significant higher absolute and body weight relative kidney weights, compared to control, were apparent for males at the end of the treatment period.

Following the four week recovery period at 450 mg/kg bw/day, statistically significant differences from control for organ weights was restricted to increased liver weight for females.  

Histopathology

Administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5) at dose levels of 30 mg/kg bw/day (Low Dose Group), 150 mg/kg bw/day (Intermediate Dose Group) and 450 mg/kg bw/day (High Dose Group) resulted in treatment-related changes in the following tissues:

Adrenals– hypertrophy (zona fasciculate) was identified in seven non-recovery high dose males.

Kidney– an increase in hyaline droplets was detected in all non-recovery male test groups. This condition was associated with pathological changes, proteinaceous casts and basophilic tubules with immunohistochemical staining of these animals proving positive for α2-microglobulin. However, no increase in hyaline droplets was detected in recovery high dose group males and the associated pathological renal changes had largely reversed with only a minor increase in the appearance of basophilic tubules at a minimal severity remaining.

Liver– centrilobular hypertrophy of hepatocytes was detected in three intermediate dose group males and in six males and all females from the non-recovery high dose group. This condition persisted in three recovery high dose group females.

Thyroid Gland– hypertrophy of the follicular cells was present in five non-recovery high dose group females and was no longer detectable in recovery high dose females examined at the end of the treatment-free period.

Spleen– at 450 mg/kg bw/day, increased hematopoiesis was apparent in all non-recovery animals at the end of the treatment period but was not present for recovery animals at this dosage at the end of the treatment-free recovery period. Increased hemosiderin was observed for the spleens for these recovery animals but this was considered to reflect slow clearance of the pigments after the increase in erythrocyte production during the treatment period. Four intermediate dose group females also showed a minor increase in hematopoiesis at the end of the treatment period but in these animals this finding was considered to be of questionable significance.

Stomach– mild or moderate hyperplasia in the non-glandular stomach region was identified in all but one of the non-recovery high dose group animals with similar levels of this condition detected in four male and three females from the intermediate dose group. Only three recovery high dose group males showed minimal levels of hyperplasia indicating an almost complete recovery in the males and complete reversibility in females.

Duodenum– mild mucosal hypertrophy was present in one non-recovery control female and in six non-recovery high dose group females. This condition had fully reversed in animals examined at the end of the twenty-eight day treatment-free period.

Thymus– atrophy (minimal) was present in six non-recovery high dose group males and in five females from this test group and in two control females. This change had completely reversed in the recovery animals.

Conclusion

Oral administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS#5809-08-5) to male and female Wistar Han™:RccHan™:WIST strain rats for ninety consecutive days at dose levels of 30, 150 and 450 mg/kg bw/day followed by a 28 day treatment free period in sub-groups of animals previously dosed at 450 mg/kg bw/day resulted in treatment related changes in all male dose groups and in females that received 450 or 150 mg/kg bw/day precluding identification of a No Observed Effect Level (NOEL) in animals at these dosages. However, under the conditions of this study the NOEL for females was considered to be 30 mg/kg bw/day. At 30 mg/kg bw/day, only the presence of hyaline droplets in the kidney precluded this dosage from being a NOEL for the male rat and this finding has no relevance in the assessment of potential hazard to human health. Excluding effects associated with hyaline droplets in the male kidney, the No Observed Adverse Effect Level (NOAEL) for local toxicity for the male and females rat within this study is considered to be 30 mg/kg bw/day, due to local irritant effects of the Test Item to the stomach, and the NOAEL for systemic toxicity for both sexes is considered to be 150 mg/kg bw/day.  

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed according to current guidelines under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
male body weights, room temperature and humidity, pituitary and thyroid weights not recorded
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 acclimatised for six days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 173 to 192g (see Deviations from Study Plan), the females weighed 143 to 166g, and were approximately six to eight weeks old. The animals were housed in groups of five 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. A certificate of analysis of the batch of diet used is given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). 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 computerised system, and print-outs of hourly temperatures and humidities are included in the study records

Study Plan target ranges for temperature and humidity were 22 ± 3ºC and 50 ± 20% RH respectively. Achieved ranges were 20-22ºC and 41-80% RH; the deviations from the target ranges for relative humidity were transient in nature and were considered to have had no impact on 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 randomisation 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 randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The vehicle used for the preliminary study was Arachis Oil and as dosing formulations proved satisfactory in that study the same vehicle was employed for this main 28 Day Toxicity Study using a treatment volume of 4 ml/Kg bw. The test item was administered daily, for twenty-eight 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 Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as an emulsion in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least nineteen days. Formulations were therefore prepared in two batches each of 14 days and made one day in advance of the first day of use and stored at approximately 4ºC in the dark.
Samples of each test item formulation were taken and analysed for concentration of
1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) at Harlan Laboratories Ltd.,
Shardlow, UK, Analytical Services. The method used for analysis of formulations and
the results obtained are given in Appendix 16. The results indicate that the prepared
formulations were within 99-103%% of the nominal concentration and were considered.
acceptable for the purpose of this study.

Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
five
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages were selected in collaboration with the sponsor based on available toxicity data including a preliminary seven day range-finder investigation (Harlan Study No. 41104764) in the rat. In the preliminary study, treatment at 500 mg/kg bw/day was generally well tolerated but a dosage of 750 mg/kg bw/day was shown to be unsuitable for any long term investigation of toxicity. A high dosage of 600 mg/kg bw/day was therefore chosen as the high dosage for this investigation of toxicity, with 100 and 300 mg/kg bw/day being selected for the low and intermediate dosages.
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin colour
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour
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 Behavioural 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 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.
The animal was pulled by the base of the tail until its grip was broken. 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 Behavioural Assessments and Sensory Reactivity
Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalisation Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

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

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

Water Consumption
Water intake was measured and recorded daily for each cage group.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from
each test and control group at the end of the study (Day 28). Blood samples were
obtained from the lateral tail vein. Where necessary repeat samples were obtained by
cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.
The methods used for haematological and blood chemical investigations are given in
Addendum 2 and normal ranges are shown in Addendum 4.

Haematology
The following parameters were measured on blood collected into tubes containing
potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
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 Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)
Bile acids

Sacrifice and pathology:
Pathology
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.

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
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Thyroid/Parathyroid
Uterus with Cervix

It was intended that pituitary and thyroid/parathyroid weights would be recorded postfixation. Due to a technical error these weights were not recorded (see Deviations from Study Plan). Normal ranges for these organ weights are given in Addendum 5.

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
Salivary glands (submaxillary)
Caecum
Sciatic nerve
Colon
Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides ♦
Skin (hind limb)
Eyes (fixed in Davidson's fluid)
Spinal cord (cervical, mid-thoracic
Gross lesions and lumbar)
Heart Spleen
Ileum Stomach
Jejunum
Testes ♦
Kidneys Thymus
Liver Thyroid/Parathyroid
Lungs (with bronchi). Lungs were inflated to approximately normal respiratory volume with buffered 10% formalin before immersion in fixative.
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus & Cervix
Muscle (skeletal)
Vagina
Oesophagus

♦ preserved in Bouin's fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later.

All tissues were despatched to the histology processing Test Site Harlan Laboratories Ltd., Itingen, Switzerland) for processing (Principal Investigator: S Gähler later replaced by W Henderson). The tissues shown in bold from all control and 600 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 100 and 300 mg/kg bw/day dose group animals. In addition, sections of testes and epididymides from all Control and 600 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Pituitary and thyroid/parathyroid weights were not recorded in error, histopathological examinations of these tissues was therefore extended to include animals from the low and intermediate groups.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidneys, stomach and adrenals from all animals from the low and intermediate groups. Microscopic examination was conducted by the Study Pathologist (Principal Investigator: Dr K. Heider) at AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland. A complete histopathology phase report, including methods, is presented in Appendix 15.
Statistics:
Evaluation of Data
Data were processed to give summary incidence or group mean and standard deviation values were appropriate. All data were summarised in tabular form. 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, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed 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 analysed 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 (nonparametric).

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):
Transient post-dosing salivation at all doses.
Mortality:
mortality observed, treatment-related
Description (incidence):
Transient post-dosing salivation at all doses.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: lower weight gain first week of dosing at 600 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower in males and females at 600 mg/kg/day
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased in males and females at 600 mg/kg/day and in males at 300 mg/kg/day
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Decreased Hgb, RBC, MCHC and Hct at 300 and 600; increased reticulocytes at 600.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased liver, kidney, spleen and adrenal weights at 300 and 600 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Thickening of glandular stomach in males and females at 600 mg/kg/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatocellular hypertrophy, extramedullary hematopoiesis, nephropathy in males and gastric hyperkeratosis at 300 and 600 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths on the study.
Clinical signs amongst treated animals were restricted to transient post-dosing salivation. At 300 and 600 mg/kg bw/day all animals were affected with the sign first being apparent on the second day of the study. At 100 mg/kg bw/day only three males and one female were affected with the sign first being apparent on Day 24 of the study. Post dose salivation is frequently observed when animals are dose via the oral gavage route and is
generally considered to reflect distaste of the dosing formulation rather than a systemic effect of the test item.

BODY WEIGHT AND WEIGHT GAIN
At 600 mg/kg bw/day mean body weight gain of males was lower than control during the first week of dosing, with differences attaining statistical significance. Thereafter, there were no statistically significant differences from control although overall body weight gain was generally slightly lower than control.
Body weight gain for either sex at 100 and 300 mg/kg bw/day or for females at 600 mg/kg bw/day was unaffected by treatment. For females at 300 mg/kg bw/day, body weight gain was statistically significantly higher than control during Week 3 but these differences were considered to reflect normal biological variation and were unrelated to treatment.

FOOD CONSUMPTION AND FOOD EFFICIENCY
At 600 mg/kg bw/day food intake for both sexes was lower than control during the first week of treatment; subsequent food intake was similar to control. At 100 and 300 mg/kg bw/day there were no effects of treatment on food consumption during the study. There was no effect of treatment on food conversion efficiency 100, 300 or 600 mg/kg bw/day.


WATER CONSUMPTION
At 600 mg/kg bw/day, water consumption for both sexes was higher than control throughout most of the study.
At 300 mg/kg bw/day water consumption for males was higher than control throughout most of the study. For females at this dosage, the pattern of water intake appeared inconsistent, however higher water was generally apparent during the final week of treatment. At 100 mg/kg bw/day there was no clear effect of treatment on water consumption for either sex.

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
For males at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day, mean haemoglobin levels and erythrocyte count were lower than control. Additionally at 300 and 600 mg/kg bw/day, mean cell haemoglobin concentration for both sexes and haematocrit values for females were lower than control and mean cell volume was increased for females. At 600 mg/kg bw/day, these changes were accompanied by an increased reticulocyte count for both sexes.

At all dosages, mean neutrophils counts for both sexes were higher than control.

CLINICAL CHEMISTRY
There were no differences from control that were considered to represent an adverse effect of treatment.

URINALYSIS

NEUROBEHAVIOUR
No neurological effects of treatment at 100, 300 or 600 mg/kg bw/day were apparent.

ORGAN WEIGHTS

GROSS PATHOLOGY
At 600 mg/kg bw/day, three males and four females showed thickening and sloughing of the non-glandular region of the stomach, with thickening of the non-glandular region being apparent for remaining males. At 300 mg/kg bw/day, one male showed thickening of the non-glandular region of the stomach and another male showed sloughing of the non-glandular region of the stomach. One male at 100 mg/kg bw/day and three males at 300 and at 600 mg/kg bw/day showed a mottled appearance of the kidneys at terminal necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination of the liver revealed minimal to slight centrilobular hepatocellular hypertrophy in some males and females at 300 mg/kg bw/day and in all males and females at 600 mg/kg bw/day. In males, the liver cell hypertrophy was associated with glycogen depletion. In addition, the age-related minimal to slight focal to multifocal bile duct hypertrophy/hyperplasia was slightly increased at 300 mg/kg bw/day and moderately at 600 mg/kg bw/day.

Histopathological examination of the thyroid revealed minimal diffuse follicular cell hypertrophy for 3/5 females at 600 mg/kg bw/day.

Histopathological examination of the spleen revealed that extramedullary hematopoiesis was minimally increased in severity in males and females at 300 mg/kg bw/day and slightly increased in severity in males and females at 600 mg/kg bw/day.

Histopathological examination of the kidneys revealed nephropathy in males in all treated groups. This finding was characterized by minimal focal/multifocal and mainly bilateral tubular degeneration/regeneration in renal proximal tubules was noted in all males at 100 mg/kg bw/day, minimal to slight multifocal and bilateral tubular degeneration/regeneration in all males at 300 mg/kg bw/day, and minimal to moderate mainly multifocal and bilateral in 4/5 males at 600 mg/kg bw/day. These tubular findings were associated with an increased severity in tubular hyaline droplets and often also with granular casts in tubules of the inner cortex.

Histopathological examination of the forestomach (non-glandular stomach) revealed a minimal diffuse hyperkeratosis in all males and 2/5 females at 100 mg/kg bw/day. The diffuse hyperkeratosis was noted at a slight severity in all animals at 300 mg/kg bw/day and at moderate severity in all animals at 600 mg/kg bw/day. A minimal to slight and mainly diffuse squamous hyperplasia was present in all animals at 300 mg/kg bw/day and a slight to moderate diffuse squamous hyper-plasia occurred in all snimals at 600 mg/kg bw/day. A focal to multifocal minimal to moderate vesicle formation in the keratin layer was diagnosed in all animals at 600 mg/kg bw/day. In all animals with epithelial squamous hyperplasia, minimal to slight multifocal submucosal inflammation was also noted.

Histopathological examination of the adrenal cortices revealed a slightly increase in incidence and minimally in severity for the often seen diffuse fatty change in the zona fasciculata for males at 600 mg/kg bw/day.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS
Key result
Dose descriptor:
LOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Within the context of this study it was not possible to establish a No Observed Adverse Effect Level (NOAEL) for the oral administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) to the rat.
Executive summary:

The study was designed to investigate the systemic toxicity of 1,1,3,3-tetramethylbutyl hydroperoxide, CAS 5809-08-5 according to the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 100, 300 and 600 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP) over the same treatment period. Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Histopathological examinations were extended to include the low and intermediate dosages for the liver, kidneys, thyroids, spleen, stomach and adrenals.

There were no unscheduled deaths on the study. Clinical signs were restricted to post-dosing salivation for a few animals at 100 mg/kg bw/day and all animals at 300 and 600 mg/kg bw/day. There were no treatment-related neurological effects on behaviour, functional performance or sensory reactivity at any dose tested.

At 600 mg/kg bw/day body weight gain of males was lower than control during Week 1. At 600 mg/kg bw/day, food consumption for males was lower than control during Week 1. Food conversion efficiency was unaffected by treatment at 100, 300 or 600 mg/kg bw/day. At 600 mg/kg bw/day, water consumption for increased for both sexes compared to control throughout most of the study. At 300 mg/kg bw/day, water intake was higher than control throughout most of the study and for females during the final week of the study.

For males at 100 mg/kg bw/day and both sexes at 300 and 600 mg/kg bw/day, mean haemoglobin levels and erythrocyte count were lower than control. Additionally at 300 and 600 mg/kg bw/day, mean cell haemoglobin concentration for both sexes and haematocrit values for females were lower than control and mean cell volume was increased for females. At 600 mg/kg bw/day, these changes were accompanied by an increased reticulocyte count for both sexes. At all dosages, mean neutrophil counts for both sexes were higher than control.

There were no treatment-related effects on blood chemistry at any dose tested.

Necropsy. Thickening and sloughing of the non-glandular region of the stomach was apparent in both sexes at 600 mg/kg bw/day. Two males at 300 mg/kg bw/day showed similar findings.

Organ Weights. At 300 and 600 mg/kg bw/day, absolute and body weight relative liver weights were increased for both sexes. Absolute and body weight relative kidney weights were increased for males at 100 and 300 mg/kg bw/day and both sexes at 600 mg/kg bw/day. Absolute and body weight relative spleen weights were increased for females at 300 mg/kg bw/day and both sexes at 600 mg/kg bw/day. Absolute and body weight relative adrenal weights were increased for males at 100, 300 and 600 mg/kg bw/day.

Histopathology. The following treatment related microscopic findings were detected:

Liver: centrilobular hepatocellular hypertrophy in both sexes at 300 and 600 mg/kg bw/day with associated glycogen depletion for males. Increased age-related focal/multifocal bile duct hypertrophy/hyperplasia was observed at 300 and 600 mg/kg bw/day. Thyroid: Minimal diffuse follicular cell hypertrophy for females at 600 mg/kg bw/day.

Spleen: Increased in severity for extramedullary hematopoiesis for both sexes at 300 and 600 mg/kg bw/day.

Kidneys: Nephropathy, characterized by tubular degeneration/regeneration in renal proximal tubules for males at 100, 300 and 600 mg/kg bw/day, with an associated increased severity in tubular hyaline droplets, often with granular casts in tubules of the inner cortex.

Stomach: Hyperkeratosis of the non-glandular region in both sexes at 100, 300 and 600 mg/kg bw/day, with diffuse squamous hyperplasia and submucosal inflammation also present at 300 and 600 mg/kg bw/day. Minimal to moderate vesicle formation in the keratin layer was observed at 600 mg/kg bw/day.

Adrenals: Slightly increased incidence and severity for diffuse fatty change in the zona fasciculata of the adrenal cortices for males at 600 mg/kg bw/day.

Within the context of this study it was not possible to establish a No Observed Adverse Effect Level (NOAEL) for the oral administration of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) to the rat. For both sexes, adverse histopathological changes were observed for the non-glandular region of the stomach which extended down to the low dosage of 100 mg/kg bw/day. In the absence of any equivalent to the non-glandular region of the stomach for the rat in humans this finding may have limited relevance in the assessment of human health, although it does indicate an irritant effect of the test item. Additionally for males, kidney changes associated with hyaline droplets and granular casts also extended down to 100 mg/kg bw/day. Such kidney changes are well documented as being peculiar to the male rat in response to some hydrocarbons and this finding is also considered to represent limited relevance to humans. These histopathological changes were accompanied by increased numbers of neutrophils, and for males lower haemoglobin levels and erythrocyte counts, at this low dosage.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Adequate; the study was performed under GLP according to currrent guidelines.

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

Justification for classification or non-classification

The NOAEL is 150 mg/kg bw/day and based on this no classification has been assigned for repeated dose toxicity.