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Description of key information

Introduction

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

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

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

The study design also took into account elements from the following guideline:

·        In vivoMammalian Alkaline Comet Assay” (Adopted 26 September 2014)

Methods

The test item was administered by oral gavage to three groups of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, at dose levels of 10, 100 or 200/300 mg/kg bw/day. Animals from the high dose group were dosed at 200 mg/kg bw/day up to Day 30 (females) or Day 31 (males) with the dose level increased to 300 mg/kg bw/day thereafter; as these animals were dosed at 300 mg/kg bw/day for approximately 2/3 of the dosing period, the dose level for this group will generally be referred to as 300 mg/kg bw/day throughout this study report. A control group of ten males and ten females was dosed with the vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (200/300 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days. The first five males from each non-recovery dose group (Groups 1 to 4) were dosed for ninety-one consecutive days whilst the remaining males and females from these dose groups were dosed for ninety consecutive days. An additional group of five males (Group 5), used as positive control for comet assay assessment, was administered with 25 mg/kg bw/day N-Nitroso-N-methylurea by oral gavage on Days 90 and 91. Animals treated on Days 90 and 91 were dosed approximately twenty-seven and three hours (respectively) before euthanasia and selected tissues from these animals were used for comet assay assessment.

Clinical signs, functional observations, body weight change and dietary intake were monitored during the study. Hematology and blood chemistry were evaluated for all Groups 1 to 4 animals at the end of treatment and treatment-free periods. Urinalysis was performed for all Groups 1 to 4 non-recovery animals during Week 12 of dosing and for all recovery animals during the last week of the treatment-free period. Ophthalmoscopic examination was also performed on all Groups 1 to 4 animals prior to the start of treatment and on all non-recovery control and high dose animals during Week 12 of the study. Additionally, the stage of estrous was monitored for all non-recovery females during the last three weeks 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 (Groups 1 and 4) as well as any gross lesions from Groups 1 to 4 animals was performed in the first instance. As there were treatment-related findings in the kidneys (males only) and the stomach (males and females), examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups as well as recovery groups.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Throughout the treatment period, there were no clinical signs indicative of test item toxicity.

Behavioral Assessment

Behavioral assessment scores across the test-item treated animals of either sex remained similar to controls.

Functional Performance Tests

There were no treatment-related changes in functional performance for animals of either sex at any dose level.

Sensory Reactivity Assessments

Sensory reactivity scores were comparable across all dose groups including controls.

Body Weight

There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex. Overall body weight gain in non-recovery males treated with 300 mg/kg bw/day was approximately 13% lower than control at the end of dosing, but recovery was evident during the treatment-free period, and this finding was deemed to be related to irritant properties of the test item and not an indication of its systemic toxicity.

Food Consumption

There was no adverse effect of treatment with the test item at any dose level on food consumption or food conversion efficiency in animals of either sex. Marginally lower dietary intake in males treated with 300 mg/kg bw/day from Week 6 of dosing was considered to be due to irritant properties of the test item rather than an indication of its systemic toxicity.


Water Consumption

When compared with controls, visual inspection of water bottles did not reveal any intergroup differences in animals of either sex receiving the test item.

Estrous Cycling

There was no adverse effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the last three weeks of dosing.

Ophthalmoscopy

Ophthalmoscopic examination of non-recovery males and females from the control and 300 mg/kg bw/day dose groups during Week 12 of dosing did not reveal any treatment-related differences.

Hematology

Hematology evaluations at the end of the treatment or treatment-free periods did not reveal any toxicologically significant effects in animals of either sex resulting from treatment with the test item.

Blood Chemistry

Blood chemistry evaluations at the end of the treatment or treatment-free periods did not indicate any effects of toxicological relevance in animals of either sex resulting from test item administration.

Urinalysis

Urinalytical evaluations did not identify any treatment-related effects in males or females receiving the test item up to a dose level of 300 mg/kg bw/day.

Necropsy

There were no macroscopic observations at terminal necropsy considered to be related to treatment with the test item.

Sperm Analysis

At the end of the treatment or treatment-free periods, sperm concentration, motility and progressive motility across all test item-treated male dose groups were similar to controls. Morphological sperm assessment also did not identify any treatment-related differences between non-recovery males from the control and 300 mg/kg bw/day dose groups.

Organ Weights

Increased kidney weights in non-recovery males from the 300 mg/kg bw/day dose groups, which persisted in the recovery males previously receiving 300 mg/kg bw/day albeit to a lower extent, were considered to be associated with α-2u-globulin nephropathy syndrome in these males. The increase in liver weights observed in non-recovery animals of either sex receiving 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed complete reversibility in recovery animals and was not associated with any histopathology findings.

Histopathology

Treatment-related findings were recorded in the kidneys of males and the stomach of males and females. Some of the changes in the kidneys persisted after the twenty-eight day recovery period but the changes in the stomach had resolved. The histopathology findings were as following:

Kidneys

At the end of the dose administration period, hyaline droplets were present in males treated with 100 or 300 mg/kg bw/day. Multifocal basophilic tubules and proteinacious casts were also observed in these males. Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels in males treated with 100 or 300 mg/kg bw/day. At the end of the recovery phase, males previously treated with 300 mg/kg bw/day still showed proteinaceous casts and chronic nephropathy (basophilic tubules, fibrosis, tubular atrophy, tubular dilation). Immunohistochemical staining was positive for α-2u-globulin in all recovery males with males previously receiving 300 mg/kg bw/day showing increased staining levels relative to controls.

Stomach

At the end of the dosing period, minimal hyperplasia of the non-glandular region was noted in most males and some females treated with 300 mg/kg bw/day. This change had resolved after the recovery period.

Conclusion

Excluding the findings from the comet assay assessment, the oral (gavage) administration of bisisobutyryl peroxide (CAS# 3437-84-1) to male and female Wistar Han:RccHan:WIST strain rats at dose levels of 10, 100 or 300 mg/kg bw/day was well tolerated. There were no findings of toxicological significance in animals of either sex and a dose level of 300 mg/kg bw/day could therefore be established as a ‘No Observed Adverse Effect Level’ (NOAEL) for general systemic toxicity within the confines of this type of study (OECD 408).

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: 20 January 2016 Experimental Completion Date: 02 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Identification : Bisisobutyryl peroxide (CAS# 3437-84-1)
Physical State/Appearance : Clear colorless liquid
Purity : Diisobutyryl peroxide (38.91%)
Batch Number : BYK004856-171
Date Received : 25 November 2015
Storage Conditions : Approximately -20 °C
Expiry Date : 30 April 2016


Reference Item and Supporting Information
Identification : N-Nitroso-N-methylurea
Physical State/Appearance : Off-white powder
Purity : 90%
Batch Number : P102-01944
Date Received : 13 April 2015
Storage Conditions : Approximately 4 °C
Expiry Date : 12 April 2017
A purity adjustment (for 90% purity) was made when preparing the dosing formulations.


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 appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system 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 at least nine days during which time their health status was assessed. A total of one hundred and twenty-five animals (sixty-five males and sixty females) were accepted into the study. At the start of treatment the males weighed 181 to 227g, the females weighed 153 to 204g, and were approximately six to eight weeks old.

Animal Care and Husbandry
Groups 1 to 4 animals were housed in groups of three or four by sex whilst Group 5 animals were housed in groups of two or three 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. Certificates of analysis of the batches of diet used are given in Annex 6; expiry date for the batch number 070715MA was extended to 07 July 2016. 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; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Details on route of administration:
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.
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the test item formulations were determined as part of Envigo Study Number 41502236 where formulations were shown to be stable for at least four hours when kept on crushed ice. Formulations were therefore prepared daily and kept on crushed ice before and during use.
Samples were taken from test item formulation on various occasions during the study and were analyzed for concentration of Bisisobutyryl peroxide (CAS# 3437-84-1) at Envigo Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicated that the test item formulations were inside the acceptance criteria of ±20% on most occasions; see deviations from Study Plan.
4.4 Reference Item Preparation and Analysis
For the purpose of this study, N-Nitroso-N-methylurea was prepared at the appropriate concentration as a solution in distilled water.
Fresh formulations were prepared and used within two hours of being prepared; it is assumed this formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of N-Nitroso-N-methylurea formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an esternal 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 yeild a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.

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

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


Preparation of test samples
The formulations received were kept on ice and extracted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to extract. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

The cocentration of test item in the final solution was quantified by HPLC using UV detection as detailed in the instrument paraneters below:

HPLC: Agilent Technologies 1200, incorporating autosampler and work station
Column: Luna 5µ C18 (150 x 2 mm id)
Column temperature: 7°C
Autosampler temperature: 5°C
Mobile phase: acetonitrile:water (70:30 v/v)
Flow-rate: 0.4 mL/min
UV detector wavelength: 230 nm
Injection volume: 5 µL
Retention time: ~2.5 mins
Duration of treatment / exposure:
90 days
The first five males from each non-recovery dose group (Groups 1 to 4) were dosed for ninety-one consecutive days whilst the remaining males and females from these dose groups were dosed for ninety consecutive days. An additional group of five males (Group 5), used as positive control for comet assay assessment, was administered with 25 mg/kg bw/day N-Nitroso-N-methylurea by oral gavage on Days 90 and 91.
Frequency of treatment:
Daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
Low dose level treatment group. Concentration: 2 mg/mL
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose level treatment group. Concentration: 20 mg/mL
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
High dose level treatment group. Concentration: 400 mg/mL.
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
High dose level treatment group.
Dose level increased to 300 mg/kg bw/day from Day 32 (males) and Day 31 (females)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Recovery control
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Recovery group - high
Dose level increased to 300 mg/kg bw/day from Day 32 (males) and Day 31 (females)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Positive control
No. of animals per sex per dose:
10 males/ 10 females
Control animals:
yes, concurrent vehicle
other: Positive control
Details on study design:
The dose levels and dose volume were selected in collaboration with the Sponsor and were based on available toxicity data including a 28 Day toxicity study in the rat (Notox Project: 498267) and a pre-natal development toxicity study in the rat (Envigo Study Number: 41502236). In the 28 day study, dose levels up to 1000 mg/kg/day did not result in any toxicologically relevant effects on body weight development or food consumption for animals of either sex. Treatment at 300 or 1000 mg/kg/day resulted in histopathological changes in the forestomach in both sexes that were considered to be reflective of irritant properties of the test item. Diffuse hepatocellular hypertrophy, associated with increased liver weights, was also observed in the liver of both sexes receiving 1000 mg/kg/day and was considered to be indicative of adaptive hepatic changes. In the pre-natal development toxicity study, however, a dose level of 300 mg/kg bw/day resulted in four premature deaths with further deaths at higher dose levels. Based on these results, a dose level of 200 mg/kg bw/day was considered to be a suitable high dose for investigation in the present study together with 10 and 100 mg/kg bw/day as the low and intermediate dose levels, respectively. The dose level of 200 mg/kg bw/day appeared to be tolerated well and in consultation with the Sponsor it was increased to 300 mg/kg bw/day from Day 32 (males) and Day 31 (females).
Positive control:
N-nitroso-N-methylurea
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
Groups 1 to 4 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, Groups 1 and 4 animals were observed daily. Group 5 animals were observed daily over Days 1 to 89 and on Days 90/91 individual clinical observations for these animals were performed immediately before dosing, up to thirty minutes after dosing and approximately one hour after dosing. All observations were recorded; see deviations fron Study Plan.

Body Weight
Individual body weights for Groups 1 to 4 animals were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Individual body weights for Group 5 animals were recorded on Day 1 and at weekly intervals thereafter and on Day 90. Body weights were also recorded at terminal kill. As the body weight data for Group 5 animals were primarily for health monitoring purpose and dose volume calculation, these body weights have not been reported in this study report.

Food Consumption
Food consumption was recorded for each Groups 1 to 4 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 Groups 1 to 4 non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all non-recovery Groups 1 to 4 animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery Groups 1 to 4 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 Groups 1 to 4 animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each non-recovery Groups 1 to 4 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 non-recovery Groups 1 to 4 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 Groups 1 to 4 animals were examined pre-treatment. During Week 12, the eyes of all non-recovery control and high dose animals (Groups 1 and 4, respectively) were examined. Examinations included observation of the anterior structures of the eye and 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 ophthalmoscope was performed.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery Groups 1 to 4 animals from each test and control group at the end of the study (Day 90) and on all recovery Groups 1 and 4 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 and 119; see deviations from Study Plan. Animals were not fasted prior to sampling.

Urinalytical investigations were performed on all non-recovery Groups 1 to 4 animals during Week 12 and on all recovery Groups 1 and 4 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.


Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)

Platelet count (PLT)
Reticulocyte count (Retic) - methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

Urinalysis
The following parameters were measured on collected urine:
Volume
Ketones
Specific Gravity
Bilirubin
pH
Urobilinogen
Protein
Blood
Glucose
Appearance
Sacrifice and pathology:
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 animals were killed by intravenous overdose of a suitable barbiturate agent 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 Groups 1 to 4 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 sperm concentration, 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. The cauda epididymis was also separated from the body of the epididymis, weighed and stored frozen at approximately -20°C. As there were no treatment-related effects on sperm concentration or motility, these tissues were not further analyzed for homogenization resistant spermatid count.

Morphological assessment was performed on a sample of a minimum of 200 sperm from non-recovery Groups 1 and 4 males to determine the number with apparent structural anomalies. As there were no treatment-related effects in Group 4, this was not extended to males from other dose groups.

Comet Assay
For the first five non-recovery males from Groups 1 to 4 as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed appropriately by Envigo Research Ltd., Shardlow, UK Cell and Molecular Sciences Department to provide slides for the comet assay assessment; full details of the method are provided in the comet assay report presented in Annex 3.

Organ Weights
The following organs, removed from Groups 1 to 4 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
Right/left Epididymis
Right/left Testis
Heart
Thymus
Kidneys
Uterus
Liver
Left Cauda


Histopathology
Samples of the following tissues were removed from all Groups 1 to 4 animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)•
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Right Epididymis ♦
Skin
Esophagus
Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Right Testis ♦
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 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of approximately 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. Additionally, sections of the right testis were stained with Periodic Acid Schiff stain and microscopically examined. Immunohistochemical staining of the male kidneys for α-2U-globulin was also performed.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the kidneys (males only) and stomach (males and females) 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 (V Mowat) at the Test Site (Envigo CRS Limited, Huntingdon).

Statistics:
Please see section "Any other information on materials and methods"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs considered to be related to the toxicity of the test item.

Sporadic episodes of increased post-dose salivation were detected in animals of either sex treated with 300 mg/kg bw/day from Week 2 and persisted throughout the dosing period. Individual males and females from the 100 mg/kg bw/day also showed isolated instances of increased post-dose salivation during Week 6. Such observations are common in this type of study and were considered likely to reflect an irritant nature of the test item and/or formulation. Three males and one female treated with 300 mg/kg bw/day also showed some instances of noisy respiration. These observations may be related to the irritant nature of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. This was substantiated by histopathological examination of the stomach from non-recovery animals of either sex in the 300 mg/kg bw/day dose group, which showed minimal hyperplasia in the non-glandular region, an anatomical area not present in man and as such of no toxicological relevance for risk assessment. One male from the 300 mg/kg bw/day dose group also showed staining around the snout during Week 12; due to the isolated nature of this observation, it was considered likely to be incidental.

There were no clinical signs for any of the animals receiving the test item at a dose level of 10 mg/kg bw/day. None of the positive control males showed any clinical signs throughout the study.

During the treatment-free period, there were no clinical signs for any of the recovery animals.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex throughout the study.

At 300 mg/kg bw/day, males showed statistically significantly lower group mean body weight gains in Weeks 6, 7, 10 and 12 of dosing when compared with control (p<0.01-0.05). Overall body weight gain in these males at the end of the dosing period was approximately 13% lower than controls. Following the cessation of dosing, recovery was evident, with slightly higher body weight gains seen in males previously treated with 300 mg/kg bw/day than controls over the first two weeks; statistical significance was achieved during Week 2 (p<0.05).

Males treated with 100 mg/kg bw/day also showed minor fluctuations in weekly body weight gains achieving statistical significance in some instances. There was generally no dose-relationship and although the overall body weight gain in these males at the end of the treatment period was approximately 9% lower than controls, this was considered likely to be due to normal biological variation. Occasional fluctuations in weekly group mean body weight gains were also observed in males treated with 10 mg/kg bw/day and females from all test item-treated dose groups achieving statistical significance in some instances. There was generally no dose-dependence and overall body weight gains in these animals were comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment with the test item at any dose level on food consumption or food conversion efficiency throughout the study.

At 300 mg/kg bw/day, males showed marginally lower dietary intake from Week 6 of dosing in relation to controls. Recovery was evident during the treatment-free period with food consumption for males previously treated with 300 mg/kg bw/day being generally comparable with controls. Dietary intake for males treated with 100 or 10 mg/kg bw/day and females from all test item-treated dose groups was generally comparable with controls throughout the study.

Occasional fluctuations in food conversion efficiency were evident at all dose levels and were deemed to be due to minor intergroup differences in body weight gains and/or food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not reveal any inter-group differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmoscopic examination of the non-recovery animals of either sex from the control and 300 mg/kg bw/day dose groups did not indicate any treatment-related differences.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, males treated with 300 mg/kg bw/day showed statistically significantly lower red blood cell count in relation to controls (p<0.05), but all individual values from the high dose animals were within the historical control data ranges. Group mean leukocyte counts in males treated with 100 or 300 mg/kg bw/day were also statistically significantly lower than controls (p<0.05 and p<0.01, respectively), which was mainly due to statistically significantly lower lymphocyte counts in these animals (p<0.05 and p<0.01, respectively). A dose-relationship was evident for either parameter, but most individual values and in particular from the high dose group were within the historical control data ranges. The corresponding values in recovery males previously treated with 300 mg/kg bw/day were similar to controls and in the absence of any effect on related parameters or any histopathology correlates in the non-recovery animals, these observations were considered to be of no toxicological significance.

At all dose level, non-recovery females showed statistically significant lower prothrombin and activated partial thromboplastin times when compared with controls (p<0.05 and p<0.01, respectively). These intergroup differences were without any dose-dependence and the majority of individual values including all values from the high dose females were within the historical control data ranges. In recovery females previously given 300 mg/kg bw/day, these parameters were similar to controls, and in the absence of any histopathology correlates in non-recovery animals at the end of the dosing period, these observations were considered to be of no toxicological relevance.

Group mean corpuscular hemoglobin and mean corpuscular volume in non-recovery females treated with 10 mg/kg bw/day were statistically significantly lower than controls (p<0.05). The corresponding values in females from the 100 or 300 mg/kg bw/day dose groups were similar to controls and these findings were considered to be incidental.

At the end of the twenty-eight day treatment-free period, statistically significant intergroup differences in males previously treated with 300 mg/kg bw/day included lower mean corpuscular hemoglobin concentration (p<0.01) whilst the corresponding females showed lower neutrophil and higher hemoglobin, red blood cell count and haematocrit level (p<0.05). The corresponding parameters in non-recovery animals on Day 90 of dosing were similar to controls and these intergroup differences were regarded as of no toxicological importance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood Chemistry

At the end of the dosing period, group mean bile acid concentrations in males receiving 100 or 300 mg/kg bw/day were statistically significantly lower than controls (p<0.01). A dose-relationship was evident, but most individual values for these test item-treated animals were within the historical control data ranges. It is also worth noting that some individual control values exceed the top historical control data range, which is likely to have exaggerated the intergroup differences. Non-recovery males from all dose groups also showed statistically significantly higher phosphorus levels (p<0.01) but without any dose-dependence and most individual values in particular from the high dose males were within the historical data ranges. Group mean bile acid and phosphorus level in recovery males previously given 300 mg/kg bw/day were similar to controls and these intergroup differences in non-recovery males were considered to be of no toxicological relevance.

At all dose levels, non-recovery females showed statistically significantly higher urea levels when compared with controls (p<0.05). There was no dose-relationship and creatinine levels in these animals remained similar to controls. Females treated with 100 or 300 mg/kg bw/day also showed dose-related statistically significant reductions in alkaline phosphatase activities (p<0.01-p<0.05) and potassium levels (p<0.05) in relation to controls, but individual values in most animals remained within the historical control data limits. These parameters in recovery females previously receiving 300 mg/kg bw/day were similar to controls, and in the absence of any histopathology correlates in non-recovery animals these findings were considered to be of no toxicological significance.

Any other intergroup differences attaining statistical significance in non-recovery animals were not seen in the high dose animals and were considered to be incidental.

At the end of the twenty-eight day treatment-free period, statistically significant intergroup differences for animals previously treated with 300 mg/kg bw/day included higher glucose and chloride levels in males (p<0.05 and p<0.01, respectively) and lower phosphorus concentration in females (p<0.05). The corresponding parameters in non-recovery animals of the relevant sex on Day 90 of dosing were similar to controls and these intergroup differences were considered to be of no toxicological importance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment with the test item at any dose level in animals of either sex on urinalysis parameters evaluated towards the end of the treatment or treatment-free periods.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in the behavioral parameters considered to be related to treatment with the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, non-recovery males showed statistically significant increases in absolute and body weight-related kidney weights in comparison with controls (p<0.01). The majority of individual values from these test item-treated animals exceeded the historical control data ranges and this finding was considered likely to be associated with microscopic changes in the kidneys which were indicative of α-2u-globulin nephropathy, a species- and sex-specific condition in the male rat. Following the dose-free period of four weeks, increased kidney weights were still apparent in males previously receiving 300 mg/kg bw/day (p<0.05), but to a lower extent and hyaline droplet accumulation in these males appeared to have reversed to background levels although resultant pathology was still apparent indicating partial recovery.

At the end of the treatment period, animals of either sex given the test item at 300 mg/kg bw/day and females receiving 100 mg/kg bw/day showed statistically significantly higher absolute and body weight-related liver weights in relation to controls (p<0.01 and p<0.05, respectively). A dose-relationship was evident between 100 and 300 mg/kg bw/day females for the liver weights and individual values in particular the body weight-related weights from the affected test item-treated animals were generally above the historical control data ranges. The corresponding values in recovery males and females previously given 300 mg/kg bw/day were comparable with controls and in the absence of any histopathology correlates, this finding was considered to be of no toxicological significance.

At the end of the treatment-free period, recovery females previously receiving 300 mg/kg bw/day showed statistically significantly higher absolute and body weight-related adrenal weights in relation to controls (p<0.05). Group mean absolute and body weight-related thymus weights in these females were statistically significantly lower than controls (p<0.05). The corresponding values in males from this dose groups were similar to controls whilst no statistically significant intergroup differences were detected for these parameters in non-recovery animals of either sex at the end of dose administration period. There were no associated histopathology observations in non-recovery animals and these findings were considered not to represent an effect of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reddened lungs were observed in a number of non-recovery males and females across all dose groups including controls. No dose-relationship was evident and recovery animals of either sex from control and test item-treated dose groups also showed similar findings. Such observations are common in this type of study and were considered unlikely to be related to treatment with the test item. Other incidental findings included incidences of increased pelvic space in one or both kidneys in one control female, two females treated with 10 mg/kg bw/day and one male and one female treated with 300 mg/kg bw/day and small right testis and fluid filled left horn of the uterus in one male and female treated with 100 mg/kg bw/day respectively.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were observed in the kidneys (males only) and the stomach (males and females). The findings were as follows:
Kidney
• Multifocal basophilic tubules (degenerating/regenerating, minimal-moderate) and increased hyaline droplets (mild or moderate) were recorded in 10/10 Group 4 males.
• Proteinaceous casts (minimal to moderate) were recorded in 9/10 Group 4 males.
• In Group 3 males multifocal basophilic tubules (minimal or mild) were present in 5/10 animals with increased hyaline droplets in 4/10 of these males. Proteinaceous casts (minimal) were present in 2/10 animals.
• The Group 2 animals were comparable to controls.
• Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels in animals from Groups 3 and 4.
• In recovery males in Group 4 changes persisted with proteinaceous casts present in 8/10 animals and chronic nephropathy (basophilic tubules, fibrosis, tubular atrophy, tubular dilation) minimal or mild in 8/10 animals.
• Immunohistochemical staining was positive for α-2u-globulin in all males but increased staining levels in Group 4 persisted in males previously treated with the test item relative to controls.
Stomach
• Minimal hyperplasia of the non-glandular region was noted in 6/10 males and 3/10 females in Group 4 only and had resolved after the recovery period.
No other findings were present at histopathology which correlated with in-life changes or showed relationship to the test item. In particular, the right testis and epididymis of males in Group 4 were similar to control animals at a histological level, including following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related abnormalities in the integrity of the various cell types present within the different stages of sperm cycle).
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.

Functional performance evaluations during Week 12 of dosing identified females from all dose groups showing statistically significantly lower hindlimb grip strength when compared with controls (p<0.01). As there was no dose-relationship and this finding was only evident in 1/3 tests, it was considered to be unrelated to treatment with the test item. Motor activity assessment during this period also identified statistically significantly higher overall activity for females treated with 10 or 100 mg/kg bw/day in relation to controls (p<0.05). The corresponding values for females treated with 300 mg/kg bw/day and males from all test item-treated dose groups were similar to controls and in the absence of any apparent clinical signs of neurotoxicity, this observation was deemed likely to be incidental.


Estrous Cycling
There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the last three weeks of dosing.


Sperm Analysis
At all dose levels, there were no differences in group mean sperm concentration and motility at the end of treatment or treatment-free periods. An evaluation of sperm morphology in non-recovery males from the control and 300 mg/kg bw/day dose groups also did not reveal any treatment-related differences.


Comet Assay Assessment
The following findings were noted in non-recovery males:

Liver
At all dose levels, small increases in the mean and median percentage tail intensities were noted in males relative to vehicle controls achieving statistical significance in most instances at 100 and 300 mg/kg bw/day (p<0.001-p<0.01). There was no strict dose-relationship but most individual values exceeded the historical vehicle control data ranges. Although the overall results were considered not to fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to prove that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ruled out.

There were no appreciable dose-related increases in the percentage hedgehog frequencies in the liver.

Glandular Stomach
At 10 or 300 mg/kg bw/day, small increases in the mean and median percentage tail intensities were observed in non-recovery males when compared with vehicle controls. There was no dose-relationship and the mean percentage tail intensity value in the 10 mg/kg bw/day only marginally exceeded the historical vehicle control data range whilst the median percentage tail intensity for this dose group was at the upper limit of this range. Although the data from the 300 mg/kg bw/day dose group showed appreciable increases over vehicle controls, these were mainly attributed to one animal showing considerably higher values than the remaining animals in the group; when values for this animal were excluded from the data, the mean percentage tail intensity for the group was within the historical control data ranges and the test item was considered not to induce DNA damage to the glandular stomach tissue.

There were no appreciable dose-related increases in the percentage hedgehog frequencies in the glandular stomach.

Jejunum
Treatment with the test item up to a dose level of 300 mg/kg bw/day did not induce any treatment-related changes in the percentage mean or median tail intensities in the male jejunum when compared with vehicle control.

No dose-related increases in the percentage hedgehog frequencies were observed at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Please refer to "Remarks"
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
other: Please refer to "Any other information on results"
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Kidneys

At the end of the dose administration period, hyaline droplets were present in males treated with 100 or 300 mg/kg bw/day. Multifocal basophilic tubules and proteinacious casts were also observed in these males. Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels in males treated with 100 or 300 mg/kg bw/day. At the end of the recovery phase, males previously treated with 300 mg/kg bw/day still showed proteinaceous casts and chronic nephropathy (basophilic tubules, fibrosis, tubular atrophy, tubular dilation). Immunohistochemical staining was positive for α-2u-globulin in all recovery males with males previously receiving 300 mg/kg bw/day showing increased staining levels relative to controls.

Group Mean Hematological Values

                                                                                                                                                      

                                       Group          Hb     RBC     Hct   MCH    MCV   MCHC   WBC      Neut                                         

                                      (Sex)          g/dl  10^12/l  %     pg     fl     g/dl  10^9/l   10^9/l                                        

                                                                                                                                                              

                                      1(M)   Mean   16.56   9.281  48.70 17.85  52.48  34.01   8.24    1.777                                        

                                              S.D.    0.58   0.350   1.71  0.61   0.94   0.68   1.15    0.547                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      2(M)   Mean   16.97   9.288  49.56 18.29  53.40  34.25   7.86    1.492                                        

                                              S.D.    0.39   0.309   1.26  0.69   1.78   0.57   1.65    0.476                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      3(M)   Mean   16.76   9.161  49.34 18.31  53.90  33.97   7.05*   1.592                                        

                                              S.D.    0.59   0.345   1.45  0.82   1.78   0.51   1.39    0.701                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      4(M)   Mean   16.15   8.872* 47.38 18.22  53.44  34.09   6.53**  1.551                                        

                                              S.D.    0.46   0.366   1.42  0.48   1.43   0.47   1.06    0.518                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                                                                                                                                              

   

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                                

 

(continued)           Group Mean Hematological Values

                                                                                                                                                             

                                       Group         Lymph     Mono    Eos    Bas      CT      PLT    APTT                                            

                                      (Sex)          10^9/l   10^9/l  10^9/l 10^9/l  Seconds  10^9/l Seconds                                          

                                                                                                                                                              

                                      1(M)   Mean    6.337    0.015   0.114  0.000n    9.39   616.8   14.59                                          

                                              S.D.    0.784    0.032   0.101  0.000     0.62    73.7    1.98                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      2(M)   Mean    6.325    0.007   0.063  0.000n    9.33   632.8   15.85                                          

                                              S.D.    1.516    0.022   0.096  0.000     0.70    76.5    1.02                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      3(M)   Mean    5.413*   0.007   0.038  0.000n    8.87   594.4   14.77                                          

                                              S.D.    1.257    0.022   0.055  0.000     1.17   124.6    2.00                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      4(M)   Mean    4.928**  0.014   0.040  0.000n    9.68   673.0   15.27                                          

                                              S.D.    0.777    0.030   0.057  0.000     0.36    58.8    1.23                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                                                                                                                                              

              

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                       

 


(continued)           Group Mean Hematological Values

                                                                                                                                                             

                                       Group          Hb     RBC     Hct   MCH    MCV   MCHC   WBC      Neut                                         

                                      (Sex)          g/dl  10^12/l  %     pg     fl     g/dl  10^9/l   10^9/l                                        

                                                                                                                                                              

                                      1(F)   Mean   15.62   8.208  46.11 19.05  56.19  33.87   4.53    0.883                                        

                                              S.D.    0.40   0.271   1.10  0.64   1.44   0.69   1.26    0.398                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      2(F)   Mean   15.12   8.194  44.65 18.42* 54.49* 33.83   4.89    1.211                                        

                                              S.D.    1.80   0.950   5.37  0.49   1.19   0.37   0.82    0.352                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      3(F)   Mean   15.72   8.282  46.44 18.97  56.08  33.83   4.52    1.102                                        

                                              S.D.    0.56   0.332   1.54  0.64   1.29   0.66   1.00    0.265                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                      4(F)   Mean   15.44   8.290  45.75 18.62  55.22  33.74   4.14    1.172                                        

                                              S.D.    0.47   0.291   1.67  0.40   1.08   0.34   1.62    0.427                                        

                                              N       10    10       10    10     10     10    10       10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                        

                                                                                                                                                              

          

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day

        (continued)           Group Mean Hematological Values

                                                                                                                                                             

                                       Group         Lymph     Mono    Eos    Bas      CT      PLT    APTT                                            

                                      (Sex)          10^9/l   10^9/l  10^9/l 10^9/l  Seconds  10^9/l Seconds                                          

                                                                                                                                                              

                                      1(F)   Mean    3.606    0.004n  0.039  0.000n    8.72   652.4   15.05                                          

                                              S.D.    0.932    0.013   0.055  0.000     0.44    96.6    0.74                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      2(F)   Mean    3.649    0.000n  0.032  0.000n    8.38*  668.0   12.79**                                        

                                              S.D.    0.646    0.000   0.051  0.000     0.24   102.0    0.96                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      3(F)   Mean    3.387    0.000n  0.030  0.000n    8.18*  685.6   11.97**                                        

                                              S.D.    0.820    0.000   0.037  0.000     0.24    97.0    1.03                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                      4(F)   Mean    2.930    0.015n  0.028  0.000n    8.50*  665.1   14.98**                                        

                                              S.D.    1.268    0.020   0.040  0.000     0.30    74.1    0.85                                          

                                              N       10       10      10     10       10       10     10                                             

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

           

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                         

 


(continued)           Group Mean Hematological Values

Recovery                                                                                                                                                      

                                       Group          Hb      RBC     Hct    MCH   MCV  MCHC     WBC    Neut                                         

                                      (Sex)          g/dl   10^12/l  %      pg    fl    g/dl    10^9/l 10^9/l                                        

                                                                                                                                                              

                                      1(M)   Mean   16.31    9.144  48.64  17.86 53.20 33.55     7.02  1.103                                        

                                              S.D.    0.43    0.265   1.25   0.69  1.64  0.32     0.75  0.222                                        

                                              N       10     10       10     10    10    10      10     10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒƒ ƒƒƒƒƒƒ                                        

                                      4(M)   Mean   16.10    9.279  48.66  17.33 52.44 33.08**   6.55  1.320                                        

                                              S.D.    0.53    0.348   1.56   0.41  0.95  0.32     1.57  0.884                                        

                                              N       10     10       10     10    10    10      10     10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒƒ ƒƒƒƒƒƒ                                        

                                            

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                       

 

(continued)           Group Mean Hematological Values

Recovery                                                                                                                                                     

                                          Group         Lymph   Mono    Eos    Bas      CT     PLT    APTT                                            

                                         (Sex)          10^9/l 10^9/l  10^9/l 10^9/l  Seconds 10^9/l Seconds                                          

                                                                                                                                                              

                                         1(M)   Mean    5.863  0.000n  0.056  0.000n    9.38  615.5   15.07                                          

                                                 S.D.    0.738  0.000   0.055  0.000     0.50   51.4    1.24                                          

                                                 N       10     10      10     10       10      10     10                                             

                                          ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                         4(M)   Mean    5.146  0.000n  0.087  0.000n    9.57  618.4   14.77                                          

                                                 S.D.    1.121  0.000   0.101  0.000     0.67   72.4    1.54                                          

                                                 N       10     10      10     10       10      10     10                                             

                                          ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                                                  

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                 

 

(continued)           Group Mean Hematological Values

Recovery                                                                                                                                                     

                                       Group          Hb      RBC     Hct    MCH   MCV  MCHC     WBC    Neut                                         

                                      (Sex)          g/dl   10^12/l  %      pg    fl    g/dl    10^9/l 10^9/l                                        

                                                                                                                                                              

                                      1(F)   Mean   15.08    7.863  44.61  19.28 57.02 33.82     4.00  0.716                                        

                                              S.D.    1.39    1.005   4.16   1.05  2.77  0.38     0.78  0.306                                        

                                              N       10     10       10     10    10    10      10     10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒƒ ƒƒƒƒƒƒ                                        

                                      4(F)   Mean   16.09*   8.606* 47.62* 18.72 55.39 33.78     3.33  0.445*                                       

                                              S.D.    0.55    0.419   1.65   0.76  1.73  0.45     0.92  0.169                                        

                                              N       10     10       10     10    10    10      10     10                                           

                                       ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ  ƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒƒ ƒƒƒƒƒƒ                                        

                                                                            

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                     

 

continued)           Group Mean Hematological Values

Recovery                                                                                                                                                     

                                          Group         Lymph   Mono    Eos    Bas      CT     PLT    APTT                                            

                                         (Sex)          10^9/l 10^9/l  10^9/l 10^9/l  Seconds 10^9/l Seconds                                          

                                                                                                                                                              

                                         1(F)   Mean    3.249  0.000n  0.037  0.000n    8.95  619.5   13.82                                          

                                                 S.D.    0.812  0.000   0.041  0.000     0.76  103.9    1.30                                          

                                                 N       10     10      10     10       10      10     10                                             

                                          ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

                                         4(F)   Mean    2.850  0.000n  0.037  0.000n    8.98  604.6   15.01                                          

                                                 S.D.    0.817  0.000   0.038  0.000     1.14   57.8    1.69                                          

                                                 N       10     10      10     10       10      10     10                                             

                                          ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒƒ                                          

            

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                 

 

Group Mean Blood Chemical Values

                                                                                                                                                              

                                                                  Tot.                                                                                       

                                  Group         Urea   Glucose Prot. Albumin  A/G   Na+     K+      Clƒ     Ca++                                    

                                 (Sex)          mg/dl  mg/dl   g/dl  g/dl    Ratio mmol/l mmol/l   mmol/l  mmol/l                                   

                                                                                                                                                              

                                 1(M)   Mean    40.0    166.5 6.647    3.84 1.374  147.1  4.494    102.6   1.744                                   

                                         S.D.     3.9     16.4 0.181    0.10 0.091    1.6  0.280      1.5   0.049                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 2(M)   Mean    41.9    160.2 6.694    3.77 1.314  147.3  5.554**  102.1   1.675                                   

                                         S.D.     5.3     23.7 0.983    0.42 0.172    3.4  0.881      1.5   0.111                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 3(M)   Mean    42.2    169.4 6.237    3.64 1.404  146.8  4.456    100.6*  1.700                                   

                                         S.D.     3.5     18.4 0.363    0.24 0.087    2.0  0.361      1.5   0.060                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 4(M)   Mean    40.0    162.7 6.569    3.90 1.471  147.8  4.826    103.2   1.715                                   

                                         S.D.     5.4     16.5 0.268    0.15 0.089    2.4  0.512      1.9   0.087                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

       

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                    

 

(continued)           Group Mean Blood Chemical Values

                                                                                                                                                             

                                      Group           P      ASAT  ALAT   AP     Creat Chol  Bili   Bile Acid                                         

                                     (Sex)          mmol/l   IU/l  IU/l  IU/l    mg/dl mg/dl mg/dl  µmol/l                                           

                                                                                                                                                              

                                     1(M)   Mean     1.61    74.7  52.5 169.7   0.709  90.7 0.124     15.08                                         

                                             S.D.     0.36    13.1   5.1  22.4   0.067  13.5 0.028      7.08                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     2(M)   Mean     2.16**  90.5  51.7 172.7   0.670  91.9 0.071*    11.66                                         

                                             S.D.     0.37    24.5   6.9  65.5   0.133  10.8 0.063      5.27                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     3(M)   Mean     2.14**  71.0  54.6 159.7   0.673  79.3 0.136      8.54**                                       

                                             S.D.     0.18    14.9  10.6  42.3   0.052  10.8 0.041      4.44                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     4(M)   Mean     1.80**  75.5  48.2 160.9   0.734  81.7 0.102      7.63**                                       

                                             S.D.     0.16    12.1   5.1  24.8   0.134  11.0 0.025      2.92                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

           

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                     

 


(continued)           Group Mean Blood Chemical Values

                                                                                                                                                             

                                                                  Tot.                                                                                       

                                  Group         Urea   Glucose Prot. Albumin  A/G   Na+     K+      Clƒ     Ca++                                    

                                 (Sex)          mg/dl  mg/dl   g/dl  g/dl    Ratio mmol/l mmol/l   mmol/l  mmol/l                                   

                                                                                                                                                              

                                 1(F)   Mean    41.6    148.2 7.421    4.60 1.637  149.4  4.377    104.5   2.062                                   

                                         S.D.     4.7     18.9 0.374    0.29 0.125    2.3  0.255      2.9   0.087                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 2(F)   Mean    47.3*   154.9 7.132    4.53 1.731  148.3  4.243    104.4   1.983                                   

                                         S.D.     7.2     18.9 0.353    0.22 0.105    2.9  0.347      1.8   0.099                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 3(F)   Mean    48.3*   146.0 7.314    4.57 1.669  149.2  4.104*   103.7   2.012                                   

                                         S.D.     4.4     15.9 0.275    0.20 0.127    2.9  0.254      2.8   0.122                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

                                 4(F)   Mean    46.7*   141.9 7.532    4.78 1.737  149.4  4.055*   104.0   2.060                                   

                                         S.D.     6.4     15.2 0.342    0.30 0.129    2.0  0.223      2.1   0.091                                   

                                         N       10       10    10     10     10     10    10        10     10                                      

                                  ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ  ƒƒƒƒƒƒ                                   

            

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day                      

 


(continued)           Group Mean Blood Chemical Values

                                                                                                                                                             

                                      Group           P      ASAT  ALAT   AP     Creat Chol  Bili   Bile Acid                                         

                                     (Sex)          mmol/l   IU/l  IU/l  IU/l    mg/dl mg/dl mg/dl  µmol/l                                           

                                                                                                                                                              

                                     1(F)   Mean     1.40    79.0  52.3  85.0   0.933 106.7 0.093     15.53                                         

                                             S.D.     0.35    20.7   8.7  18.7   0.070  13.5 0.016     12.40                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     2(F)   Mean     1.46    68.3  50.6  74.7   0.966  97.7 0.089     12.33                                         

                                             S.D.     0.20    10.7   9.2  33.3   0.101  25.2 0.014      6.40                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     3(F)   Mean     1.78    73.0  53.6  67.0*  0.880  98.0 0.095     17.40                                         

                                             S.D.     0.50    13.3  13.1  18.6   0.097  13.4 0.034      9.46                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

                                     4(F)   Mean     1.56    74.1  48.3  55.8** 0.951  94.4 0.090     14.89                                         

                                             S.D.     0.40    17.2   8.8   8.6   0.064  17.9 0.015      8.73                                         

                                             N       10       10    10    10      10    10    10       10                                            

                                      ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ   ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ  ƒƒƒƒƒƒƒƒ                                         

   

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 10 mg/kg bw/day   Group 3 ƒ 100 mg/kg bw/day   Group 4 ƒ 200/300 mg/kg bw/day             

 

(continued)           Group Mean Blood Chemical Values

Recovery                                                                                                                                                      

                                                                   Tot.                                                                                      

                                   Group         Urea  Glucose  Prot. Albumin  A/G   Na+     K+    Clƒ      Ca++                                    

                                  (Sex)          mg/dl mg/dl    g/dl  g/dl    Ratio mmol/l mmol/l mmol/l   mmol/l                                   

                                                                                                                                                              

                                  1(M)   Mean    46.9   160.1  6.884    3.63 1.118  143.3  4.791  104.0    1.766                                   

                                          S.D.     5.1    14.9  0.274    0.13 0.039   18.8  0.203    1.4    0.046                                   

                                          N       10      10     10     10     10     10    10      10      10                                      

                                   ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                   

                                  4(M)   Mean    48.4   176.2* 6.899    3.64 1.126  149.6  4.899  105.9**  1.753                                   

                                          S.D.     4.1    14.5  0.378    0.15 0.083    1.5  0.299    1.4    0.042                                   

                                          N       10      10     10     10     10     10    10      10      10                                      

                                   ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                   

                                                        

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                               

 

(continued)           Group Mean Blood Chemical Values

Recovery                                                                                                                                                      

                                         Group           P     ASAT  ALAT   AP   Creat Chol  Bili  Bile Acid                                          

                                        (Sex)          mmol/l  IU/l  IU/l  IU/l  mg/dl mg/dl mg/dl µmol/l                                            

                                                                                                                                                              

                                        1(M)   Mean     1.69   94.8  66.1 113.2 0.725  93.3 0.101     8.01                                          

                                                S.D.     0.19   22.8  18.9  23.3 0.041   9.7 0.023     5.92                                          

                                                N       10      10    10    10    10    10    10      10                                             

                                         ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒƒ                                          

                                        4(M)   Mean     1.62   99.0  71.5 123.8 0.791  86.8 0.101     6.61                                          

                                                S.D.     0.14   43.5  30.9  23.9 0.134  12.6 0.020     2.59                                          

                                                N       10      10    10    10    10    10    10      10                                             

                                         ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒƒ                                          

                                                                        

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                     

 

(continued)           Group Mean Blood Chemical Values

Recovery                                                                                                                                                      

                                                                   Tot.                                                                                      

                                   Group         Urea  Glucose  Prot. Albumin  A/G   Na+     K+    Clƒ      Ca++                                    

                                  (Sex)          mg/dl mg/dl    g/dl  g/dl    Ratio mmol/l mmol/l mmol/l   mmol/l                                   

                                                                                                                                                              

                                  1(F)   Mean    41.2   144.9  7.022    4.12 1.418  145.7  5.469  102.2    1.697                                   

                                          S.D.     4.8    18.5  0.594    0.40 0.054    3.1  3.179    3.0    0.161                                   

                                          N       10      10     10     10     10     10    10      10      10                                      

                                   ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                   

                                  4(F)   Mean    45.6   150.0  7.449    4.29 1.356  148.3  4.230  104.3    1.797                                   

                                          S.D.     7.2    18.9  0.513    0.36 0.080    3.1  0.429    3.0    0.058                                   

                                          N        9       9      9      9      9      9     9       9       9                                      

                                   ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ   ƒƒƒƒƒƒ                                   

                                                                      

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                     

 

(continued)           Group Mean Blood Chemical Values

Recovery                                                                                                                                                     

                                         Group           P     ASAT  ALAT   AP   Creat Chol  Bili  Bile Acid                                          

                                        (Sex)          mmol/l  IU/l  IU/l  IU/l  mg/dl mg/dl mg/dl µmol/l                                            

                                                                                                                                                              

                                        1(F)   Mean     1.35   88.6  49.4  50.1 0.781  84.7 0.081     8.52                                          

                                                S.D.     0.16   28.5   6.0  14.6 0.116  17.2 0.045     6.16                                          

                                                N       10      10    10    10    10    10    10      10                                             

                                         ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒƒ                                          

                                        4(F)   Mean     1.12*  74.9  55.7  58.6 0.833  88.1 0.079     7.37                                          

                                                S.D.     0.29   13.1   9.0  12.4 0.074  12.0 0.015     5.15                                          

                                                N        9       9     9     9     9     9     9       9                                             

                                         ƒƒƒƒƒ ƒƒƒƒƒƒ ƒƒƒƒƒƒ  ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒ ƒƒƒƒƒƒƒƒ                                          

                                                                                

Dose Levels: Group 1 ƒ 0 (Control)   Group 4 ƒ 200/300 mg/kg bw/day                    

Conclusions:
Excluding the findings from the comet assay assessment, the oral (gavage) administration of bisisobutyryl peroxide (CAS# 3437-84-1) to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels of 10, 100 or 300 mg/kg bw/day was well tolerated. There were no findings of toxicological significance in animals of either sex and a dose level of 300 mg/kg bw/day could therefore be established as a ‘No Observed Adverse Effect Level’ (NOAEL) for general systemic toxicity within the confines of this type of study (OECD 408).

The comet assessment in male rats treated with the test item up to a dose level of 300 mg/kg bw/day resulted in possible treatment-related increases in the mean and median tail intensities in the liver. Although these observations did not fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to show that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ignored. Taking these results into consideration, a NOAEL cannot be established in the male within the constraints of the study design where comet assay evaluations were only performed in males.

Executive summary:

Introduction

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

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

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

The study design also took into account elements from the following guideline:

·        In vivoMammalian Alkaline Comet Assay” (Adopted 26 September 2014)

Methods

The test item was administered by oral gavage to three groups of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, at dose levels of 10, 100 or 200/300 mg/kg bw/day. Animals from the high dose group were dosed at 200 mg/kg bw/day up to Day 30 (females) or Day 31 (males) with the dose level increased to 300 mg/kg bw/day thereafter; as these animals were dosed at 300 mg/kg bw/day for approximately 2/3 of the dosing period, the dose level for this group will generally be referred to as 300 mg/kg bw/day throughout this study report. A control group of ten males and ten females was dosed with the vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (200/300 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days. The first five males from each non-recovery dose group (Groups 1 to 4) were dosed for ninety-one consecutive days whilst the remaining males and females from these dose groups were dosed for ninety consecutive days. An additional group of five males (Group 5), used as positive control for comet assay assessment, was administered with 25 mg/kg bw/day N-Nitroso-N-methylurea by oral gavage on Days 90 and 91. Animals treated on Days 90 and 91 were dosed approximately twenty-seven and three hours (respectively) before euthanasia and selected tissues from these animals were used for comet assay assessment.

Clinical signs, functional observations, body weight change and dietary intake were monitored during the study. Hematology and blood chemistry were evaluated for all Groups 1 to 4 animals at the end of treatment and treatment-free periods. Urinalysis was performed for all Groups 1 to 4 non-recovery animals during Week 12 of dosing and for all recovery animals during the last week of the treatment-free period. Ophthalmoscopic examination was also performed on all Groups 1 to 4 animals prior to the start of treatment and on all non-recovery control and high dose animals during Week 12 of the study. Additionally, the stage of estrous was monitored for all non-recovery females during the last three weeks 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 (Groups 1 and 4) as well as any gross lesions from Groups 1 to 4 animals was performed in the first instance. As there were treatment-related findings in the kidneys (males only) and the stomach (males and females), examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups as well as recovery groups.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Throughout the treatment period, there were no clinical signs indicative of test item toxicity.

Behavioral Assessment

Behavioral assessment scores across the test-item treated animals of either sex remained similar to controls.

Functional Performance Tests

There were no treatment-related changes in functional performance for animals of either sex at any dose level.

Sensory Reactivity Assessments

Sensory reactivity scores were comparable across all dose groups including controls.

Body Weight

There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex. Overall body weight gain in non-recovery males treated with 300 mg/kg bw/day was approximately 13% lower than control at the end of dosing, but recovery was evident during the treatment-free period, and this finding was deemed to be related to irritant properties of the test item and not an indication of its systemic toxicity.

Food Consumption

There was no adverse effect of treatment with the test item at any dose level on food consumption or food conversion efficiency in animals of either sex. Marginally lower dietary intake in males treated with 300 mg/kg bw/day from Week 6 of dosing was considered to be due to irritant properties of the test item rather than an indication of its systemic toxicity.


Water Consumption

When compared with controls, visual inspection of water bottles did not reveal any intergroup differences in animals of either sex receiving the test item.

Estrous Cycling

There was no adverse effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the last three weeks of dosing.

Ophthalmoscopy

Ophthalmoscopic examination of non-recovery males and females from the control and 300 mg/kg bw/day dose groups during Week 12 of dosing did not reveal any treatment-related differences.

Hematology

Hematology evaluations at the end of the treatment or treatment-free periods did not reveal any toxicologically significant effects in animals of either sex resulting from treatment with the test item.

Blood Chemistry

Blood chemistry evaluations at the end of the treatment or treatment-free periods did not indicate any effects of toxicological relevance in animals of either sex resulting from test item administration.

Urinalysis

Urinalytical evaluations did not identify any treatment-related effects in males or females receiving the test item up to a dose level of 300 mg/kg bw/day.

Necropsy

There were no macroscopic observations at terminal necropsy considered to be related to treatment with the test item.

Sperm Analysis

At the end of the treatment or treatment-free periods, sperm concentration, motility and progressive motility across all test item-treated male dose groups were similar to controls. Morphological sperm assessment also did not identify any treatment-related differences between non-recovery males from the control and 300 mg/kg bw/day dose groups.

Comet Assay

Treatment with the test item up to a dose level of 300 mg/kg bw/day was considered not to induce any treatment-related increases in the mean or median percentage tail intensities in the jejunum or the glandular stomach from the male rat and as such was considered to be non-genotoxic to these tissues. 

At all dose levels, increases in the mean and median percentage tail intensities in the liver were observed achieving statistical significance in most instances at 100 and 300 mg/kg bw/day. Although the overall results from comet assay assessment were considered not to fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to prove that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ruled out.

Organ Weights

Increased kidney weights in non-recovery males from the 300 mg/kg bw/day dose groups, which persisted in the recovery males previously receiving 300 mg/kg bw/day albeit to a lower extent, were considered to be associated with α-2u-globulin nephropathy syndrome in these males. The increase in liver weights observed in non-recovery animals of either sex receiving 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed complete reversibility in recovery animals and was not associated with any histopathology findings.

Histopathology

Treatment-related findings were recorded in the kidneys of males and the stomach of males and females. Some of the changes in the kidneys persisted after the twenty-eight day recovery period but the changes in the stomach had resolved. The histopathology findings were as following:

Kidneys

At the end of the dose administration period, hyaline droplets were present in males treated with 100 or 300 mg/kg bw/day. Multifocal basophilic tubules and proteinacious casts were also observed in these males. Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels in males treated with 100 or 300 mg/kg bw/day. At the end of the recovery phase, males previously treated with 300 mg/kg bw/day still showed proteinaceous casts and chronic nephropathy (basophilic tubules, fibrosis, tubular atrophy, tubular dilation). Immunohistochemical staining was positive for α-2u-globulin in all recovery males with males previously receiving 300 mg/kg bw/day showing increased staining levels relative to controls.

Stomach

At the end of the dosing period, minimal hyperplasia of the non-glandular region was noted in most males and some females treated with 300 mg/kg bw/day. This change had resolved after the recovery period.

Conclusion

Excluding the findings from the comet assay assessment, the oral (gavage) administration of bisisobutyryl peroxide (CAS# 3437-84-1) to male and female Wistar Han:RccHan:WIST strain rats at dose levels of 10, 100 or 300 mg/kg bw/day was well tolerated. There were no findings of toxicological significance in animals of either sex and a dose level of 300 mg/kg bw/day could therefore be established as a ‘No Observed Adverse Effect Level’ (NOAEL) for general systemic toxicity within the confines of this type of study (OECD 408).

The comet assessment in male rats treated with the test item up to a dose level of 300 mg/kg bw/day resulted in possible treatment-related increases in the mean and median tail intensities in the liver. Although these observations did not fully meet the requirements of the OECD 489 guideline for a positive response, in the absence of any evidence to show that the effect was due to anything other than DNA damage, a relationship to treatment with the test item cannot be ignored. Taking these results into consideration, a NOAEL cannot be established in the male within the constraints of the study design where comet assay evaluations were only performed in males.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 November 2011 - 06 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 150-179 grams; females: 118-147 grams)
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 – 21.8
- Humidity (%): 38 – 61 (90 on 22 December 2011)
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests and/or ophthalmoscopic examinations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 9 December 2011 - 06 January 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance and vehicle were kept on crushed ice as much as possible prior to weighing (the test substance was not acclimated to environmental conditions). Formulations (w/w) were prepared daily immediately prior to dosing, and both vehicle for the control group and formulations were kept on crushed ice during/after formulation until dosing. No correction was made for the purity of the test substance. Adjustment was made for specific gravity of the vehicle and density of the test substance (assumed to be 1 g/mL under cooled conditions since the density of 0.830 g/mL at 20°C supplied by the sponsor was considered not representative for the lower temperature of the test substance used in this study).

On Day 1, the vehicle for group 1 was not placed on ice during dosing. Evaluation: Not critical for maintaining the integrity of the vehicle. As this occurred only once, this does not adversely affect interpretation of the study results.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX and on information from the Sponsor.

DOSE VOLUME:
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX project 498271). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 0.25 hours on ice under normal laboratory light conditions was also determined (highest and lowest concentration). Separate formulations were prepared for analytical sampling, to allow the formulations prepared for dosing to be used immediately after formulation. The formulations for analytical sampling were prepared in a similar manner as those intended for dosing, and were kept on crushed ice until analytical sampling. Analytical sampling was conducted as soon as possible after formulation. After analytical sampling, these formulations were discarded. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%, i.e. 95-101%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when kept on crushed ice under normal laboratory light conditions for at least 15 minutes.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 d/w.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels are based on results of a 7-day dose range finding study with Bisisobutyryl peroxide (NOTOX project 498268).
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: At least twice daily. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals before dosing, clinical observations were conducted outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded.
Arena observations at the end of weeks 1, 2 and 3 were conducted before dosing instead of after dosing, and no arena observation was conducted at the end of week 4. In the dose range finding study, no clear peak effect of occurrence of clinical signs was noted Therefore, the timing of conduct of the arena observations was not critical. Based on the conducted arena observations, an adequate interpretation of the study results could still be made.

BODY WEIGHT: yes
- Time schedule for examinations: Weekly.

FOOD EFFICIENCY: yes
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY:
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY:
According to test guidelines
One of the coagulating glands of one animal of Group 4 was not available for histopathology. Sufficient data was available for evaluation.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY AND CLINICAL SIGNS
One female at 1000 mg/kg was found dead on Day 16. No further mortality occurred during the study period.
No toxicologically relevant clinical signs were noted during the observation period. Salivation seen after dosing among all animals at 1000 mg/kg, and several males and all females at 300 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.
Incidental findings that were noted included hunched posture (one male at 1000 mg/kg on the last two treatment days only), scabs (one male and two females of the control group, and one male at 300 mg/kg). These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were noted. The statistically significant lower body weight gain of males at 1000 mg/kg was minor in nature and was not consistently seen over the treatment period. No toxicological relevance was therefore ascribed to this change.

FOOD EFFICIENCY
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematology parameters were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained essentially within the range considered normal for rats of this age and strain. These changes consisted of higher red blood cell counts of males at 300 mg/kg and of females at 100 mg/kg, higher haemoglobin and haematocrit levels of males at 300 mg/kg, lower platelet counts of males at 100 mg/kg, and lower mean corpuscular haemoglobin (MCH) level of females at 100 mg/kg.

CLINICAL CHEMISTRY
Males at 1000 mg/kg showed a statistically significant higher urea and creatinine level. The statistically significant higher alanine aminotransferase activity (ALAT) in males at 1000 mg/kg was related to a slightly higher value of a single animal. A value of similar magnitude was also found in one male at 300 mg/kg. Since a group response was absent, these variations were considered to be of no toxicological relevance. The statistically significant lower aspartate aminotransferase activity (ASAT) of females at 1000 mg/kg was slight in nature and the opposite effect (i.e. an increase) would be expected in case of target organ toxicity. The statistically significant higher total protein level of females at 100 and 1000 mg/kg and the lower total bilirubin level of females at 100, 300 and 1000 mg/kg occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance.

NEUROBEHAVIOUR
Motor activity (total movements and ambulations) of females at 1000 mg/kg was reduced with statistical significance (approximately 35% lower than control means). Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
Males and females at 1000 mg/kg showed a statistically significant higher liver weight and liver to body weight ratio. Absolute liver weights were increased by approximately 29 and 38% for males and females respectively, compared to controls. Other statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance since a dose-related trend was absent and means were essentially within the range considered normal for rats of this age and strain. These changes consisted of higher kidney weight and kidney to body weight ratio of males at 300 mg/kg, lower spleen weight and spleen to body weight ratio of females at 100 mg/kg, higher uterus weight and uterus to body weight ratio of females at 300 mg/kg (corresponding to the statistically significant higher incidence of fluid in the uterus noted at necropsy), and higher brain to body weight ratio of females at 300 mg/kg.

GROSS PATHOLOGY
One male at 1000 mg/kg showed enlargement of the liver and an irregular surface of the forestomach. Enlargement of the liver was also noted for the female at 1000 mg/kg found dead on Day 16. The incidence of other findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included tan discolouration of preputial or clitoral glands, gray-white foci on the adrenal glands or kidneys, red discolouration of the mandibular lymph nodes or thymus, pelvic dilation of the kidneys, enlargement of the mandibular lymph nodes, a yellowish soft nodule on the epididymides, alopecia, fluid in the uterus (statistically significant at 300 mg/kg), and red foci on the thymus or glandular mucosa of the stomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related microscopic findings were present in:
- Stomach:
− lymphogranulocytic forestomach inflammation was present in 1/5 300 mg/kg (slight) treated male rats and in 5/5 1000 mg/kg (2 minimal, 2 slight, 1 moderate) treated male and 4/5 1000 mg/kg (2 minimal, 1 slight, 1 moderate) treated female rats.
− hyperplasia of the forestomach squamous epithelium was present in 2/5 300 mg/kg (1 minimal, 1 slight) treated male and in 3/5 300 mg/kg (2 minimal, 1 slight) treated female rats and in 5/5 1000 mg/kg (1 slight, 3 moderate, 1 marked) treated male and 5/5 1000 mg/kg (2 minimal, 2 slight, 1 marked) treated female rats. This corresponded to the macroscopic finding of irregular surface of the forestomach.
− forestomach edema was present in 1/5 300 mg/kg (slight) treated male rats and in 1/5 1000 mg/kg (moderate) treated male and 1/5 1000 mg/kg (slight) treated female rats.
− forestomach erosion was present in 2/5 1000 mg/kg (slight) treated male and in 1/5 1000 mg/kg (slight) treated female rats.
− diffuse hyperkeratosis was present in 1/5 300 mg/kg (minimal) treated male and in 3/5 300 mg/kg (minimal) treated female rats and in 5/5 1000 mg/kg (4 slight, 1 moderate) treated male and 5/5 1000 mg/kg (4 slight, 1 moderate) treated female rats.
- Liver:
− diffuse hepatocellular hypertrophy was present in 5/5 1000 mg/kg (4 minimal, 1 slight) treated male and 5/5 1000 mg/kg (minimal) treated female rats. This finding corresponded to the macroscopic finding of enlarged liver.
- Kidneys:
− hyaline droplets were present at increased incidence and severity at 300 mg/kg (5/5 males: 2 slight, 3 moderate) and 1000 mg/kg (5/5 males: 1 slight, 1 moderate, 3 marked), compared to 4/5 control (3 minimal, 1 slight) and 3/5 100 mg/kg (2 minimal, 1 slight) treated male rats.
− corticomedullary tubular basophilia was present at increased incidence and severity at 1000 mg/kg (5/5 males: 1 minimal, 3 slight, 1 moderate), compared to 2/5 control (2 minimal), 3/5 100 mg/kg (minimal) and 3/5 300 mg/kg (minimal) treated male rats.
All other microscopic findings recorded were considered to be within the normal range of background
pathology encountered in rats of this age and strain and dosing method.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on treatment-related microscopic findings present in both sexes in the stomach at 300 and 1000 mg/kg and in the liver at 1000 mg/kg and in the kidneys of male rats at 300 and 1000 mg/kg.
Critical effects observed:
not specified
Conclusions:
Based on treatment-related microscopic findings present in both sexes in the stomach at 300 and 1000 mg/kg and in the liver at 1000 mg/kg and in the kidneys of male rats at 300 and 1000 mg/kg, a No Observed Adverse Effect Level (NOAEL) for Bisisobutyryl peroxide of 100 mg/kg was established for the rat. However, based on the disscussion in the executive summary, the NOAEL,for human hazard assessment is considered 1000 mg/kg/day.

The NOAEL in the 28 -day oral gavage study was considered 100 mg/kg/day for the rat. This was based on (1) forestomach effects, (2) the increase in incidence and severity of cortical hyaline droplets in the kidneys of males and (3) diffuse hepatocellular hypertrophy. These effects were considered not relevant in assessing human health hazards.

"Chemical administration by oral gavage is also likely to irritate the epithelial lining of the forestomach of the rat following the deposition of high doses directly in contract with the forestomach epithelium for long periods of tiem, which is neither representative of natural pathways of exposure nor relevant to human exposure conditions" (Proctor, D.M et al, Toxicological Sciences 98(2), 313 -326, 2007).

The increase in incidence and severity of cortical hyaline droplets in the kidneys of males at 300 and1000 mg/kg was considered to represent alpha2μglobulin, a normal protein in male rats whichundergoes reabsorption in the proximal cortical tubules (Alden, CL and Frith CH. Urinary System, Ch. 15, pp 315-387. In: Handbook of Toxicologic Pathology Eds. Haschek WM and Rousseaux CG. Academic Press, San Diego. 1991.).

Diffuse hepatocellular hypertrophy was noted in the liver of both sexes at 1000 mg/kg (generally of aminimal degree), and corresponded to the higher liver weights (approximately 30% higher thancontrols). In the absence of microscopic changes in the liver suggestive of hepatotoxicity, these
findings were suggestive of adaptive hepatic changes in response to xenobiotic administration.

Therefore, the NOAEL, for human hazard assessment is considered 1000 mg/kg/day.
Executive summary:

Wistar rats were treated with Bisisobutyryl Peroxide for 28 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg in accordance with OECD 407. Treatment up to 1000 mg/kg was well tolerated during the exposure period; no toxicologically relevant clinical signs, or changes in body weight or food intake were noted. Females at 1000 mg/kg did show a reduced motor activity during functional observations, but this was not apparent from the clinical observations. Histopathological examination of the female at 1000 mg/kg found dead on Day 16 showed a moderate granulocytic inflammation with moderate ulceration of the trachea. This indicates that test substance had entered the trachea (either through aspiration or gavage-related), since test substance has strongly irritating properties (see histopathological stomach effects). No signs of imminent death were shown by this animal, nor did the other animals of the same dose group show signs of moribundity. This strongly suggests that this death is to be considered incidental in nature, and not directly related to the test substance administration via the gavage route of exposure. Treatment resulted in forestomach effects in both sexes at 300 and 1000 mg/kg, which were considered reflective of irritant properties of the test substance. These lesions consisted of erosion of the forestomach at 1000 mg/kg, along with lymphogranulocytic inflammation, squamous epithelium hyperplasia and edema of the forestomach and diffuse hyperkeratosis in the stomach at 300 and 1000 mg/kg. The increase in incidence and severity of cortical hyaline droplets in the kidneys of males at 300 and 1000 mg/kg was considered to represent alpha2μglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (ref. 6). A range of chemicals are known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium which may then result in tubular epithelial cell damage (hyaline droplet nephropathy) which was evident in this study as an increase in the severity of corticomedullary tubular basophilia at 1000 mg/kg. Also, the higher urea and creatinine level in males at 1000 mg/kg suggest that renal filtration was affected. Diffuse hepatocellular hypertrophy was noted in the liver of both sexes at 1000 mg/kg (generally of a minimal degree), and corresponded to the higher liver weights (approximately 30% higher than controls). In the absence of microscopic changes in the liver suggestive of hepatotoxicity, these findings were suggestive of adaptive hepatic changes in response to xenobiotic administration. Based on treatment-related microscopic findings present in both sexes in the stomach at 300 and 1000 mg/kg and in the liver at 1000 mg/kg and in the kidneys of male rats at 300 and 1000 mg/kg, a No Observed Adverse Effect Level (NOAEL) for Bisisobutyryl Peroxide of 100 mg/kg was established for the rat.

The NOAEL in the 28 -day oral gavage study was considered 100 mg/kg/day for the rat. This was based on (1) forestomach effects, (2) the increase in incidence and severity of cortical hyaline droplets in the kidneys of males and (3) diffuse hepatocellular hypertrophy. These effects were considered not relevant in assessing human health hazards.

"Chemical administration by oral gavage is also likely to irritate the epithelial lining of the forestomach of the rat following the deposition of high doses directly in contract with the forestomach epithelium for long periods of time, which is neither representative of natural pathways of exposure nor relevant to human exposure conditions" (Proctor, D.M et al, Toxicological Sciences 98(2), 313 -326, 2007).

The increase in incidence and severity of cortical hyaline droplets in the kidneys of males at 300 and1000 mg/kg was considered to represent alphaglobulin, a normal protein in male rats whichundergoes reabsorption in the proximal cortical tubules (Alden, CL and Frith CH. Urinary System, Ch. 15, pp 315-387. In: Handbook of Toxicologic Pathology Eds. Haschek WM and Rousseaux CG. Academic Press, San Diego. 1991.).

Diffuse hepatocellular hypertrophy was noted in the liver of both sexes at 1000 mg/kg (generally of aminimal degree), and corresponded to the higher liver weights (approximately 30% higher thancontrols). In the absence of microscopic changes in the liver suggestive of hepatotoxicity, these

findings were suggestive of adaptive hepatic changes in response to xenobiotic administration.

Therefore, the NOAEL,for human hazard assessement is considered 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A K1 90-day study is avaialble via the oral route, this study is supported by a K1 28-day study.
System:
other: No adverse effects observed at the highest dose level

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 in the 90-day oral gavage study was considered 10 mg/kg/day for the rat. This was based on (1) forestomach effects, (2) the increase in incidence and severity of cortical hyaline droplets in the kidneys of males. These effects were considered not relevant in assessing human health hazards.

"Chemical administration by oral gavage is also likely to irritate the epithelial lining of the forestomach of the rat following the deposition of high doses directly in contract with the forestomach epithelium for long periods of time, which is neither representative of natural pathways of exposure nor relevant to human exposure conditions" (Proctor, D.M et al, Toxicological Sciences 98(2), 313 -326, 2007).

The increase in incidence and severity of cortical hyaline droplets in the kidneys of males at 100 and 300 mg/kg was considered to represent alphaglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules (Alden, CL and Frith CH. Urinary System, Ch. 15, pp 315-387. In: Handbook of Toxicologic Pathology Eds. Haschek WM and Rousseaux CG. Academic Press, San Diego. 1991.).

In the 28 -day study also diffuse hepatocellular hypertrophy was noted in the liver of both sexes at 1000 mg/kg (generally of aminimal degree), and corresponded to the higher liver weights (approximately 30% higher than controls). In the absence of microscopic changes in the liver suggestive of hepatotoxicity, these findings were suggestive of adaptive hepatic changes in response to xenobiotic administration. This was not obseved in the 90 -day study.

Therefore, the NOAEL, for human hazard assessment is considered 300 mg/kg/day based on a 90 -day oral study in rats. These results do not lead to calssification and labeling for the repeated dose endpoint.