Registration Dossier

Administrative data

Description of key information

Oral 28 -day and 90 -day repeat dose OECD 408 studies available.

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:
This study was conducted between 21 September 2015 and 23 March 2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, 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).
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 190-233g: Females: 152-190g
- Fasting period before study:
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used.
Mains drinking water was supplied from polycarbonate bottles attached to the cage

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3”C
- Humidity (%): 50 ± 20%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 October 2015 To: 05 February 2016
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals
Vehicle:
arachis oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an extgernal standard technique. the test item gave a chromatographic profile consisting of a profile of multiple peaks.

Analytical Procedure
Preparation of Standard Solutions
Stock solutions of test item in acetonitrile 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 acetonitrile to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.1 mg/mL. The standard solutions contained the equivalent amount of vehicle to that of the relevant standards.

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

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

Preparation of Accuracy Samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the test samples above

Preparation of Linearity Standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.108 mg/mL by serial dilution covering the concentration range 0.0554 mg/mL to 0.133 mg/mL

Instrumental Parameters
GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : ZB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program: oven: 100°C, for 1 minute with 10°C/minute to 260°C, for 10 minutes
Injection temperature: 250 ºC
Flame ionisation detector temperature: 250 °C
Injection volume : 1 mL
Retention time: ~ 2 to 6 mins

Results
Validation of Analytical Method
Specificity
The control dose samples and an analyzed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have a linear correlation within the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

Accuracy
The fortified samples of Arachis Oil BP were found to have a recovery value of ± 10% of the fortification.

Test Item Formulations
The formulations investigated during the study were found to comprise test item in the range of 95% to 100% and, thus, the required content limit of ±10% with reference to the nominal content was met.

In addition, the test item was found to be stable in the formulations when kept 21 days in the refrigerator (4” C) due to results which met the variation limit of 10% from the time-zero mean.

In conclusion, the results indicate the accurate use of the test item and Arachis Oil BP as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once Daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed according to the study plan and was designed to investigate the systemic toxicity of the test item, by repeated oral administration to the Wistar Han™:RccHan™:WIST strain rat for a period of ninety consecutive days at dose levels of 10, 100 or 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (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 dose levels were chosen in consultation with the Study Monitor based on previous toxicity work including a 28 day toxicity study in the rat (Harlan Study Number 41103960). In this 28 day study, administration of the test item to animals of either sex at dose levels of 30, 300 or 1000 mg/kg bw/day resulted in treatment-related findings for both sexes at all dose levels and a No Observed Effect Level could not be identified. The effects detected in females were considered to relate to adaptive microscopic liver, thyroid and spleen changes and therefore 1000 mg/kg bw/day may be considered a No Observed Adverse Effect Level (NOAEL) for females. Similar microscopic liver, thyroid and spleen changes were apparent for males however microscopic kidney changes and a concomitant increase in neutrophils were also apparent at all dosages and a NOAEL for the male rat could not be established. Kidney changes were characterized by hyaline droplets, increased tubular degeneration/ regeneration and granular casts and at 1000 mg/kg bw/day interstitial inflammatory infiltrates. The hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect was, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings were more likely to be correlated to the same condition as the hyaline droplet accumulation and were therefore considered to represent limited relevance to
humans. Excluding these kidney changes, the No Observed Adverse Effect Level (NOAEL) for males was considered to be 300 mg/kg bw/day. Taking into consideration these results and the duration of dosing in the present study, a dose level of 300 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 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.

Justification
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.
Positive control:
Not relevant
Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. ; see deviations from Study Plan. During the treatment-free period, animals were observed daily.
All observations were recorded.

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

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

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

Specialist Evaluations

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

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

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

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. 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 nonrecovery 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 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 non-recovery control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

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

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 118). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 119. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during Week 16. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

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 males, dissected from connective tissue and weighed separately.
For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.
For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. The tissue was later thawed and homogenized in a suitable saline/detergent mixture. Samples of the homogenate were examined to determine the number of homogenization resistant spermatids present; see deviations from the Study Plan.
The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C. The tissue was later thawed and homogenized in an appropriate saline/detergent to determine the numbers of homogenization resistant spermatids.
Morphological assessment was performed on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies.
Assessment of homogenization resistant spermatids was only performed for control and 300 mg/kg bw/day males. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.

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

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Right Epididymis ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
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

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were dispatched to the Test Site (Envigo CRS Limited, Eye) 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. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the kidneys (males only), the liver and the thyroid gland (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 at the Test Site (Envigo CRS Limited, Huntingdon).
Other examinations:
Data Evaluation
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.
Statistics:
Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA or ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Urine volume and specific gravity as well as sperm analysis parameters were statistically analyzed using the R Environment for Statistical Computing. Distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test. Where parametric analysis of the data was unsuitable, non-parametric analysis was performed incorporating the Kruskal- Wallis test followed by the Mann-Whitney "U" test.
Clinical signs:
no effects observed
Description (incidence and severity):
Throughout the study, there were no clinical signs considered to be related to the toxicity of the test item.
During the dosing phase of the study, sporadic instances of increased post-dose salivation were detected for animals of either sex given the test item at 300 mg/kg bw/day from Week 3 of dosing. Individual males and females from the 100 mg/kg bw/day and control dose groups also showed isolated instances of increased post-dose salivation. Such observations are common in this type of study and may reflect an irritant nature of the test item and/or formulation. Other clinical observations included one male from the 10 mg/kg bw/day dose group showing clinical signs of chromodacryorrhea during Week 9 whilst one female given 300 mg/kg bw/day exhibited generalized fur loss towards the end of the treatment period.
Due to the isolated nature of these observations, they were deemed to be unrelated to treatment with the test item.
During the treatment-free period, no clinical observations were detected for any of the recovery animals.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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.
Occasional fluctuations in weekly group mean body weight gains were observed in animals of either sex achieving statistical significance in some instances. There was generally no dose-dependence and overall body weight gains for animals of either sex treated with 300 mg/kg bw/day were comparable with controls.
Food consumption and compound intake (if feeding study):
no effects observed
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 for animals of either sex during the treatment or treatment-free periods.
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 intergroup differences
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmoscopic examination of the non-recovery animals of both sexes 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 mean hemoglobin than controls (p<0.01), but the majority of individual values were within the historical control data ranges. Group mean hematocrit for these males was also slightly lower than controls albeit without achieving statistical significance. Group mean cell hemoglobin concentrations in males from all dose groups and females receiving 100 or 300 mg/kg bw/day were statistically significantly lower than controls (p<0.01). A dose-relationship was apparent in females, but a number of individual values in particular for the control animals of either sex exceeded the historical data ranges which may have contributed to these differences. At the end of the treatment-free period, group mean hemoglobin and hematocrit values in males previously treated with 300 mg/kg bw/day were statistically significantly lower than controls (p<0.05) whilst the corresponding females showed higher red blood cell count and lower mean cell hemoglobin in relation to controls attaining statistical significance in either instance (p<0.05); most individual values for these parameters, however, remained within the historical control data ranges. Whilst these
findings may indicate some minor perturbations in red blood cells for males treated with 300 mg/kg bw/day, the affected parameters were generally not consistent between the two sexes and, these observations were considered not to be of toxicological importance.
Group mean leukocyte count in males treated with 300 mg/kg bw/day was statistically significantly higher than controls (p<0.05) which was considered primarily to be due to a higher neutrophil count in these animals (p<0.05). The corresponding values in females from this dose group were also higher than controls but statistical significance was only attained for neutrophil count (p<0.05). Most individual values from animals receiving the test item as well as some control animals exceeded the background data ranges. Additionally, males treated with 100 or 300 mg/kg bw/day showed statistically significantly higher platelet counts in relation to controls at the end of the dosing phase (p<0.01). A dose-relationship was evident, but most individual values remained within the background data ranges. There were no corresponding intergroup differences for recovery animals of either sex at the end of the treatment-free period and in the absence of any associated histopathological findings, these observations were considered to be of no toxicological relevance.
Group mean activated partial thromboplastin time in males from the 300 mg/kg bw/day was also statistically significantly shorter than controls (p<0.01) with most individual values from the test item-treated males slightly outside the control data ranges. This change was not observed in any of the female dose groups and was completely reversible in recovery males, and whilst it may have been influenced by slight metabolic perturbations in the liver, it was deemed not to be of an adverse nature. See Table 1.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the dosing period, group mean plasma concentrations of cholesterol, bile acids and bilirubin in males receiving 300 mg/kg bw/day were statistically significantly lower than controls (p<0.05 for bile acids and p<0.01 in the remaining instances) whilst group mean albumin/globulin ratio in these males was statistically significantly higher than controls (p<0.05). Females treated with 300 mg/kg bw/day also showed statistically significantly higher albumin concentration than controls (p<0.05) although total protein and albumin/globulin ratio in these females remained unaffected. All individual values from test item-treated animals of the relevant sex were within the background data ranges. The corresponding values in recovery animals of either sex previously treated with 300 mg/kg bw/day were also similar to controls. Taking into view the histopathology findings from the liver and thyroid, these intergroup differences may indicate minor perturbation in metabolism, but they were considered unlikely to be of any toxicological relevance.
At all dose levels, non-recovery females exhibited statistically significantly lower alkaline phosphatase activities in relation to controls (p<0.05). Whilst 5/10 control females showed values which slightly exceeded the historical data ranges, the corresponding values from most test item-treated females were within these ranges and these differences were regarded likely to be incidental. At the end of the twenty-eight day dose-free period, the corresponding values in females previously given 300 mg/kg bw/day were comparable with controls.
When compared with controls, non-recovery animals of either sex treated with 300 mg/kg bw/day and males given 100 or 10 mg/kg bw/day showed statistically significantly higher plasma levels of calcium (p<0.05). There was no dose-related trend and most individual values including controls were outside the background data ranges. In recovery animals of both sexes previously receiving 300 mg/kg bw/day, the corresponding values were comparable with the respective controls whilst group mean phosphorus levels in these males were statistically significantly higher than controls (p<0.05). These observations were deemed unlikely to be of any toxicological importance. SeeTable 2.
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 treatmentfree periods.
In recovery females previously receiving 300 mg/kg bw/day, specific gravity was statistically significantly higher than controls (p<0.05). The remaining urinalysis parameters for these animals were similar to controls and this finding was considered likely to be due to normal biological variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments
There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level.

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 males from all dose groups showing statistically significantly lower hindlimb grip strength when compared with controls (p<0.05). 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 lower overall activity for males treated with 10 mg/kg bw/day relative to controls (p<0.01). The corresponding values from the remaining dose groups were similar to controls and in the absence of any apparent clinical signs of neurotoxicity, this observation was considered to be incidental.

Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, males treated with 300 mg/kg bw/day showed statistically significant reduction in absolute and body weight-related left cauda weights when compared with controls (p<0.05). Although there was no effect of treatment with the test item at any dose level on group mean right/left testis and right/left epididymis weights in these males and there was no associated histopathology, taking into the view the overall results from sperm analysis, the toxicological significance of this finding was deemed unclear. At the end of the recovery period, the effect on sperm parameters in some males previously treated with 300 mg/kg bw/day persisted, however, absolute and body weight-related cauda epididymis weights in this group of males were similar to controls.
At the end of the treatment period, animals of either sex given the test item at 300 mg/kg bw/day showed statistically significantly higher absolute and body weight-related liver weights when compared with controls (p<0.01). A number of individual values from the test item-treated animals exceeded the historical control data ranges and these observations correlated with the microscopic finding of centrilobular hepatocyte hypertrophy seen in the liver from some animals in this dose group. These organ weight differences were no longer apparent in recovery males and a microscopic examination of liver from these animals also indicated complete recovery. These results were considered to reflect an adaptive nature of the alterations in the liver in response to xenobiotic metabolism.
At 100 or 300 mg/kg bw/day, non-recovery males showed dose-related statistically significant increases in absolute and body weight-related kidneys weights in comparison with controls (p<0.01). The majority of individual values from test item-treated animals exceeded the background data ranges and these findings were 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 male rats. Following the dose-free period of four weeks, increased kidneys weights were still apparent in males previously receiving
300 mg/kg bw/day; however, hyaline droplet accumulation in these males had reversed to background levels but resultant pathology was still apparent indicating partial recovery.
When compared with controls, non-recovery males from the 100 or 300 mg/kg bw/day dose groups showed dose-related statistically significant increases in absolute and body weight-related heart weights (p<0.01) with recovery animals of either sex also showing similar increases (p<0.05). Most individual values were within the historical control data ranges and in the absence of any histopathology correlates, these findings were considered not to be of any toxicological significance.
At the end of dosing phase, males from the 300 mg/kg bw/day dose group showed a reduction in absolute and body weight related adrenal weights (p<0.05). There was no doserelationship or any histopathology correlates. The corresponding values in recovery males previously treated with the test item were comparable controls whereas recovery females showed statistically significantly lower absolute and body weight-related adrenal weights; these intergroup differences were deemed most likely to be due to individual control animals showing atypically high adrenal weights. See Table 3.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the dosing period, 9/20 males from the 300 mg/kg bw/day dose group showed enlarged kidneys with kidneys from 2/10 of these animals exhibiting mottled appearance. These observations were associated with increased kidneys weights and a perceived α-2uglobulin nephropathy syndrome observed at histopathology examination.

1 non-recovery male treated with 300 mg/kg bw/day showed small testes and epididymides, which correlated with marked atrophy (right testis) and aspermia (right epididymis); only the right testis and the right epididymis were histopathologically examined. Such observations are often seen in controls populations of this strain of rat and, this finding was considered likely to be incidental.

A number of animals across most dose group including controls showed reddened lungs.
Doses: 0, 10, 100, 300 mg/kg bw
Females: 5/20, 1/10, 2/10, 3/20
Males: 2/20, 1/10, 0/10, 0/10
Such findings are common in this type of study and were considered unrelated to treatment with the test item.
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 liver and the thyroid gland in males and females. The findings were as following:
Kidneys
Non-Recovery Animals
 Hyaline droplets at a mild level were present in 9/10 Group 2 (10 mg/kg bw/day) and at moderate level in all Group 3 and 4 males (100 and 300 mg/kg bw/day, respectively).
 Multifocal basophilic tubules were present in 1/10 Group 2, 8/10 Group 3 and all Group 4 males, minimal to moderate.
 Proteinacious casts were present in 1/10 Group 2, 8/10 Group 3 and 9/10 Group 4 males from minimal to moderate.
 Lymphocytic infiltration was present in 2/10 Group 4 males.
 Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels from animals from groups treated with the test item.

Recovery Animals
 Multifocal basophilic tubules or multifocal nephropathy (defined as some or all of tubular basophilia, tubular atrophy, thickening of basement membranes, interstitial thickening/fibrosis) was present in all Group 4 males, minimal to moderate.
 Proteinacious casts were present in all Group 4 males, minimal to moderate.
 Immunohistochemical staining was positive for α-2u-globulin in all males with levels similar in control and Group 4 animals.

Liver
 At the end of the dosing period, centrilobular hepatocyte hypertrophy was present at a minimal level in 2/10 Group 3 and 4/10 Group 4 males and in 7/10 females in Group
4.
 Changes in Group 4 animals had resolved by the end of the treatment-free period and no findings related to the administration of the test item were apparent.

Thyroid Gland
 Hypertrophy of the follicular epithelium was present in 3/10 males from control and Group 2, 6/10 males from Group 3 and 9/10 males from Group 4. In females, it was
present in 3/10 Group 3 and 9/10 Group 4 animals. In all cases the change was minimal except one control male where the change was mild.
 Changes in Group 4 animals had resolved by the end of the treatment-free period and no findings related to the administration of the test item were apparent.

No other findings were present at histopathology, which were considered to be related to administration of the test item. In particular, the testes and epididymides 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 testis (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).

One male from Group 4 showed marked tubular atrophy and aspermia in the right testis and the right epididymis, respectively, which was associated with macroscopic findings of small testes and epididymides seen at necropsy. Such findings are occasionally observed in this strain of rat and, in isolation, are considered to be incidental.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm Analysis

At the end of the dosing period, males treated with 100 or 300 mg/kg bw/day showed reduced sperm concentration and motility in relation to controls. Although statistical significance was not attained for these intergroup differences, 4/10 males treated with 300 mg/kg bw/day (including Male 67 showing small testes and epididymides at necropsy) did not show any motile sperm. Progressive motility at all dose levels was statistically significantly lower than controls (p<0.05) in a dose-related manner. At 300 mg/kg bw/day, most males showed a marked increase in the number of abnormal sperm with 1/10 male from the 100 mg/kgbw/day dose group also showing a slight increase. The sperm abnormalities included misshapen sperm, sperm with abnormal hook, reverse head, head only, no head and short or kinked tail. At the end of the treatment-free period, recovery males previously given 300 mg/kg bw/day exhibited a reduction in sperm concentration and motility with 2/10 males showing none or close to none motile sperm and a marked increase in the number of abnormal sperm; these abnormalities were confined to sperm with no head or with tail only.
Although, these effects were not associated with any treatment-related histopathological findings in the relevant tissues from non-recovery males treated with 300 mg/kg bw/day, taking into account the overall sperm analysis results, the observations at 100 and 300 mg/kg bw/day were considered to be of an adverse nature. Although group mean progressive motility at 10 mg/kg bw/day was statistically significantly lower than controls, all individual values remained within the control ranges (Group 1) and in the absence of any associated changes this observation was considered unlikely to be of any toxicological significance.
At 300 mg/kg bw/day, group mean homogenization resistant spermatid counts in cauda was slightly lower than controls, but without attaining statistical significance. It is worth noting, however, that only 2/10 test item-treated males (including Male 67 showing small testes and epididymides at necropsy) showed individual values below the control ranges (Group 1). As the macroscopic/microscopic observations for the reproductive tissues from Male 67 were considered unlikely to be treatment-related, excluding the individual values for this male resulted in only minor intergroup difference in homogenization resistant spermatid counts between the test item-treated and control males, which were considered most likely to be due to biological variation. See Tables 4 to 6.

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.
One female treated with 300 mg/kg bw/day remained in diestrus throughout the estrous cycling assessment phase. Histopathological examination of the reproductive tissues from this animal identified uterine deciduoma and vaginal mucification. Uterine deciduoma is sometimes associated with pseudopregnancy, but due to the isolated nature of this observation, it was deemed unlikely to be of any toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
<= 10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities
Key result
Dose descriptor:
NOAEL
Effect level:
<= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no changes of toxicological significance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
other: toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

For all tables:

N = No animals = 10 unless indicated

Group 1 = Control; Group 2 = 10 mg/kg bw/day; Group 3 = 100 mg/kg bw/day; Group 4 = 300 mg/kg bw/day

Statistical Footnotes:

*       Significantly different from control group p<0.05

**       Significantly different from control group p<0.01

***       Significantly different from control group p<0.001

n       Data not appropriate for statistical analysis

Table 1 Group Mean Hematological Values

 Parameter  Group 1 (Male)     Group 2 (Male)     Group 3 (Male)     Group 4 (Male)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 HB (g/dl)  16.25  10.8  16.56  0.61  16.06  0.71  15.19**  0.41
 RBC (10^12/l)  8.619  0.579  8.888  0.512  8.560  0.502  8.243  0.328
 Hct (%)  45.41  2.66  46.97  2.04  45.50  1.95  43.67  1.29
 MCH (pg)  18.88  0.69  18.64  0.59  18.79  0.88  18.44  0.58
 MCV (fl)  52.73  1.31  52.88  1.4  53.25  2.26  53.01  1.19
 MCHC (g/dl)  35.80  0.54  35.24**  0.37  35.29**  0.24  34.76**  0.38
 WBC (10^9/l)  7.37  1.87  7.18  1.2  7.60  1.19  8.88*  1.25
 Neut (10^9/l)  1.347  0.553  1.228  0.469  1.264  0.599  1.988*  0.789
 Lymph (10^9/l)  5.906  1.611  5.869  1.204  6.228  0.848  6.771  1.035
 Mono (10^9/l)  0.000n  0.000  0.000n  0.000  0.018n  0.032  0.000n  0.000
 Eos (10^9/l)  0.121  0.111  0.082  0.074  0.095  0.104  0.120  0.125
 Bas (10^9/l)  0.000n  0.000  0.000n  0.000  0.000n  0.000  0.000n  0.000
 Ct (seconds)  8.61  1.12  8.68  0.96  8.50  0.79  9.56  1.38
 PLT (10^9/l)  564.2  79.1  602.7  41.2  655.7**  86.6  668.9**  43.1
 APTT (seconds)  15.62  2.22  14.68  1.54  14.73  1.71  13.18**  0.96

 Parameter  Group 1 (Female)     Group 2 (Female)     Group 3 (Female)     Group 4 (Female)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 HB (g/dl)  14.99

 0.47

 14.74  1.45  15.36  0.57  15.03  0.58
 RBC (10^12/l)  7.645  0.352  7.282  0.803  7.883  0.501  7.795  0.282
 Hct (%)  41.73  1.39  41.07  4.25  43.32  1.64  42.78

 1.66

 MCH (pg)  19.63  0.72  20.27  0.90  19.51  1.6  19.29  0.45
 MVC (pg)  5455.60  1.71  56.48  2.30  55.06  2.51  54.92  0.90
 MCHC (g/dl)  35.97  0.038  35.92  0.50  35.43**  0.44  35.12**  0.39
 WBC (10^9/l)  5.07  1.11  4.77  1.64  5.65  1.29  6.05  1.88
 Neut (10^9/l)  0.734  0.288  0.742  0.261  0.852  0.478  1.053*  0.503
 Lymph (10^9/l)  4.314  0.929  3.994  1.527  4.743  1.098  4.822  1.874
 Mono (10^9/l)  0.000n  0.000  0.000n  0.000  0.000n  0.000  0.006  0.019
 Eos (10^9/l)  0.023  0.57  0.036  0.041  0.057  0.061  0.069  0.067

 Bas (10^9/l0)

 0.000n

 0.000

 0.000n

 0.000

 0.000n

 0.000

 0.000n

 0.000

 CT (seconds)  8.42  0.71  8.58  0.71  8.68  0.72  8.80  0.79
 PLT (10^9/l)  637.9  56.8  566.9  179.1  548.1  191.2  639.7  119.2
 APTT (seconds)  15.75  1.49  14.76  1.59  16.41  1.98  15.30  1.69

Recovery Groups

 Parameter  Group 1 (Male)     Group 4 (Male)     Group 1 (Female)     Group 4 (Female)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 HB (g/dl)  16.52 0.42 16.07*  0.29 14.91 1.11 15.42 0.75
 RBC (10^12/l)  9.231 0.479 9.076  0.289 7.842  0.620  8.373*  0.403
 Hct (%)  48.75  1.30  47.54*  0.84 43.19 3.33 45.02  2.23
 MCH (pg)  17.91  0.88 17.71 0.56  19.05 0.66 18.45* 0.42
 MCV (fl)  52.89 0.98

52.42

1.32

 55.11

1.62

53.75

1.25 

 MCHC (g/dl)

 33.83

 0.45

33.79

0.34

 34.54

0.37 

34.27

0.25 

 WBC (10^9/l)

4.99 

1.75 

5.80 

1.55 

3.62

0.94

3.64

0.62

 Neut (10^9/l)

0.867

0.314

 0.932

0.549

0.509 

0.140 

0.539 

0.267

 Lymph (10^9/l)

4.056

 0.524

 4.798

1.205

 3.077

1.000

3.069

0.670

 Mono (10^9/l)

 0.000n

0.000

0.000n 

0.000

0.000n

0.000 

0.003n 

0.009 

 Eos (10^9/l)

 0.070

0.049 

 0.059

 0.079

0.035

0.033 

0.030 

0.024 

 Bas (10^9/l)

 0.000n

 0.000

 0.000n

0.000

0.000n

0.000

0.000n 

0.000 

 Ct (seconds)

8.68

 0.84

9.32

 0.85

 8.79

0.79 

8.86 

0.83 

 PLT (10^9/l)

 541.8

58.4

 573.8

84.1

640.3

96.3 

579.0 

77.1

 APTT (seconds)

 16.95

1.52

16.04 

 1.98

 14.83

1.87 

15.22

1.35

Table 2 Group Mean Blood Chemical Values

 Parameter  Group 1 (Male)     Group 2 (Male)     Group 3 (Male)     Group 4 (Male)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 Urea (mg/dl) 47.2   6.5  46.0  2.6  46.4  6.2  45.9  12.1
 Glucose (mg/dl)  172.7  37.7  158.4  14.1  172.9  25.2  162.  20.4
 Total Protein (g/dl)  7.186  0.451  7.383  0.309  7.206  0.318  6.955  0.605
 Albumin (g/dl) 3.98  0.18  4.11  0.13  3.99  0.13  6.96  0.19
 A/G ratio  1.242  0.069  0.268  0.065  1.241  0.057  1.317*  0.053
 Na+ (mmol/l)  149.3  2.7  149.6  2.1  148.6  1.8  148.4  3.0
 K+ (mmol/l)  4.703  0.645  4.335  0.365  4.340  0.298  4.914  1.133
 CL- (mmol/l)  102.9  14  102.3  1.6  102.1  1.5  102.5  1.9
 Ca++ (mmol/l)  2.004  0.076  2.103*  0.062  2.046*  0.055  2.065*  0.068
 P (mmol/l)  2.21  0.28  2.08  0.49  2.11  0.21  2.60  0.38
 ASAT (IU/l)  79.8  15.6  90.6  24.5  97.5  30.5  102.0  29.6
 ALAT (IU/l)  60.8  45.3  62.4  9.7  64.1  16.1  73.6  13.4
 AP (IU/l)  129.7  33.1  142.0  26.5  141.8  33.9  151.0  31.3
 Creat (mg/dl)  0.858  0.134  0.840  0.075  0.805  0.111  0.996  0.329
 Chol (mg/dl)  104.7  16.7  97.7  12.8  95.6  14.7  72.3**  9.3
 Bili (mg/dl)  0.113  0020  0.104  0.021  0.102  0.020  0.086**  0.018
 Bile Acid (µmol/l)  13.50  6.56  12.34  6.10  12.75  9.16  6.66*  1.55

 Parameter  Group 1 (Female)     Group 2 (Female)     Group 3 (Female)     Group 4 (Female)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
 Urea (mg/dl) 51.1  6.7 45.7 11.3 52.8 8.9  52.5  10.9
 Glucose (mg/dl)  154.3  15.5  151.2  14.0  168.8  19.6  169.4  28.9
 Total Protein (mg/dl)  7.514  0.535  7.441  0.542  7.587  0.367  7.909  0.600
 Albumin (g/dl)  4.43  0.33  4.42  0.27  4.50  0.18  4.74*  0.35
 A/G Ratio  1.445  0.070  1.469  0.091  1.468  0.096  1.497  0.103
 Na+ (mmol/l)  147.3  1.1  148.8  2.1  149.8  2.6  148.3  1.8
 K+ (mmol/l)  4.161  0.401  4.111  0.339  4.485  0.499  4.181  0.341
 Cl- (mmol/l)  103.6  1.8  104.3  2.1  104.4  2.5  103.2  1.0
 Ca++ (mmol/l)  2.008  0.083  2.023  0.069  2.022  0.079  2.094*  0.071
 P (mmol/l)  1.43  0.30  1.41  0.29  1.88  0.63  1.50  0.22
 ASAT (IU/l)  85.6  14.3  81.0  13.0  113.1  81.5  83.5  20.2
 ALAT (IU/l)  57.6  11.7  59.1  14.4  54.0  11.8  61.8  17.8
 AP (IU/l)  95.9  45.4  68.9*  18.8  62.9*  17.5  61.9*  18.7
 Creat (mg/dl)  0.798  0.099  0.864  0.241  0.917  0.096  0.916  0.105
 Chol (mg/dl)  86.1  14.7  85.1  15.4  98.9  27.9  98.1  8.2
 Bili (mg/dl)  0.082  0.039  0.085  0.038  0.063  0.025  0.087  0.013
 Bile acid (µmol/l)  18.61  16.22  17.61  11.80  13.58  9.02  18.02  16.42

Recovery Groups

 Parameter  Group 1 (Male)     Group 4 (Male)     Group 1 (Female)     Group 4 (Female)   
   Mean  S.D.  Mean  S.D.  Mean  S.D.  Mean  S.D.
Urea (mg/dl)  40.7 5.1 37.9 7.0 38.7 8.1 38.8 7.1
 Glucose (mg/dl)  197.1  32.5  174.8  18.0  146.2  12.6  144.5  22.2
 Total Protein (mg/dl)  6.933  0.722  6.904  0.279  6.769  0.621  6.750  0.686
 Albumin  3.93  0.38  3.91  0.14  4.16  0.54  4.13  0.42
 A/G ratio  1.318  0.072  1.306  0.104  1.588  0.160  1.597  0.098
 Na+ (mmol/l)  147.4  2.5  147.7  2.3  144.8  1.4  145.1  3.5
 K+ (mmol/l)  4.301  0.334  4.766  0.944  4.323  0.609  4.182  0.176
 Cl- (mmol/l)  104.5  2.2  104.9  1.2  101.4  1.6  101.7  1.5
 Ca++ (mmol/l)  1.747  0.107  1.783  0.080  1.722  0.254  1.767  0.086
 P (mmol/l)  1.30  0.35  1.71*  0.49  1.28  0.22  1.37  0.26
 ASAT (IU/l)  112.5  415  97.7  60.6  85.1  24.1  88.7  50.9
 ALAT (IU/l)  65.9  15.6  61.4  19.6  48.8  1.7  50.1  8.9
 AP (IU/l)  116.4  45.2  98.5  25.9  41.9  17.6  48.6  11.0
 Creat (mg/dl)  0.763  0.047  0.724  0.044  0.755  0.101  0.781  0.099
 Chol (mg/dl)  73.6  17.3  69.8  12.4  76.0  13.8  66.8  16.1
 Bili (mg/dl)  0.082  0.011  0.074  0.029  0.099  0.041  0.098  0.018
 Bile Acid  6.24  3.54  6.39  5.60  6.58  5.44  8.80  5.25

Table 3       Group Mean Organ Weights

 Tissue    Male           Female         
   Dose (mg/kg bw/day)  0  10  100  300  0  10  100  300
 Terminal Bodyweight  Mean (g)  393.2  385.1  400.6  393.6  239.4  235.5  236.4  245.7
   S.D.  44.1  33.9  29.9  27.3  19.8  18.3  17.4  22.3
 Adrenals  Mean (g)  0.07980  0.08233  0.08640  0.06866*  0.07818  0.10006  0.09778  0.07980
   S.D.  0.01479  0.01175  0.01531  0.01293  0.02655  0.03825  0.01573  0.01795
 Brain (including Cerebrum, Cerebellum and Pons)  Mean (g)  1.89365  1.91020  1.93339  1.92746  1.77113  1.81747  1.81551  1.81046
   S.D.  0.13622  0.06586  0.09580  0.12126  0.12974  0.09806  0.05739  0.06702
 Righ Epididymis  Mean (g)  0.78236  0.082926  0.83452  0.69620        
   S.D.  0.10743  0.16942  0.13315  0.11533        
 Heart  Mean (g)  1.538886  1.14014  1.29112**  1.39023**  0.7922  0.81823  0.87777  0.92832
   S.D.  0.19198  0.26242  0.18387  0.13923  0.15565  0.18607  0.13307  0.10163
 Kidneys  Mean (g)  2.16918  2.21135  2.61359**  3.02505**  1.50533  1.52857  0.59569  1.65444
   S.D.  0.28820

 0.26897

 0.34843

 0.43037

 0.09673

 0.54807

 0.17128

 0.19146

 Liver  Mean(g)  12.7775  12.4620  13.5479  15.2125**  8.85870  8.70670  9.27943  11.4179**
   S.D.

 1.70573

 1.33822  1.70747  1.42997  0.74942  1.09903  1.16000  0.91001
 Ovaries  Mean (g)          0.10322  0.09689  0.11546  0.1117
   S.D.          0.2399  0.01957  0.01592  0.01496
 Spleen  Mean (g)  0.72515  0.69845  0.75756  0.73488  0.58444  0.59865  0.66342  0.64041
   S.D.  0.15222  0.15271  0.10884  0.08336  0.07260  0.12436  0.14548  0.12523
 Right Testis  Mean (g)  1.81400  1.86567  1.87340  1.75821        
   S.D.  0.11886  0.12472  0.19073  0.29198        
 Thymus  Mean (g)  0.36646  0.41043  0.41699  0.37084  0.31762  0.35237  0.34521  0.34546
   S.D. 0.04633  0.09343  0.07242  0.05664  0.07798  0.06096  0.06592  0.05865
 Uterus (with Cervix)  Mean (g)          0.68419  0.91903  0.78165  0.80295
   S.D.          0.17005  0.30259  0.15734  0.24029
 Left Epididymis  Mean (g)  0.79058  0.87026  0.83667  0.73897        
   S.D.

 0.06381

 0.14578  0.16768  0.15812        
 Left Testis  Mean (g)  1.79762  1.88442  1.1307  1.66436        
   S.D. 0.14790  0.13917  0.20230  0.36720        
 Left Cauda  Mean (g)  0.30859  0.33272  0.31043  0.25772*        
 

 S.D.

 0.02789  0.05855  0.06310  0.05400        

                   

Recovery Groups

 Tissue    Male     Female   
   Dose (mg/kg bw/day)  0 (Control)  300  0 (Control)  300
 Terminal Bodyweight  Mean (g)  416.5  414.0  254.0  243.1
   S.D.  33.8  43.1  15.2  16.1
 Adrenals   Mean (g)  0.07946  0.06736  0.14234  0.07724*
   S.D.  0.02049  0.00884  0.14845  0.01200
 Brain (Including Cerebrum, Cerebellum and Pons)  Mean (g)  2.00568  1.1975  1.77325  1.82358
   S.D.  0.07466  0.15545  0.14095  0.06342
 Right Epdidymis  Mean (g)  0.3696  0.82013    
   S.D.  0.8742  0.10486    
 Heart  Mean (g)  1.15631  1.29997*  0.77270  0.86163*
   S.D.

 0.12746

 0.21948

 0.07387  0.08048
 Kidneys  Mean (g)  2.17802  2.76464**  1.57023  1.63175
   S.D.  0.23854  0.31759  0.13253  0.24527
 Liver  Mean (g)  12.8845  13.2434  8.74326  8.80434
   S.D.  1.87863  1.80336  1.33215  0.95937
 Ovaries  Mean (g)      0.10731  0.11218
   S.D.      0.03252  0.01520
 Spleen  Mean (g)  0.74969  0.79163  0.51717  0.53107
   S.D.

 0.09420

 0.13701

 0.07301

 0.07471
 Right Testis  Mean (g)  1.83938  1.81500    
   S.D.  0.13894  0.15302    
 Thumus  Mean (g)  0.34407  0.39252  0.28648  0.36509
   S.D.

 0.07746

 0.07794  0.10034  0.09077
 Uterus (with Cervix)  Mean (g)    

 0.89979

0.92101
   S.D.      0.24723  0.36245
 Left Epididymis  Mean (g)  0.84712  0.82741    
   S.D.  0.13087  0.06187    
 Left Testis  Mean (g)  1.87318  1.82123    
   S.D.  0.12715  0.16502    
 Left Cauda  Mean (g)  0.33080  0.29908    
   S.D.  0.07129  0.02795    

Table 4 Sperm Concentration and Motility - Group Mean Values

 Group (sex)    Concentration (M/ml)  Motility (%)  Progressive Motility (%)
 1 (M)  Mean  160.5  85.3  15.3
   S.D.  52.2  6.0  4.3
 2 (M) Mean  177.3  82.4  11.3*
   S.D.  58.9  7.2  1.9
 3 (M)  Mean  130.4  65.8  10.1*
   S.D.  75.7  23.5  5.9
 4 (M)  Mean  125.3  47.1  7.8*
   S.D.  106.2  43.1  7.2
 Recovery 1 (M)  Mean  206.5  82.0  11.2
   S.D.  55.6  12.6  3.2
 Recovery 4 (M)  Mean  149.8  57.3  10.3
   S.D.  111.0  35.4  6.5

Table 5 Sperm Morphology - Group Mean Values

 Group (Sex)    Number Counted  Number Normal  Number Abnormal  % Normal  % Abnormal
 1 (M)  Mean  200  199.9  0.1  100.0  0.1
   S.D.  0.0  0.3  0.3  0.2  0.2
 2 (M)  Mean  200  200.0  0.0  100.0  0.0
   S.D.  0.0  0.0  0.0  0.0  0.0
 3 (M)  Mean  200  199.2  0.8  99.6  0.4
   S.D.  0.0  1.6  1.6  0.8  0.8
 4 (M)  Mean  183  169.9***  13.2***  92.9***  7.1***
   S.D.  53.4  52.0  16.2  7.9  7.9
 Recovery 1 (M)  Mean  200.0  199.9  0.1  100.0  0.1
   S.D.  0.0  0.3  0.3  0.2  0.2
 Recovery 4 (M)  Mean  200.0  174.8  25.5  67.3  12.7
   S.D.  0.9  52.4  53.0  26.3  26.3

Table 6 Homogenisation Resistant Spermatid Counts - Group Mean Values

 Group (Sex)    Testis (million/gram  Cauda Epididymis (million/gram)
 1 (M)  Mean  57.9600  462.3300
   S.D.  8.9594  108.0776
 4 (M)  Mean  53.9000  380.6400
   S.D.  11.3105  151.7929
Conclusions:
The oral (gavage) administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS#51240-95-0) to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 100 or 300 mg/kg bw/day resulted in toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities. These were not reversible in males previously treated with 300 mg/kg bw/day after a twenty-eight day treatment-free period. It is therefore considered that a dose level of 10 mg/kg bw/day could be established as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the male within the confines of this type of study. In contrast, there were no changes of toxicological significance in the females up to a dose level of 300 mg/kg bw/day, which could therefore be established as a NOAEL in the female within the confines of this type of study.
Executive summary:

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 "Repeated Dose 90-Day Oral Toxicity Study in Rodents” (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).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 or 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (300 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.

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

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all non-recovery control and high dose animals as well as any gross lesions was performed in the first instance. As there were treatment-related findings in the kidneys (males only), the liver and the thyroid gland (males and females), examination of these tissues was subsequently extended to include relevant animals from the remaining dose groups on the study.

Statistical analysis

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

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

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

The homogeneity of variance from mean values was analyzed using Bartlett’s test.

Intergroup variances were assessed using suitable ANOVA or ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Urine volume and specific gravity as well as sperm analysis parameters were statistically analyzed using the R Environment for Statistical Computing. Distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test. Where parametric analysis of the data was unsuitable, non-parametric analysis was performed incorporating the Kruskal- Wallis test followed by the Mann-Whitney "U" test.

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 the respective controls.

Functional Performance Tests

There were no treatment-related changes in functional performance for animals of both sexes 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.

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.

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.

Ophthalmoscopy

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

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.

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

Urinalysis evaluations towards the end of the treatment or treatment-free periods did not identify any effects of toxicological importance in males or females receiving the test item up to a dose level of 300 mg/kg bw/day.

Necropsy

At the end of the treatment period, most males from the 300 mg/kg bw/day dose group showed enlarged kidneys; 2/10 males had kidneys of mottled appearance. These findings correlated with increased kidneys weights and a perceived α-2u-globulin nephropathy syndrome. At the end of the dose-free period, the macroscopic appearance of the kidneys from males previously given 300 mg/kg bw/day was similar to controls.

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

Sperm Analysis

Sperm evaluation at the end of the treatment period identified reduced sperm concentration and motility which was associated with an increase in sperm morphological abnormalities in males treated with 100 or 300 mg/kg bw/day. These observations persisted in some recovery males previously given 300 mg/kg bw/day after a treatment-free period of twenty-eight days. Although microscopic examination of the right testis or the right epididymis did not identify any treatment-related alteration, the effect on sperm parameters was considered to be of an adverse nature.

Organ Weights

At the end of the dosing period, group mean absolute and body weight-related left cauda epididymis weights in males from the 300 mg/kg bw/day dose group were statistically significantly lower than controls. This was no longer apparent in the recovery males. Although there were no histopathology correlates, taken into consideration the effect of treatment with the test item at a dose level of 100 or 300 mg/kg bw/day on sperm concentration, motility and morphology, the toxicological relevance of this finding was deemed unclear.

The increase in liver weights observed in non-recovery males and females from the 300 mg/kg bw/day dose groups showed complete reversibility in recovery animals and was considered to be associated with adaptive microscopic alterations detected in the livers from the non-recovery group animals. Increased kidneys weights in non-recovery males from the 100 or 300 mg/kg bw/day dose groups which persisted in the recovery males previously receiving 300 mg/kg bw/day were considered to be associated with α-2u-globulin nephropathy syndrome.

Histopathology

Treatment-related findings were recorded in the kidneys of males as well as the liver and thyroid gland of males and females dosed with the test item. Some of the changes in the kidneys persisted after the recovery period but all other changes had resolved. The histopathology findings were as following:

Kidneys

At the end of the dosing period, hyaline droplets were present in most males from all dose groups receiving the test item. Multifocal basophilic tubules and proteinacious casts were also observed in individual males given 10 mg/kg bw/day and most males from the remaining dose groups. In addition, Lymphocytic infiltration was present in 2/10 males from the 300 mg/kg bw/day dose group. Immunohistochemical staining was positive for α-2u-globulin in males from all groups with an indication of increased staining levels from test item-treated animals.

At the end of the recovery phase, multifocal basophilic tubules or multifocal nephropathy and proteinacious casts were detected in all males previously receiving the test item at a dose level of 300 mg/kg bw/day. Immunohistochemical staining was positive for α-2u-globulin in all these males with levels similar in control and Group 4 animals.

Liver

At the end of the dosing period, minimal centrilobular hepatocyte hypertrophy was detected in a number of individual males treated with 100 or 300 mg/kg bw/day (dose-related) and most females receiving 300 mg/kg bw/day. At the end of the treatment-free period, no findings related to the administration of the test item were apparent in recovery animals of either sex.

Thyroid Gland

At the end of the treatment period, minimal hypertrophy of the follicular epithelium was observed in animals of either sex receiving 100 or 300 mg/kg bw/day in a dose-related manner. Changes in recovery males and females previously receiving 300 mg/kg bw/day had resolved by the end of the treatment-free period.

Conclusion

The oral (gavage) administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 100 or 300 mg/kg bw/day resulted in toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities. These were not reversible in males previously treated with 300 mg/kg bw/day after a twenty-eight day treatment-free period. It is therefore considered that a dose level of 10 mg/kg bw/day could be established as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the

male within the confines of this type of study. In contrast, there were no changes of toxicological significance in the females up to a dose level of 300 mg/kg bw/day, which could therefore be established as a NOAEL in the female within the confines of this type of study.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 17 January 2012 (formulation chemistry) and 23 February 2013. The in-life phase of the study was conducted between 14 February 2012 (first day of treatment) and 13 March 2012 (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 407).
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
The weight range of the animals at the start of the treatment were outside those detailed in the study plan. This was concluded to have no impact on the purpose or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
The weight range of the animals at the start of the treatment were outside those detailed in the study plan. This was concluded to have no impact on the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female rats were obtained from Harlan Laboratories U.K. Ltd. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for six days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 196 to 226 g, the females weighed 165 to 183 g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A pelleted diet was used. A certificate of analysis of the batch of diet used is given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd. Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve Study Plan target values of 22 ± 3ºC and 50 ± 20% respectively, there were no deviations from these target ranges.

The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The test item was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Twenty-eight consecutive days.
Frequency of treatment:
Daily.
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
The test item was administered to three groups, each of five male and five female rats at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).
Control animals:
yes, concurrent vehicle
Details on study design:
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.

The dose levels were chosen based on the results from previous toxicity work including a preliminary seven day range-finder investigation.

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Positive control:
No data
Observations and examinations performed and frequency:
Clinical Observations:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. All observations were recorded.

Functional Observations:
Prior to the start of treatment (Day -1) and on Days 7, 14, 21 and 27, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

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

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

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

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

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

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

The following parameters were observed:

Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

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

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

Water Consumption:
As a possible increase in water consumption was observed in the preliminary study, formal gravimetric measurement of water intake was performed on a daily basis throughout the study.
Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

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

Thyroid Hormone Assessment:
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20°C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were not analysed and were discarded.

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

Adrenals
Brain
Epididymides
Heart
Kidneys
Pituitary (post-fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Liver
Ovaries
Spleen
Testes
Thymus
Thyroid/Parathyroid
Uterus with Cervix

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

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

♦ = preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
* = eyes fixed in Davidson’s fluid
# = Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to the histology processing Test Site for processing. The tissues shown with the following symbol after their name ■ from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides were stained with Periodic Acid-Schiff (PAS) stain and examined. Any macroscopically observed lesions in all dosage groups were also similarly processed, together with the liver and spleen from all 30 and 300 mg/kg bw/day dose group animals.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidneys and thyroids from all animals from the 30 and 300 mg/kg bw/day groups.

Microscopic examination was conducted by the Study Pathologist.
Other examinations:
Laboratory Investigations:
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Haematology:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)

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

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

Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarised in tabular form.

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

Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:

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

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>=0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no unscheduled deaths on the study. Clinical signs were restricted to post-dosing salivation for both sexes at 300 and 1000 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no unscheduled deaths on the study. Clinical signs were restricted to post-dosing salivation for both sexes at 300 and 1000 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day mean body weight gain of males tended to be slightly lower than control throughout the study, although differences only attained statistical significance during Week 3.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day food intake for males was slightly lower than control during Week 1 and Week 3.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, water consumption for both sexes was notably higher than control throughout the study. At 300 mg/kg bw/day water consumption of males tended to be higher than control during the first two weeks of treatment.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See below
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, four males showed dark liver at necropsy, one of these males also showed pallor of the kidneys and a small prostate/seminal vesicles. At 300 mg/kg bw/day, one male showed pallor of the kidneys.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality:
There were no unscheduled deaths on the study.

Clinical Observations:
Clinical signs were restricted to post-dosing salivation for both sexes at 300 and 1000 mg/kg bw/day.

There were no clinical signs apparent for either sex at 30 mg/kg bw/day.

Functional Observations
Behavioural Assessments:
Assessment of the animals in a standard arena did not reveal any obvious neurological effects of treatment at 30, 300 or 1000 mg/kg bw/day.

A higher incidence of urination and defecation was noted for males at 1000 mg/kg bw/day during the last week of the study, although no similar findings were apparent for these animals during routine daily assessment of clinical signs, or for females at this dosage. The increased incidence of urination probably reflects the higher water consumption and, possibly also, the microscopic kidney changes observed for males at this dosage. In isolation, these findings were considered not to indicate any underlying behavioural or neurological effect of treatment.

Functional Performance Tests:
Assessment of grip strength or motor activity did not reveal any consistent pattern of results that indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

For females at 1000 mg/kg bw/day lower hind limb grip strength during Test 2 attained statistical significance when compared with control. No statistically significant differences from control were apparent during the other assessment of grip strength at this dosage and, in isolation, this finding was considered incidental and unrelated to treatment.

For femles at 30 mg/kg bw/day lower fore limb grip strength during Test 1 attained statistical significance when compared to control. This isolated finding, in the absence of similar effects at higher dosages, was considered incidental and unrelated to treatment.

Sensory Reactivity Assessments:
Sensory reactivity to different stimuli did not reveal any adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Body Weight:
At 1000 mg/kg bw/day mean body weight gain of males tended to be slightly lower than control throughout the study, although differences only attained statistical significance during Week 3. By termination, overall body weight gain for the treatment period was only 81% of the concurrent control.

There was no obvious effect of treatment on body weight performance for females at 1000 mg/kg bw/day or for either sex at 30 and 300 mg/kg bw/day.

Food Consumption:
At 1000 mg/kg bw/day food intake for males was slightly lower than control during Week 1 and Week 3.

Although there was a suggestion of lower food intake during Week 1 for females at this dosage, the differences from control were slight and probably reflect normal biological variation. Subsequent food intake for the remainder of the study was essentially similar or slightly superior to control.

At 30 and 300 mg/kg bw/day there were no obvious adverse effects of treatment on food consumption during the study.

Food conversion efficiency was considered to be unaffected by treatment at 30, 300 or 1000 mg/kg bw/day.

Water Consumption:
At 1000 mg/kg bw/day, water consumption for both sexes was notably higher than control throughout the study.

At 300 mg/kg bw/day water consumption of males tended to be higher than control during the first two weeks of treatment; thereafter water intake was generally similar to control. Water consumption of females at this dosage appeared unaffected by treatment.

At 30 mg/kg bw/day water consumption for both sexes was considered to have been unaffected by treatment. For females at this dosage, water consumption was noticeably higher than control on some occasions but in the absence of any similar increases for females at 300 mg/kg bw/day, these differences were considered to be incidental and unrelated to treatment.

Laboratory Investigations
Haematology:

Males
At 300 and 1000 mg/kg bw/day lower haemoglobin, erythrocyte count and haematocrit values attained statistical significance compared with control. At 1000 mg/kg bw/day, mean values and the majority of individual values were also lower than the historical control range. At 300 mg/kg bw/day, while three of the five values for erythrocyte count were lower than the historical control range, the mean value was still within this control range and, for haemoglobin and haematocrit mean values and the majority of individual values were within the historical control range.

Additionally at 1000 mg/kg bw/day, mean corpuscular haemoglobin was also statistically significantly lower than control. This seems to represent high values within the control rather than any adverse effect of treatment, as all values at 1000 mg/kg bw/day were within the historical control range while three controls values exceeded this historical range.

At 1000 mg/kg bw/day mean platelet count was higher than control, with differences attaining statistical significance. All individual values at this dosage exceeded the historical control range, but two of the five control values also exceeded this range.

At all dosages mean corpuscular haemoglobin concentration was lower than control with differences attaining statistical significance. Once again this seems to represent high control values, with three controls exceeding the historical control range compared to none within the treatment groups.

At all dosages, mean total leucocyte count was higher than concurrent control principally due to an increase in the mean number of neutrophils; these differences showed a dosage relationship and attained statistical significance. With the exception of one male at 30 mg/kg bw/day, the individual neutrophil counts for treated males exceeded the historical control range and this range was also exceeded for total leucocyte count by the majority of males at 1000 mg/kg bw/day.

At all dosages, mean prothrombin (CT) and activated partial thromboplastin (APTT) times were statistically significantly lower than control: individual and mean values were within the control range and no clear dosage relationship was apparent for prothrombin times.

Females
At 1000 mg/kg bw/day, a lower mean haemoglobin value attained statistical significance when compared to control; values for four of the five females at this dosage were lower than the historical control range. Additionally at this dosage, lower mean corpuscular haemoglobin concentration also attained statistical significance when compared to control. However, as previously observed for males, this may represent high control values as values for all females at 1000 mg/kg bw/day were within historical control range while the control values exceeded this range.

At 30 mg/kg bw/day, lower lymphocyte counts attained statistical significance when compared to control but the mean and all individual values were within the historical control range. In the absence of any similar decrease at higher dosages, this finding was considered fortuitous and unrelated to treatment.

At all dosages higher neutrophil counts attained statistical significance when compared with control. The majority of individual values for treated animals exceeded the historical control range but for mean values no consistent dosage relationship was apparent.

Blood Chemistry:

Males
At 1000 mg/kg bw/day a higher mean albumin value attained statistical significance when compared to control; the majority of individual values and the group mean value at this dosage exceeded the historical control range.

At all dosages, higher values for albumin/globulin ratio compared to control attained statistical significance and appeared to show a dosage relationship. However, all individual and mean values for treated groups were within the historical control range, while the majority of individual control values and the control group mean were below this historical range. There were no corresponding effects observed for levels of total protein and only higher albumin levels for males at 1000 mg/kg bw/day attained statistical significance when compared to control.

At all dosages, higher mean calcium levels showed a dosage relationship and attained statistical significance; all group mean values and the majority of individual values were within the historical control range. At 1000 mg/kg bw/day higher mean sodium levels also attained statistical significance when compared with control, but all individual values at this high dosage were within the historical control range.

At 300 and 1000 mg/kg bw/day, lower mean total cholesterol levels attained statistical significance compared with control. This may reflect high control levels as all control values and values at 30 mg/kg bw/day exceeded the historical control range, while only one value at 300 mg/kg bw/day and no values at 1000 mg/kg bw/day were outside the normal control range.

At all dosages, mean values for total bilirubin were statistically significantly lower than control but differences showed no obvious dosage relationship. Again this may reflect high control levels as only one value (occurring at 1000 mg/kg bw/day) marginally exceeded the historical control range for treated males compared to three out of five for the control group.

At 1000 mg/kg bw/day, bile acid values were statistically significantly lower than control.

Females
At all dosages, higher levels of total protein and albumin attained statistical significance when compared to control; although the highest values occurred at the high dosage of 1000 mg/kg bw/day, there was no dosage relationship at lower dosages. For total protein, mean values and the majority of individual values at 30 and 1000 mg/kg bw/day exceeded the historical control range, mean values and the majority of individual values for control and at 300 mg/kg bw/day were within this control range. For albumin, mean values and the majority of individual values at 1000 mg/kg bw/day exceeded the historical control range, but mean values and the majority of individual values at lower dosages were within this control range.

At all dosages, higher calcium levels showed a dosage relationship and attained statistical significance when compared with control, however only one individual value at 1000 mg/kg bw/day exceeded the historical control range.

At 300 and 1000 mg/kg bw/day, aspartate aminotransferase levels were statistically significantly lower than control but differences showed no dosage-relationship. This finding seems to reflect unusually high values within the control group as three of the five values exceeded the historical control range. A reduction in aspartate aminotransferase levels is unlikely to indicate an adverse effect of treatment.

Pathology
Necropsy:
At 1000 mg/kg bw/day, four males showed dark liver at necropsy, one of these males also showed pallor of the kidneys and a small prostate/seminal vesicles.

At 300 mg/kg bw/day, one male showed pallor of the kidneys.

No macroscopic findings were apparent at necropsy for both sexes at 30 mg/kg bw/day or females 300 and 1000 mg/kg bw/day.

Organ Weights:
Absolute and body weight-relative liver weights were increased for males at all dosages and females at 300 and 1000 mg/kg bw/day compared to control; with differences showing a dosage relationship and attaining statistical significance.

Absolute and body weight-relative kidney weights were increased for males at 300 and both sexes at 1000 mg/kg bw/day compared to control; differences attaining statistical significance and for males showed a dosage relationship.

Absolute and body weight-relative spleen weights were increased for males at 300 and 1000 mg/kg bw/day compared to control; differences showing a dosage relationship and attaining statistical significance.

At 1000 mg/kg bw/day, both sexes showed a statistically significantly increase in mean absolute and body weight-relative adrenal and thyroid weights compared with control. Males at this dosage also showed statistically significant lower absolute and body weight-relative prostate/seminal vesicle weights compared with control.

At 1000 mg/kg bw/day, females showed an increase in absolute and body weight-relative heart weights compared to control. However, all individual values at this dosage were within the historical control range and, in the absence of any histopathological correlates, this finding was considered to be incidental and unrelated to treatment.

Histopathology:
At all dosages, both sexes showed centrilobular to diffuse hepatocellular hypertrophy of minimal to moderate severity in the liver. This finding was not paralleled by increased inflammatory and/or degenerative lesions and was, therefore was considered to represent an adaptive change due to increased metabolism of the xenobiotic. The finding is expected to be reversible upon cessation of treatment. Minimal degrees of bile concrements were found in four males receiving 1000 mg/kg bw/day but this was not paralleled by degenerative of proliferative changes of the bile ducts and was deemed to represent a secondary/metabolic change.

At all dosages, minimal/slight increased severity of extramedullary hemopoiesis was apparent in the spleen, in a dose dependent manner. This finding was considered to be an adaptive change due to increased demand.

At all dosages, hyaline droplets nephropathy in the kidneys (consisting of increased severity of hyaline droplets, increased tubular degeneration/regeneration and granular casts) was observed for males. Additionally at 1000 mg/kg bw/day this nephropathy was often associated with interstitial inflammatory infiltrates. The occurrence of this nephropathy is deemed to be related to treatment.

For males at 30 mg/kg bw/day and both sexes at 300 and 1000 mg/kg bw/day, thyroid follicular hypertrophy was observed at elevated incidence and severity when compared to controls. Follicular hypertrophy of the thyroids is deemed to be associated with the increased hepatic metabolization of thyroid hormones (T3/T4) due to the previously described hepatocellular hypertrophy and as such is considered to represent a secondary effect.

The remainder of findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The effects detected in females were considered to relate to adaptive microscopic liver, thyroid and spleen changes.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
The oral administration of 1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0) to rats for twenty-eight days at dose levels up to 1000 mg/kg bw/day resulted in treatment related findings for both sexes at 30, 300 and 1000 mg/kg bw/day and it was not possible to identify a No Observed Effect Level (NOEL) for either sex.

The effects detected in females were considered to relate to adaptive microscopic liver, thyroid and spleen changes and therefore 1000 mg/kg bw/day may be considered a No Observed Adverse Effect Level (NOAEL) for females. Similar microscopic liver, thyroid and spleen changes were apparent for males however microscopic kidney changes and a concomitant increase in neutrophils were also apparent at all dosages and a NOAEL for the male rat could not be established. Kidney changes were characterised by hyaline droplets, increased tubular degeneration/regeneration and granular casts and at 1000 mg/kg bw/day interstitial inflammatory infiltrates. The hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings are more likely to be correlated to the same condition as the hyaline droplet accumulation and are therefore considered to represent limited relevance to humans. Excluding these kidney changes the No Observed Adverse Effect Level (NOAEL) for males was considered to be 300 mg/kg bw/day due to effects on haemoglobin, erythrocyte count and haematocrit values at 1000 mg/kg bw/day, the aetiology of which was uncertain.
Executive summary:

The oral administration of 1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0) to rats for twenty-eight days at dose levels up to 1000 mg/kg bw/day resulted in treatment related findings for both sexes at 30, 300 and 1000 mg/kg bw/day and it was not possible to identify a No Observed Effect Level (NOEL) for either sex. The effects detected in females were considered to relate to adaptive microscopic liver, thyroid and spleen changes and therefore 1000 mg/kg bw/day may be considered a No Observed Adverse Effect Level (NOAEL) for females. Similar microscopic liver, thyroid and spleen changes were apparent for males however microscopic kidney changes and a concomitant increase in neutrophils were also apparent at all dosages and a NOAEL for the male rat could not be established. Kidney changes were characterised by hyaline droplets, increased tubular degeneration/regeneration and granular casts and at 1000 mg/kg bw/day interstitial inflammatory infiltrates. The hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings are more likely to be correlated to the same condition as the hyaline droplet accumulation and are therefore considered to represent limited relevance to humans. Excluding these kidney changes the No Observed Adverse Effect Level (NOAEL) for males was considered to be 300 mg/kg bw/day due to effects on haemoglobin, erythrocyte count and haematocrit values at 1000 mg/kg bw/day, the aetiology of which was uncertain.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
K1: The study was performed according to OECD guidelines and GLP.
System:
male reproductive system
Organ:
other: sperm

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

The oral administration of 1,1,3,3-Tetramethylbutyl peroxyneodecanoate (CAS Number: 51240-95-0) to rats for twenty-eight days at dose levels up to 1000 mg/kg bw/day resulted in treatment related findings for both sexes at 30, 300 and 1000 mg/kg bw/day and it was not possible to identify a No Observed Effect Level (NOEL) for either sex. The effects detected in females were considered to relate to adaptive microscopic liver, thyroid and spleen changes and therefore 1000 mg/kg bw/day may be considered a No Observed Adverse Effect Level (NOAEL) for females. Similar microscopic liver, thyroid and spleen changes were apparent for males however microscopic kidney changes and a concomitant increase in neutrophils were also apparent at all dosages and a NOAEL for the male rat could not be established. Kidney changes were characterised by hyaline droplets, increased tubular degeneration/regeneration and granular casts and at 1000 mg/kg bw/day interstitial inflammatory infiltrates. The hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings are more likely to be correlated to the same condition as the hyaline droplet accumulation and are therefore considered to represent limited relevance to humans. Excluding these kidney changes the No Observed Adverse Effect Level (NOAEL) for males was considered to be 300 mg/kg bw/day due to effects on haemoglobin, erythrocyte count and haematocrit values at 1000 mg/kg bw/day, the aetiology of which was uncertain.

The oral (gavage) administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 100 or 300 mg/kg bw/day resulted in toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities. These were not reversible in males previously treated with 300 mg/kg bw/day after a twenty-eight day treatment-free period. It is therefore considered that a dose level of 10 mg/kg bw/day could be established as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the male within the confines of this type of study. In contrast, there were no changes of toxicological significance in the females up to a dose level of 300 mg/kg bw/day, which could therefore be established as a NOAEL in the female within the confines of this type of study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
K1: The study was performed according to OECD guidelines and GLP.

Justification for classification or non-classification

Based on the result from a 90 -day oral study in rats the substance is classified as a Category 2 Reproductive Toxicant.

The oral (gavage) administration of 1,1,3,3-tetramethylbutyl peroxyneodecanoate (CAS# 51240-95-0) to male and female Wistar Han™:RccHan™:WIST strain rats at a dose level of 100 or 300 mg/kg bw/day resulted in toxicologically significant changes in sperm concentration and motility and an associated increase in sperm abnormalities. These were not reversible in males previously treated with 300 mg/kg bw/day after a twenty-eight day treatment-free period. It is therefore considered that a dose level of 10 mg/kg bw/day could be established as a No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the male within the confines of this type of study. In contrast, there were no changes of toxicological significance in the females up to a dose level of 300 mg/kg bw/day, which could therefore be established as a NOAEL in the female within the confines of this type of study.