Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
clear, colorless

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.