Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See attached document.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 30 August 2016 Experimental Completion Date: 17 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.
Test material information:
Composition 1
Specific details on test material used for the study:
Identification (chemical name) :2,2-di(tert-butylperoxy) butane
Product Name : Trigonox D-C50
CAS Number : CAS # 2167-23-9
EC Name : Di-tert-butyl sec-butylidene diperoxide
Description : Clear colorless liquid
Purity : 50.4% (in solvent, isododecane)
Batch Number : 1603442200
Label : TRIGONOX D-C50 For maximum quality 30 °C max
Date Received : 04 May 2016
Storage Conditions : Stored at ambient temperature 10 to 30 °C in the dark; formulated in the light at ambient temperature 10 to 30 °C
Expiry Date : 04 May 2017
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 314 to 350 g, the females weighed 182 to 224 g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least fourteen days. Formulations were initially prepared daily for the first two weeks and then weekly thereafter. Weekly formulations were stored at approximately 4 ºC in the dark. No correction was made for purity.

Samples of test item formulations were taken and analyzed for concentration of 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Item and Supporting Information
The test item described in the main part of this study was also used as the analytical standard.

Test Item Preparation and Analysis
Preparation of Calibration Standards
Stock slution of test item in dilution solvent were prepared for the external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentrationn of 1 mg/mL. Aliquots of this stock standard solution were used to prepare wprking standard solutions in dilution solvent with a concentration of 0.1 mg/mL.

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

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

To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 1.058 mg/mL by serial dilution covering the concentration range of 0.0529 mg/mL to 0.1587 mg/mL.

Preparation of Test Samples

The formulations recieved were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extract solvent this was then ultra-spnicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentrations.

Preparation of Accuracy and Precision 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.

The concetration of test item in the final solution was quantified by GC using FID detection as detailed in the instrument parameters below:


Instrument Parameters
GC System: Aligent Technologies 6890, incorporating autosampler and workstation
Column: RTX-1 (15 m x 0.32 nm id x 1 µm film)
Oven temperature programme:
Oven: 50°C for 2 minutes
with 6°C/minute to 100°C for 0 minutes
then 70°C/minute to 300°C for 3 minutes
Injection temperature: 110°C
Flame ionisation detector temperature: 310°C
Injection volume: 1 µL
Retention time: ~5-8 minutes


Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for the Test Item in the sample and procedural recovery chromatograms were measured. The concentration of Test Item was determined using the following calculations:

The concentration "x" of the test item in an injected sample was calculated by the following equation:

x= (y-a) / m

where:
x = concentration of the test item in the injected sample (mg/mL)
y = mean peak area response of the test item in the injected sample (counts)
m = slope of the calibration plot (counts per mg/mL)
a: y-axis intercept (counts)

The concentration of the test item in the test sample was calculated using the following equation:

c (x/mass) x F x SG

where
c= concentration of the test item in the test sample (mg/mL)
x = concentration of the test item in the injected sample solution (mg/mL)
F = sample dilution factor
SG: specific gravity (where applicable) (g/mL)
Mass = Mass taken (g)

The recovery rate (R) of the spiked recovery sample was calculated using the following equation:

R = (c/cfort) x 100

wher
R = recovery rate(%)
C = determined concentration of the test item in the spiked recovery sample (mg/mLL)
Cfort: fortified concentration of the test item in the spiked recovery sample (mg/mL)

Validation of the Analytical Procedure
The analytical procedire was validated by determining the following parameters:
The specifcity of the chromatographic analysis in control sample chromatogram
The linearity of detector response over the calibration standard cncentration range
The method accuracruy (recovery) and precision) by analyzing five reovery samples at nominal concentrations of 10 mg/mL and 125 mg/mL.
The limit of quantiifcation (LOQ) was determiined at the lowest standard concentration used during the study.

Homogeneity and Stability in Vehicle Formulations
The homogeneity and stability of Test Item in Arachis Oil BP forumlations was assessed at nominal concentrations of 10 mg/mL and 125 mg/mL during refrigerated storage.

Refrigerated storage (nominally +4°C)
The formulations were analysed and refrigerated on receipt. On day 14, the formulations were removed from storage and equilibrated to ambient temperature. the formulations were mixed as stated previously and single samples were removed fpr analysis from the top, middle and bottom of the mixed formulation.

Concentration of Dose Formulations
For each analysis occasion, freshly prepared test formulations were analysed. Duplicte samples were analysed in accordance with analytical procedure. Samples were disposed off once satisfactory results were achieved.


Duration of treatment / exposure:
up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.

iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.

viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Days 43 or 44.

ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
Not applicable
Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food
consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study. From Day 16 onwards, water intake was observed daily by visual inspection of water bottles.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

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

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

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute 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 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 following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
The methods used for hematological and blood chemical investigations are presented in Annex 6 and normal ranges are shown in Annex 9.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
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++)
Estrous cyclicity (parental animals):
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition
Sperm parameters (parental animals):
Testes weight
Testes histopathology
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data); see deviations from Study Plan.

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Days 43 or 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals
Pituitary (post-fixation)
Brain
Prostate and Seminal Vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary
Brain (including cerebrum, cerebellum and pons)
Prostate
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides ¿
Skin
Esophagus
Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Testes ¿
Jejunum
Thyroid/Parathyroid
Kidneys
Trachea
Liver
Thymus
Lungs (with bronchi)#
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus & Cervix
Mammary gland
Vagina

Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing. The tissues from five selected control and 500 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 500 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related liver, thyroid and kidney changes, examination was subsequently extended to include similarly prepared sections of the liver (both sexes), thyroids (both sexes) and kidneys (males only) from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Postmortem examinations (offspring):
All offspring were subjected to a full external and internal eamination, and any macroscopic abnormalities were recorded.
Statistics:
See below
Reproductive indices:
Reproductive Indices

Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index

The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i. Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = (Number of corpora lutea - Number of implantation on sites) Number of corpora lutea x100

Post–implantation loss (%) = Number of implantation sites - Total number of offsprinf / Number of implantation sites) x100

ii. Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offpsring alive on Day 1 / Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4 / Number of offpsring alive on Day 1) x 100

iii. Sex Ratio (% males)

Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

(Total of male offspring / Total number of offpsring) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg bw/day showed increased salivation from Day 4 onwards. Animals of either sex treated with 150 mg/kg bw/day also showed increased salivation albeit to a lesser extent. Two males treated with 500 mg/kg bw/day had pilo-erection between Days 20 and 31. No such effects were detected in animals of either sex treated with 50 mg/kg bw/day.
One female treated with 500 mg/kg bw/day had corneal opacity on Day 19 only. In isolation this finding was considered to be of no toxicological significance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 500 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Body weight gain in these males remained lower during Week 2, however, statistical significance was not achieved. Overall body weight gain for these males was 23% lower when compared to controls. A slight reduction in body weight gain was evident in females treated with 500 mg/kg bw/day during the first week of treatment, however, statistical significance was not achieved and recovery was evident thereafter. No adverse effects were evident in animals of either sex treated with 150 or 50 mg/kg bw/day.
Females treated with 150 and 50 mg/kg bw/day showed statistically significant increases in body weight gains between Days 7 and 14 of gestation, cumulative body weight gain between Days 0 and 14 of gestation and body weight on Day 4 of lactation. Females treated with 150 mg/kg bw/day also showed a statistically significant increase in cumulative body weight gain between Days 0 and 20 of gestation. An increase in body weight/body weight gain is considered not to represent an adverse effect of treatment therefore these intergroup differences were considered of no toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 500 mg/kg bw/day showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter.
No adverse effects were evident in males treated with 150 or 50 mg/kg bw/day or in treated females during maturation, gestation or lactation.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food conversion efficiency was slightly reduced in animals of either sex treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter and the intergroup difference was considered to reflect the change in body weight gain seen in these animals during this period.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males treated with 500 mg/kg bw/day showed an increase (30%) in overall water consumption during the pre-pairing phase. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.
Males treated with 500 mg/kg bw/day showed statistically significant reductions in hemoglobin, erythrocyte count and hematocrit. Four out of the five individual values were within historical control ranges and no associated histopathological changes were observed. The reduced group means were therefore most likely to be the result of one individual animal, whose values were below the historical control ranges and in isolation was considered of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in alanine aminotransferase. Males from this treatment group also showed an increase in aspartate aminotransferase, however, statistical significance was not achieved. Females treated with 500 mg/kg bw/day also showed statistically significant increases (p<0.05-0.01) in glucose, total protein, albumin and cholesterol. The effect on total protein and cholesterol also extended to females treated with 150 mg/kg bw/day. The majority of individual values for all of these parameters were within historical control ranges and may represent an adaptive response to the associated hepatic histopathological changes. Therefore a relationship to treatment cannot be excluded.
No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.
Males treated with 500 and 150 mg/kg bw/day showed a statistically significant reduction (p<0.05) in chloride concentration, which did not follow a dose-related response. Males treated with 500 mg/kg bw/day also showed a statistically significant increase (p<0.05) in phosphorus. All of the individual values for chloride concentration and the majority of individual values for phosphorous were within the historical control ranges therefore the intergroup differences were considered of no toxicological importance.
Females from all treatment groups showed a statistically significant increase (p<0.05) in chloride concentration and a statistically significant reduction in albumin/globulin ratio. Females treated with 500 mg/kg bw/day also showed a statistically significant increase (p<0.05) in creatinine. All of the individual values for albumin/globulin ratio and the majority of individual values for creatinine were within the historical control ranges and a true dose related response was not evident for albumin/globulin ratio. The intergroup differences were therefore considered not to be of toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments
One male treated with 500 mg/kg bw/day had pilo-erection during the final week of assessments. Another male showed increased salivation during Week 2 assessments. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.
One male treated with 50 mg/kg bw/day was observed to have ptosis (left eye only) during Weeks 4, 5 and 6 assessments. In the absence of any similar findings at 150 or 500 mg/kg bw/day, this observation was considered to be unrelated to treatment.

Functional Performance Tests
There were no intergroup differences at any dose level considered to be related to treatment with the test item.
During motor activity evaluations, males treated with 500 and 150 mg/kg bw/day showed a statistically significant reduction during the final 20% of activity monitoring time (p<0.05) whilst females treated with 150 mg/kg bw/day showed a statistically significant increase during the final 20% of activity monitoring time (p<0.01) when compared to controls. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance. Females from all treatment groups showed a statistically significant increase in hindlimb grip strength (p<0.05). A true dose related response was not evident and in the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered not to be of toxicological significance.

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):
Males from all treatment groups and females treated with 500 and 150 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in liver weight both absolute and relative to terminal body weight. Males treated with 500 and 150 mg/kg bw/day also showed a statistically significant increase (p<0.05-0.01) in absolute and relative kidney weights.
No toxicologically significant effects were evident in females treated with 50 mg/kg bw/day.
Males treated with 500 mg/kg bw/day showed a statistically significant increase in spleen weight both absolute and relative to terminal body weight. Females from all treatment groups showed a statistically significant increase in absolute and relative ovary weights. The majority of individual values were within the historical control ranges and in the absence of a true dose related response in ovary weight or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Eleven males treated with 500 mg/kg bw/day had mottled kidneys. Eight of these males also had enlarged kidneys, one male also had increased pelvic space in the left kidney and the left kidney was fluid filled, one male had an enlarged and dark liver and another male had enlarged kidneys. Five males treated with 150 mg/kg bw/day had mottled kidneys and one of these males also had increased pelvic space in both kidneys. One male treated with 50 mg/kg bw/day also had mottled kidneys. No such effects were detected in treated females.
Incidental findings that were not associated with either a true dose related response or any histopathological correlates and were considered to be unrelated to treatment included gaseous distension in the caecum (one control male), a dark and enlarged liver (one 500 mg/kg bw/day male), reddened lungs (two control males, one female treated with 50 mg/kg bw/day, one male and one female treated with 150 mg/kg bw/day and one male treated with 500 mg/kg bw/day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys: An increase in hyaline droplets (mild or moderate) was evident in all males treated with 500 and 150 mg/kg bw/day and in one male treated with 50 mg/kg bw/day (mild). Basophilic tubules were present in eleven males treated with 500 mg/kg bw/day and ten males treated with 150 mg/kg bw/day. In the remaining male treated with 500 mg/kg bw/day moderate nephropathy was apparent (tubular basophilia along with dilation and interstitial changes). Proteinaceous casts were also present (minimal to moderate) in all males treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day.

Liver: Periportal pigment was present in the bile ducts of three males and two females treated with 500 mg/kg bw/day and in four males treated with 150 mg/kg bw/day. Periportal Kupffer cell pigmentation was present in four males and one female treated with 500 mg/kg bw/day and in one male treated with 150 mg/kg bw/day. Special staining carried out on one control and one male treated with 500 mg/kg bw/day was negative for bile pigment, lipofuscin and hemosiderin. Inflammatory cell infiltration in the periportal area was present in two males and one female treated with 500 mg/kg bw/day. Centrilobular hypertrophy, minimal or mild, was present in four males and four females treated with 500 mg/kg bw/day and in two males and one female treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Thyroid Gland: Minimal follicular cell hypertrophy was present in three males and two females treated with 500 mg/kg bw/day and in three males treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle). No treatment effects were observed at evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Centrilobular hypertrophy is generally considered to be due to an adaptive response to mixed function oxidase. This change results in the increased clearance of thyroid hormones and the subsequent compensatory increased production causes hypertrophy of the follicular cells (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011).
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating
There was no adverse effect of treatment on mating performance. All animals mated within the first four days after pairing.

Fertility
Fertility as assessed by pregnancy index was unaffected by treatment with the test item at any dose level.
One female treated with 500 mg/kg bw/day and one female treated with 150 mg/kg bw/day were found to be non-pregnant following positive evidence of mating. Histopathological examinations of both of these females and their respective male partners did not reveal any significant microscopic changes which could account for the lack of pregnancy therefore these were considered incidental and unrelated to treatment.

Gestation Length
Gestation lengths were between 22½ and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.
Statistical analysis of gestation lengths did not reveal any statistically significant intergroup differences.

Litter Responses
In total all females from the control group and 50 mg/kg bw/day dose group and eleven females from the 150 and 500 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
There was no effect of treatment with the test item at any dose level on the mean number of corpora lutea, implantations or pre and post-implantation losses. Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from all treated dose groups was comparable with controls indicating the lack of any effect on offspring viability.
There were no treatment-related intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated groups when compared with controls. Statistical analysis of the data did not reveal any significant intergroup differences.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
other: male rat specific alpha 2u-globulin nephropathie
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Surface righting reflex for all treated dose groups were also comparable with controls.
Clinical signs detected in pups from treated dose groups included small size, cold, no milk in stomach, physical injury and missing. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 4post partum. Litter weights for all treated dose groups were also comparable with controls.
Male and female offspring from litters treated with 500 mg/kg bw/day showed a statistically significant increase in body weight on Day 4 of lactation. This intergroup difference was considered not to represent an adverse effect of treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 50, 150 or 500 mg/kg bw/day.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed in F1 generation
Critical effects observed:
no
Reproductive effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
no

 Group Mean Body Weight Gains

                                                                  Increase in Body Weight (g)                                                                 

                                                                                                                                                              

                                                                    Day Numbers Relative to Start Date                                                      

                                                                                                     Abs      %                                      

                                                                                                     Gain   Gain                                     

                                   Group  From:     1        8     15     22     29     36      1      1                                     

                                  (Sex)    To:       8       15     22     29     36     43    43     43                                      

                                                                                                                                                       

                                  1(M)    Mean     13.4     14.5    7.7   11.2   15.3    3.0   65.0   19.7                                   

                                           S.D.       5.9      5.2    3.4    4.6    5.5    6.4   16.7    4.8                                   

                                           N         12       12     12     12     12     12     12     12                                     

                                                                                                                                                       

                                  2(M)    Mean     11.4     12.4    6.6   13.6   12.6    3.8   60.3   18.4                                   

                                           S.D.      4.7      5.9    4.2    5.7    5.4    5.0   16.8    4.9                                   

                                           N        12       12     12     12     12     12     12     12                                     

                                                                                                                                                       

                                  3(M)    Mean     12.4     11.3   11.6   11.9   14.6    6.0   67.8   20.5                                   

                                           S.D.      3.9      4.6    3.9    4.7    6.1    4.8   12.3    3.5                                   

                                           N        12       12     12     12     12     12     12     12                                     

                                                                                                                                                       

                                  4(M)    Mean      6.7**   10.9    5.6   11.4   12.5    3.0   50.1   15.0                                   

                                           S.D.      5.9      8.1    8.2    4.1    5.7    3.7   15.8    4.5                                   

                                           N        12       12     12     12     12     12     12     12                                     

                                                                                                                                             

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day                          

 


(continued)             Group Mean Body Weight Gains

                                                                  Increase in Bodyweight (g)                                                                 

                                                                                                                                                              

                                                                    Day Numbers Relative to Start Date                                                      

                                                                                   Abs      %                                                        

                                                                                   Gain   Gain                                                       

                                                     Group  From:     1     8     1      1                                                       

                                                    (Sex)    To:      8    15   15     15                                                        

                                                                                                                                                       

                                                    1(F)    Mean     7.5   5.8  13.3    6.6                                                      

                                                             S.D.     4.8   4.6   7.5    3.8                                                      

                                                             N       12    12    12     12                                                        

                                                                                                                                                       

                                                    2(F)    Mean     7.8   8.2  16.0    7.5                                                      

                                                             S.D.     4.7   3.0   5.2    2.2                                                      

                                                             N       12    12    12     12                                                        

                                                                                                                                                       

                                                    3(F)    Mean     8.8   8.9  17.8    8.6                                                      

                                                             S.D.     4.4   2.8   5.6    2.7                                                      

                                                             N       12    12    12     12                                                        

                                                                                                                                                       

                                                    4(F)    Mean     5.8   5.8  11.6    5.6                                                      

                                                             S.D.     5.1   5.8   8.5    4.1                                                      

                                                             N       12    12    12     12                                                        

                                                                                                                                                                    

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day   


(continued)             Group Mean Body Weight Gains

 

 

Increase in Body Weight (g)
Days

 

Cumulative Body Weight Change (g)
Days

 

 

Gestation

 

Lactation

 

Gestation

Group
(sex)

From:

0

7

14

 

1

 

0

0

To:

7

14

20

 

4

 

14

20

 

 

 

 

 

 

 

 

 

 

1(F)

Mean

23.7

23.2

58.0

 

8.8

 

46.8

104.8

 

S.D.

4.8

4.2

8.5

 

7.7

 

7.3

11.8

 

N

12

12

12

 

12

 

12

12

 

 

 

 

 

 

 

 

 

 

2(F)

Mean

26.4

28.8*

61.1

 

7.0

 

55.3*

116.3

 

S.D.

7.8

4.8

10.9

 

7.4

 

11.0

14.7

 

N

12

12

12

 

12

 

12

12

 

 

 

 

 

 

 

 

 

 

3(F)

Mean

25.8

29.7**

64.0

 

11.8

 

55.5*

119.5*

 

S.D.

5.4

4.8

5.7

 

9.3

 

7.5

9.6

 

N

11

11

11

 

11

 

11

11

 

 

 

 

 

 

 

 

 

 

4(F)

Mean

25.7

26.6

55.1

 

10.0

 

52.4

107.5

 

S.D.

4.0

4.7

10.8

 

7.7

 

5.4

12.1

 

N

11

11

11

 

11

 

11

11

 

                                                                                                                     

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day                                                                                                                                                                                                                                                      

Group Mean Food Consumptions

                                                                                                                                                              

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                                   Group From:    1     8    22    29    36                                                       

                                                  (Sex)   To:     8    15    29    36    43                                                       

                                                                                                                                                         

                                                  1(M)   Mean    21.0  21.6  20.8  22.1  19.9                                                     

                                                          N       12    12    12    12    12                                                       

                                                                                                                                                         

                                                  2(M)   Mean    20.2  21.4  21.0  22.2  20.6                                                     

                                                          N       12    12    12    12    12                                                       

                                                                                                                                                         

                                                  3(M)   Mean    20.5  20.5  21.5  22.6  21.2                                                     

                                                          N       12    12    12    12    12                                                       

                                                                                                                                                         

                                                  4(M)   Mean    17.0  18.6  21.7  23.0  20.8                                                     

                                                          N       12    12    12    12    12                                                       

                                                                                                                                                         

                                                                                                                                 

Food Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day   


(continued)             Group Mean Food Consumptions

                                                                                                                                                              

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                                             Group From:    1     8                                                                  

                                                            (Sex)   To:     8    15                                                                  

                                                                                                                                                         

                                                            1(F)   Mean    15.3  15.8                                                                

                                                                    N       12    12                                                                  

                                                                                                                                                         

                                                            2(F)   Mean    15.7  16.1                                                                

                                                                    N       12    12                                                                  

                                                                                                                                                         

                                                            3(F)   Mean    15.6  15.9                                                                

                                                                    N       12    12                                                                  

                                                                                                                                                         

                                                            4(F)   Mean    14.5  14.9                                                                

                                                                    N       12    12                                                                  

                                                                                                                                                         

                                                                                                                                                       

Food Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day             


(continued)             Group Mean Food Consumptions

 

 

Day Numbers

 

Gestation

Lactation

Group
(Sex)

From:

0

7

14

 

1

 

To:

7

14

20

 

4

 

 

 

 

 

 

 

 

 

1(F)

Mean

19.2

21.0

33.6

 

26.1

 

S.D.

1.9

1.7

12.7

 

5.9

 

N

12

12

12

 

12

 

 

 

 

 

 

 

 

 

2(F)

Mean

20.4

23.1

25.2

 

24.5

 

S.D.

2.7

3.1

7.6

 

5.0

 

N

12

12

12

 

12

 

 

 

 

 

 

 

 

 

3(F)

Mean

20.1

22.5

35.7

 

30.2

 

S.D.

2.1

1.9

13.0

 

4.6

 

N

11

11

11

 

11

 

 

 

 

 

 

 

 

 

4(F)

Mean

19.8

22.9

28.5

 

27.0

 

S.D.

2.3

2.1

10.3

 

5.2

 

N

11

11

11

 

11

 

 

                                                                                                                                  

Food Consumption = g/animal/day.                                                           

                                                                                                                                                             

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day 

 

                    

Group Mean Daily Water Consumptions

                                                                                                                                                              

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                            Group From:    1     2     3     4     5     6     7                                                

                                           (Sex)   To:     2     3     4     5     6     7     8                                                

                                                                                                                                                         

                                           1(M)   Mean    29    29.3  31.8  34    28.5  27.5  34.3                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           2(M)   Mean    27.3  30.5  30.3  31.8  29    30.8  35.8                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           3(M)   Mean    25.8  28.3  29.5  30.3  25.3  25.3  30.3                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           4(M)   Mean    30.8  34    46.3  46.3  35.5  42    45                                                

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                               

Water Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day          

 


(continued)           Group Mean Daily Water Consumptions

                                                                                                                                                           

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                            Group From:    8     9    10    11    12    13    14                                                

                                           (Sex)   To:     9    10    11    12    13    14    15                                                

                                                                                                                                                         

                                           1(M)   Mean    29.5  34.5  35.3  32.3  29.5  29.8  31.5                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           2(M)   Mean    30.8  34.8  33.8  33.5  30.8  30    36                                                

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           3(M)   Mean    24.8  33    31    29.8  30    29.3  30.8                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           4(M)   Mean    37.3  45    43    45    39    36.3  44                                                

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                    

Water Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day   


(continued)           Group Mean Daily Water Consumptions

                                                                                                                                                              

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                            Group From:    1     2     3     4     5     6     7                                                

                                           (Sex)   To:     2     3     4     5     6     7     8                                                

                                                                                                                                                         

                                           1(F)   Mean    23    23.3  30    25.8  20    22.8  24.8                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           2(F)   Mean    21.3  24    25    30.5  21.3  24.5  24.5                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           3(F)   Mean    21.5  23.3  24.8  25.3  21.8  22.3  22.3                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           4(F)   Mean    23    20.3  24.5  26.3  23    23.3  24                                                

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                   

Water Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day            

(continued)           Group Mean Daily Water Consumptions

                                                                                                                                                             

                                                                                                                                                              

                                                             Day Numbers Relative to Start Date                                                              

                                                                                                                                                              

                                                                                                                                                         

                                            Group From:    8     9    10    11    12    13    14                                                

                                           (Sex)   To:     9    10    11    12    13    14    15                                                

                                                                                                                                                         

                                           1(F)   Mean    20    23    25.5  26.5  24    21    25.5                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           2(F)   Mean    23.8  24.5  26.5  25.3  27.3  21.3  28.8                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           3(F)   Mean    20.8  23.8  24.3  23.3  23    13.5  24.5                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                                                                         

                                           4(F)   Mean    21.3  27.3  27    26.3  25.8  24.5  27.3                                              

                                                   N       12    12    12    12    12    12    12                                                

                                                                                                           

Water Consumption = g/animal/day.                                                            

                                                                                                                                                              

Dose Levels: Group 1 ƒ 0(Control)   Group 2 ƒ 50 mg/kg bw/day   Group 3 ƒ 150 mg/kg bw/day   Group 4 ƒ 500 mg/kg bw/day   


      

Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

                                          ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ MALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ FEMALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                          ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                               0         50         150        500         0         50         150        500                               

                                            Control            mg/kg bw/day            Control           mg/kg bw/day                             

                                          ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ                            

Terminal Bodyweight ...........  Mean (g)  395.8     379.3     399.4     381.3     253.4     269.5     263.8     261.5                               

                                 S.D.       21.9      34.6      17.0      23.1      16.2      19.4      22.0      21.8                               

                                 N           12        12        12        12        12        12        12        12                                

                                                                                                                                                              

Adrenals ......................  Mean (g) 0.09878   0.08800   0.09924   0.10390   0.11706   0.11868   0.12296   0.11704                               

                                 S.D.    0.02101   0.01525   0.03363   0.00969   0.01398   0.00977   0.01844   0.02282                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.026     0.024     0.024     0.027     0.047     0.046     0.049     0.046                              

                                 S.D.       0.006     0.005     0.008     0.003     0.004     0.003     0.009     0.008                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Brain (Including Cerebrum,                                                                                                                                    

Cerebellum And Pons) ..........  Mean (g) 1.96950   2.02096   1.95582   1.98160   1.84508   1.85782   1.84016   1.86708                               

                                 S.D.    0.07111   0.07836   0.11588   0.12485   0.05905   0.07383   0.05483   0.04740                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.511     0.561     0.482     0.509     0.748     0.722     0.730     0.731                              

                                 S.D.       0.031     0.097     0.042     0.040     0.063     0.075     0.053     0.072                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Epididymides ..................  Mean (g) 1.60024   1.47404   1.54728   1.47470                                                                           

                                 S.D.    0.21604   0.16944   0.16112   0.09319                                                                           

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

                                 Mean (%)   0.406     0.391     0.387     0.387                                                                          

                                 S.D.       0.059     0.051     0.034     0.024                                                                          

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

Heart .........................  Mean (g) 1.04534   1.14060   1.15180   1.23128   0.87732   0.87944   0.88506   0.82590                               

                                 S.D.    0.09663   0.09601   0.12407   0.35485   0.10345   0.09322   0.09887   0.12634                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.271     0.316     0.284     0.315     0.354     0.341     0.352     0.322                              

                                 S.D.       0.019     0.052     0.033     0.090     0.037     0.046     0.056     0.049                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                         

                                                                                                                                 (continued)           Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

                                         ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ MALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ FEMALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                          ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                               0         50         150        500         0         50         150        500                               

                                            Control            mg/kg bw/day            Control           mg/kg bw/day                             

                                          ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ                            

Kidneys .......................  Mean (g) 2.35436   2.48718   2.74300*  3.62548** 1.66630   1.60538   1.80654   1.79342                               

                                 S.D.    0.20817   0.11454   0.31436   0.25985   0.13948   0.18334   0.11282   0.12396                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.611     0.691     0.673*    0.932**   0.674     0.622     0.716     0.699                              

                                 S.D.       0.064     0.127     0.054     0.085     0.057     0.077     0.058     0.052                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Liver .........................  Mean (g) 11.7631   12.6853*  14.1153*  17.3418** 10.3262   10.4903   12.5855*  13.1684*                              

                                 S.D.    1.07139   1.32021   0.87226   1.83350   0.97955   1.52794   0.71778   2.18038                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   3.050     3.480*    3.471*    4.436**   4.165     4.044     5.001*    5.125*                             

                                 S.D.       0.267     0.275     0.156     0.226     0.275     0.401     0.566     0.801                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Ovaries .......................  Mean (g)                                            0.10636   0.12670*  0.13370*  0.11841*                              

                                 S.D.                                                0.02359   0.01543   0.02216   0.01581                               

                                 N                                                       12        12        11        11                                

                                                                                                                                                              

                                 Mean (%)                                               0.042     0.047*    0.051*    0.045*                             

                                 S.D.                                                   0.011     0.006     0.012     0.006                              

                                 N                                                       12        12        11        11                                

                                                                                                                                                              

Pituitary .....................  Mean (g) 0.01087   0.00888   0.01030   0.00904*  0.01193   0.01092   0.01262   0.01202                               

                                 S.D.    0.00246   0.00175   0.00218   0.00155   0.00319   0.00231   0.00312   0.00340                               

                                 N           12        12        12        11        12        11         9        10                                

                                                                                                                                                              

                                 Mean (%)   0.003     0.002     0.003     0.002*    0.005     0.004     0.005     0.004                              

                                 S.D.       0.001     0.000     0.001     0.000     0.001     0.001     0.001     0.001                              

                                 N           12        12        12        11        12        11         9        10                                

                                                                                                                                                              

                                                                                                                                     


(continued)           Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

                                         ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ MALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ FEMALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                          ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                               0         50         150        500         0         50         150        500                               

                                            Control            mg/kg bw/day            Control           mg/kg bw/day                             

                                          ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ                            

Prostate ......................  Mean (g) 0.62576   0.63303   0.70083   0.54308                                                                           

                                 S.D.    0.19166   0.12130   0.09987   0.14387                                                                           

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

                                 Mean (%)   0.158     0.169     0.176     0.142                                                                          

                                 S.D.       0.044     0.042     0.025     0.034                                                                          

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

Seminal Vesicles ..............  Mean (g) 1.98468   1.81459   1.93593   1.87379                                                                           

                                 S.D.    0.36822   0.40988   0.33176   0.42302                                                                           

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

                                 Mean (%)   0.504     0.484     0.483     0.489                                                                          

                                 S.D.       0.104     0.122     0.073     0.089                                                                          

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

Spleen ........................  Mean (g) 0.76026   0.63456   0.77052   0.85364*  0.64322   0.66908   0.73160   0.55586                               

                                 S.D.    0.06907   0.04045   0.05617   0.07420   0.05340   0.12153   0.18244   0.03042                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.197     0.175     0.190     0.219*    0.261     0.259     0.292     0.217                              

                                 S.D.       0.016     0.017     0.015     0.009     0.029     0.047     0.085     0.017                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Testes ........................  Mean (g) 3.56646   3.65411   3.76428   3.73640                                                                           

                                 S.D.    0.21649   0.44110   0.29910   0.28500                                                                           

                                 N           12        12        12        12                                                                            

                                                                                                                                                              

                                 Mean (%)   0.903     0.967     0.943     0.984                                                                          

                                 S.D.       0.060     0.113     0.068     0.104                                                                          

                                 N           12        12        12        12                                                                            

            
(continued)           Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

                                         ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ MALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ FEMALES ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                          ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ                            

                                               0         50         150        500         0         50         150        500                               

                                            Control            mg/kg bw/day            Control           mg/kg bw/day                             

                                          ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒƒƒƒƒƒƒ                            

Thymus ........................  Mean (g) 0.35550   0.33350   0.36350   0.28738   0.21644   0.22812   0.24758   0.23942                               

                                 S.D.    0.05594   0.05759   0.02571   0.04682   0.01191   0.05921   0.06879   0.05782                               

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.092     0.093     0.089     0.074     0.088     0.087     0.098     0.093                              

                                 S.D.       0.013     0.023     0.007     0.014     0.006     0.018     0.025     0.019                              

                                 N            5         5         5         5         5         5         5         5                                

                                                                                                                                                              

Thyroid/Parathyroid ...........  Mean (g) 0.01300   0.01458   0.01396   0.01580   0.02480   0.02968   0.02638   0.01952                               

                                 S.D.    0.00299   0.00417   0.00379   0.00657   0.00432   0.00329   0.01174   0.00632                               

                                 N            5         5         5         4         5         5         5         5                                

                                                                                                                                                              

                                 Mean (%)   0.003     0.004     0.003     0.004     0.010     0.012     0.011     0.008                              

                                 S.D.       0.001     0.001     0.001     0.002     0.002     0.001     0.005     0.002                              

                                 N            5         5         5         4         5         5         5         5                                

                                                                                                                                                              

Uterus & Cervix ...............  Mean (g)                                            0.67787   0.64608   0.72538   0.66220                               

                                 S.D.                                                0.11362   0.10643   0.13003   0.10689                               

                                 N                                                       12        12        11        11                                

                                                                                                                                                              

                                 Mean (%)                                               0.269     0.240     0.275     0.253                              

                                 S.D.                                                   0.052     0.037     0.057     0.053                              

                                 N                                                       12        12        11        11                                

       

Conclusions:
The oral administration of Trigonox D-C50 (50.4% 2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) to Wistar Han™:RccHan™:WIST strain rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 and 150 mg/kg bw/day. These included reduced initial body weight gains in either sex at 500 mg/kg bw/day, reduced food consumption and increased water consumption in males at 500 mg/kg bw/day, organ weight changes in either sex at 500 and 150 mg/kg bw/day, macroscopic changes in males at 500 and 150 mg/kg bw/day and microscopic changes in either sex at 500 and 150 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 50 mg/kg bw/day for either sex.
The inflammatory cell infiltration in the periportal area of the liver in either sex at 500 mg/kg bw/day was considered to represent an adverse effect of treatment. The microscopic liver changes evident in females treated with 150 mg/kg bw/day were considered to be an adaptive response to mixed function oxide induction (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011). Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for females. Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 500 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of Trigonox D-C50 on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

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

Trigonox D-C502 (50.4% 2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

Adult males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.


Results…….

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Animals of either sex treated with 500 mg/kg bw/day showed increased salivation from Day 4 onwards. Animals of either sex treated with 150 mg/kg bw/day also showed increased salivation albeit to a lesser extent. Two males treated with 500 mg/kg bw/day had pilo-erection between Days 20 and 31. No such effects were detected in animals of either sex treated with 50 mg/kg bw/day.

Behavioral Assessment

One male treated with 500 mg/kg bw/day had pilo-erection during the final week of assessments. Another male showed increased salivation during Week 2 assessments. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

Functional Performance Tests

There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex.

Sensory Reactivity Assessments

Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Body Weight

Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Overall body weight gain for these males was also reduced.  A slight reduction in body weight gain was evident in females treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. No adverse effects were evident in animals of either sex treated with 150 or 50 mg/kg bw/day.

Food Consumption

Males treated with 500 mg/kg bw/day showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. No adverse effects were evident in males treated with 150 or 50 mg/kg bw/day or in treated females during maturation, gestation or lactation. Food conversion efficiency was slightly reduced in animals of either sex treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter.

Water Consumption

Males treated with 500 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

Reproductive Performance

Mating

There was no effect of treatment on mating performance. All animals mated within four days of pairing.

Fertility

There were no treatment-related effects in conception rates for test item-treated animals in relation to controls.

Gestation Lengths

There were no differences in gestation lengths in animals receiving the test item when compared with controls.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on the mean number of corpora lutea, implantations, pre and post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age at 50, 150 or 500 mg/kg bw/day.

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights or clinical signs up to Day 5 of age at 50, 150 or 500 mg/kg bw/day.

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

Blood Chemistry

Animals of either sex treated with 500 mg/kg bw/day showed an increase in alanine aminotransferase. Males from this treatment group also showed an increase in aspartate aminotransferase. Females treated with 500 mg/kg bw/day also showed increases in glucose, total protein, albumin and cholesterol. The effect on total protein and cholesterol also extended to females treated with 150 mg/kg bw/day.

No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Pathology

Necropsy

Eleven males treated with 500 mg/kg bw/day had mottled kidneys. Eight of these males also had enlarged kidneys, one male also had increased pelvic space in the left kidney and the left kidney was fluid filled, one male had an enlarged and dark liver and another male had enlarged kidneys. Five males treated with 150 mg/kg bw/day had mottled kidneys and one of these males also had increased pelvic space in both kidneys. One male treated with 50 mg/kg bw/day also had mottled kidneys.

No such effects were detected in treated females.

Organ Weights

Males from all treatment groups and females treated with 500 and 150 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 and 150 mg/kg bw/day also showed an increase in absolute and relative kidney weights. No such effects were evident in females treated with 50 mg/kg bw/day.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Kidneys:An increase in hyaline droplets (mild or moderate) was evident in all males treated with 500 and 150 mg/kg bw/day and in one male treated with 50 mg/kg bw/day (mild). Basophilic tubules were present in eleven males treated with 500 mg/kg bw/day and ten males treated with 150 mg/kg bw/day. In the remaining male treated with 500 mg/kg bw/day moderate nephropathy was apparent (tubular basophilia along with dilation and interstitial changes). Proteinaceous casts were also present (minimal to moderate) in all males treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day.

Liver:Periportal pigment was present in the bile ducts of three males and two females treated with 500 mg/kg bw/day and in four males treated with 150 mg/kg bw/day. Periportal Kupffer cell pigmentation was present in four males and one female treated with 500 mg/kg bw/day and in one male treated with 150 mg/kg bw/day. Special staining carried out on one control and one male treated with 500 mg/kg bw/day was negative for bile pigment, lipofuscin and hemosiderin. Inflammatory cell infiltration in the periportal area was present in two males and one female treated with 500 mg/kg bw/day. Centrilobular hypertrophy, minimal or mild, was present in four males and four females treated with 500 mg/kg bw/day and in two males and one female treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Thyroid Gland: Minimal follicular cell hypertrophy was present in three males and two females treated with 500 mg/kg bw/day and in three males treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Centrilobular hypertrophy is generally considered to be due to an adaptive response to mixed function oxidase. This change results in the increased clearance of thyroid hormones and the subsequent compensatory increased production causes hypertrophy of the follicular cells (Capen C.Cet al; 2002, Cattley R.Cet al; 2002 and Zabka T.Set al; 2011).

Conclusion

The oral administration of Trigonox D-C50 (50.4% 2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) to Wistar Han™:RccHan™:WIST strain rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 and 150 mg/kg bw/day. These included reduced initial body weight gains in either sex at 500 mg/kg bw/day, reduced food consumption and increased water consumption in males at 500 mg/kg bw/day, organ weight changes in either sex at 500 and 150 mg/kg bw/day, macroscopic changes in males at 500 and 150 mg/kg bw/day and microscopic changes in either sex at 500 and 150 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 50 mg/kg bw/day for either sex.

The inflammatory cell infiltration in the periportal area of the liver in either sex at 500 mg/kg bw/day was considered to represent an adverse effect of treatment. The microscopic liver changes evident in females treated with 150 mg/kg bw/day were considered to be an adaptive response to mixed function oxidase induction (Capen C.Cet al; 2002, Cattley R.Cet al; 2002 and Zabka T.Set al; 2011). Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for females. Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 500 mg/kg bw/day.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
other: male rat specific alpha 2u-globulin nephropathie
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Executive summary:

A study was designed to investigate the systemic toxicity and potential adverse effects of Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane(CAS # 2167-23-9) in isododecane) on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996). Trigonox D-C50 was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP). Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. Adult males were terminated on Days 43 or 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths during the study. Animals of either sex treated with 500 mg/kg bw/day showed increased salivation from Day 4 onwards. Animals of either sex treated with 150 mg/kg bw/day also showed increased salivation albeit to a lesser extent. Two males treated with 500 mg/kg bw/day had pilo- erection between Days 20 and 31. No such effects were detected in animals of either sex treated with 50 mg/kg bw/day. One male treated with 500 mg/kg bw/day had pilo-erection during the final week of assessments. Another male showed increased salivation during Week 2 assessments. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day. There was no effect of treatment with the test item at any dose level on functional performance in animals of either sex. Sensory reactivity scores across all test item-treated dose groups were similar to controls. Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first two weeks of treatment. Overall body weight gain for these males was also reduced.  A slight reduction in body weight gain was evident in females treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. No adverse effects were evident in animals of either sex treated with 150 or 50 mg/kg bw/day. Males treated with 500 mg/kg bw/day showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. No adverse effects were evident in males treated with 150 or 50 mg/kg bw/day or in treated females during maturation, gestation or lactation. Food conversion efficiency was slightly reduced in animals of either sex treated with 500 mg/kg bw/day during the first week of treatment, however, recovery was evident thereafter. Males treated with 500 mg/kg bw/day showed an increase in overall water consumption during the pre-pairing phase. No such effects were evident in females treated with 500 mg/kg bw/day or in animals of either sex treated with 150 or 50 mg/kg bw/day.

There were no toxicologically significant effects detected in the hematological parameters examined. Animals of either sex treated with 500 mg/kg bw/day showed an increase in alanine aminotransferase. Males from this treatment group also showed an increase in aspartate aminotransferase. Females treated with 500 mg/kg bw/day also showed increases in glucose, total protein, albumin and cholesterol. The effect on total protein and cholesterol also extended to females treated with 150 mg/kg bw/day. No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.Eleven males treated with 500 mg/kg bw/day had mottled kidneys. Eight of these males also had enlarged kidneys, one male also had increased pelvic space in the left kidney and the left kidney was fluid filled, one male had an enlarged and dark liver and another male had enlarged kidneys. Five males treated with 150 mg/kg bw/day had mottled kidneys and one of these males also had increased pelvic space in both kidneys. One male treated with 50 mg/kg bw/day also had mottled kidneys. No such effects were detected in treated females. Males from all treatment groups and females treated with 500 and 150 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 and 150 mg/kg bw/day also showed an increase in absolute and relative kidney weights. No such effects were evident in females treated with 50 mg/kg bw/day. The following treatment-related microscopic abnormalities were detected:

Kidneys:An increase in hyaline droplets (mild or moderate) was evident in all males treated with 500 and 150 mg/kg bw/day and in one male treated with 50 mg/kg bw/day (mild).Basophilic tubules were present in eleven males treated with 500 mg/kg bw/day and ten males treated with 150 mg/kg bw/day. In the remaining male treated with 500 mg/kg bw/day moderate nephropathy was apparent (tubular basophilia along with dilation and interstitial changes). Proteinaceous casts were also present (minimal to moderate) in all males treated with 500 mg/kg bw/day and in two males treated with 150 mg/kg bw/day.

Liver:Periportal pigment was present in the bile ducts of three males and two females treated with 500 mg/kg bw/day and in four males treated with 150 mg/kg bw/day. Periportal Kupffer cell pigmentation was present in four males and one female treated with 500 mg/kg bw/day and in one male treated with 150 mg/kg bw/day. Special staining carried out on one control and one male treated with 500 mg/kg bw/day was negative for bile pigment, lipofuscin and hemosiderin. Inflammatory cell infiltration in the periportal area was present in two males and one female treated with 500 mg/kg bw/day. Centrilobular hypertrophy, minimal or mild, was present in four males and four females treated with 500 mg/kg bw/day and in two males and one female treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Thyroid Gland: Minimal follicular cell hypertrophy was present in three males and two females treated with 500 mg/kg bw/day and in three males treated with 150 mg/kg bw/day. No treatment related changes were evident in animals of either sex treated with 50 mg/kg bw/day.

Centrilobular hypertrophy is generally considered to be due to an adaptive response to mixed function oxidase. This change results in the increased clearance of thyroid hormones and the subsequent compensatory increased production causes hypertrophy of the follicular cells (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011).

The oral administration of

Trigonox D-C50 (50.4% 2,2-di(tert-butylperoxy) butane (CAS # 2167-23-9) in isododecane) to Wistar Han™:RccHan™:WIST strain rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 500 and 150 mg/kg bw/day. These included reduced initial body weight gains in either sex at 500 mg/kg bw/day, reduced food consumption and increased water consumption in males at 500 mg/kg bw/day, organ weight changes in either sex at 500 and 150 mg/kg bw/day, macroscopic changes in males at 500 and 150 mg/kg bw/day and microscopic changes in either sex at 500 and 150 mg/kg bw/day. A No Observed Effect Level (NOEL) for systemic toxicity was considered to be 50 mg/kg bw/day for either sex.

The inflammatory cell infiltration in the periportal area of the liver in either sex at 500 mg/kg bw/day was considered to represent an adverse effect of treatment. The microscopic liver changes evident in females treated with 150 mg/kg bw/day were considered to be an adaptive response to mixed function oxidase induction (Capen C.C et al; 2002, Cattley R.C et al; 2002 and Zabka T.S et al; 2011). Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for females. Although the kidney findings of tubular basophilia and proteinaceous casts in male kidneys could be considered an adverse effect, these findings were considered to be associated with alpha 2u-globulin and formation of hyaline droplets, an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, these findings observed on this study would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 150 mg/kg bw/day for males because the findings do not reflect true systemic toxicity.