Cyclohexanone, peroxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item 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…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks to males and up to eight weeks to females including a two week pre-pairing phase, pairing, gestation and early lactation, at dose levels of 30, 100 or 200 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

 

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. Female 85 treated with 200 mg/kg bw/day was killedin extremison Day 3 of dosing and Male 73 was therefore not paired with any female.

 

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 Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results…….

Mating

There was no effect of treatment on mating performance.

 

Fertility

Fertility remained unaffected by treatment with the test item at any dose level.

Conclusion

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

 

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day. 

 

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 March 2015 Experimental completion date: 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days before the start of dosing 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 313 to 362g, the females weighed 191 to 232g, and were approximately twelve weeks old.

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, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Appendix 29. 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 late 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 Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a 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:

polyethylene glycol

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 24 days when stored at approximately 4 °C, in the dark. Formulations were therefore prepared approximately fortnightly, aliquoted and stored as above before use.

Samples of the test item formulation were taken and analyzed for concentration of Cyclohexanone peroxide (CAS# 012262-58-7) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were 96 to 108% of the nominal concentration.

Details on mating procedure:

iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. Female 85 treated with 200 mg/kg bw/day was killed in extremis on Day 3 of dosing and Male 73 was therefore not paired with any female.
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, five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consiting of a single peak.

Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.2 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

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

Analysis of samples
The formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile, which was then shaken to dissolve. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lowest and hihgest anticipated dose concentrations. These samples were then prepared for analysis.

Preparation of lineraity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.047 mg/mL by serial dilution covering the conventration range 0 to 0.3141 mg/mL.

Instrumental parameters
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Zorbax Eclipse XDB 5µ C18 (150 x 4.6 mm id)
Mobile phase: Aluent A_Water Eluent B_acetonitrile
Time %B
0 55
4 55
7 100
10 100

Flow-rate: 1 mL/min
UV detector wavelength: 210 nm
Injection volume: 10 µL
Retention time: ~2 mins

Duration of treatment / exposure:

Males: approximately six weeks
Females: up to eight weeks

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. Female 85 treated with 200 mg/kg bw/day was killed in extremis on Day 3 of dosing and Male 73 was therefore not paired with any female.
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, 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 Day 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 surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males, 12 females

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); see deviations from Study Plan. All observations were recorded.

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 Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin 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

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; see deviations from Study Plan.

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 observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Female 85 treated with 200 mg/kg bw/day was killed in extremis on Day 3 of dosing and Male 73 was therefore not paired with any female. 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.

Pregnancy and Parturition
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 and public holidays. 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

Laboratory Investigations
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.

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
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded.

Sperm parameters (parental animals):

Testest weight and 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)

Postmortem examinations (parental animals):

Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43 or 44. Surviving adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on Day 26 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.

SACRIFICE AND PATHOLOGY
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 with an asterix (*) were weighed from all remaining animals:

Adrenals
Prostate
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

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 with an asterix (*) 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 (hind limb)
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes
Gross lesions*
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*
Liver
Thymus
Lungs (with bronchi)
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

Tissues were dispatched to the Test Site (Huntingdon Life Sciences Ltd.) for processing. The tissues from five selected control and 200 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy 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 200 mg/kg bw/day animals and any macroscopic abnormalities were also processed. In addition, sections of testes from all control and 200 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.

Microscopic examination was conducted by the Study Pathologist.

Postmortem examinations (offspring):

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

Statistics:

Due to the nature and quantity of this data, please see the section "Any other information on material and methods including tables"

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:

Litter Responses
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 sites) / Number of corpora lutea) x100

Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / 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 offspring alive on Day 1 / Number of offspring born) x 100

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

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Adult responses
Mortality
Please see the section "Any other information on results"

Clinical Observations
Of the animals surviving to the scheduled necropsy, 2/12 males and 4/10 females treated with the test item at a dose level of 200 mg/kg bw/day showed sporadic instances of noisy respiration with one of these males also showing clinical signs of noisy/labored respiration and decreased respiratory rate on Day 35 of dosing; the latter clinical signs were observed during behavioral assessment for this male. It is worth noting that the instances of noisy respiration for these animals were generally observed during the latter half of the treatment period although Female 89 showed noisy respiration on Days 2 and 3 of dosing. Additionally, two females each from the 100 or 200 mg/kg bw/day dose groups that were either found dead or killed in extremis due to their deteriorating clinical condition, also showed respiratory signs including noisy/labored/gasping respiration and/or decreased respiratory rate prior to death. For three of these premature decedents, the clinical signs correlated with microscopic changes in the upper respiratory tract which were considered to be likely due to an irritant nature of the test item. The remaining early decedent (Female 65 treated with 100 mg/kg bw/day) also showed clinical signs of labored respiration, piloerection, lethargy, hunched posture, pale extremities, diarrhea and chromodacryorrhea prior to death on Day 22 of gestation and these were deemed to be related to marked inflammatory change in the reproductive tract and thus parturition rather than the administration of the test item.

At 30 or 100 mg/kg bw/day, none of the animals of either sex surviving to the scheduled necropsy were observed with any clinical signs.

At 200 mg/kg bw/day, a few instances of increased post-dose salivation were observed for individual males and one female. Such observations are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered to be of no toxicological importance.

Two control males from one cage showed scab formation on some days which is likely to have resulted from fighting amongst cage animals. One control female also showed generalized fur loss during the last week of dosing and this finding was not treatment-related.

Functional Observations
Behavioral Assessments

There were no changes in the behavioral parameters measured that were considered to be related to the toxicity of the test item at any dose level.

Male 73 from the 200 mg/kg bw/day dose group and Female 68, an early decedent from the 100 mg/kg bw/day, were observed with noisy/labored respiration and decreased respiratory rate during behavioral assessment in Week 5 with noisy respiration again being evident for the male during Week 6 assessment. These observations were considered likely to be due to an irritant nature of the test item.

Functional Performance Tests

There were no intergroup differences considered to be related to treatment with the test item.

Grip strength evaluations during the last week of the treatment period revealed statistically significantly higher group mean value for hindlimb strength in males receiving the test item at 200 mg/kg bw/day relative to controls (p<0.01). It is worth noting, however, that this difference was only apparent in 1/3 grip strength tests with no statistically significant intergroup differences during the remaining 2/3 tests and, as such, this observation was considered to be incidental.

Sensory Reactivity Assessments

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

Body Weight
There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex.

At 200 mg/kg bw/day, group mean body weight gain in males over the first week of dosing was marginally lower than controls albeit without achieving statistical significance. Recovery was evident thereafter such that the weekly group mean weight gains for these males were occasionally slightly higher than controls resulting in a slightly higher overall mean body weight gain for these animals. At 30 or 100 mg/kg bw/day, body weight development in males was generally comparable with controls throughout the treatment period.

During the first week of the pre-pairing phase of the study, group mean body weight gain in females treated with 200 mg/kg bw/day was marginally lower than controls albeit without achieving statistical significance. Thereafter, improvement was apparent and group mean body weight gain over the second week of dosing for these females was comparable with controls. A high degree of inter-individual variation was evident amongst female dose groups, but body weight performance at 30 or 100 mg/kg bw/day during this phase of the study was generally similar to controls. During the gestation and lactation phases of the study, group mean body weight gains for all test item-treated female dose groups were comparable with controls.


Food Consumption
There was no adverse effect of treatment with the test item on dietary intake or food conversion efficiency in animals of either sex.

During the second week of dosing, food intake in females given 200 mg/kg bw/day was marginally lower than controls. During gestation and lactation phases of the study, however, food consumption across all test item-treated female dose groups was similar to controls and any initial effect in the 200 mg/kg bw/day dose group was considered not to be of an adverse nature. Food consumption in males from all dose groups remained comparable with controls. Additionally, food conversion efficiency values for animals of either sex were generally comparable with controls with any differences deemed to be reflective of minor intergroup differences in body weight gains.

Water Consumption
Visual inspection of water bottles did not indicate any differences for the animals given the test item in relation to controls.

Reproductive Performance
Mating
There was no effect of treatment with the test item on mating performance with most animals mating within four days after pairing. The only exception was Female 18 from the control group, which showed signs of mating after 12 days of pairing.


Fertility
Fertility as assessed by pregnancy index remained unaffected by treatment with the test item at any dose level.

Of the females reaching the scheduled termination on Day 5 post partum, two females from the control group (Females 22 and 24) and one female each treated with 30 or 100 mg/kg bw/day (Females 48 and 69, respectively) did not achieve pregnancy following evidence of positive mating. Additionally, Female 42 given 30 mg/kg bw/day showed evidence of pregnancy at necropsy but there was no evidence of littering. The three premature decedent females from the 100 or 200 mg/kg bw/day dose groups that were paired with males also showed evidence of pregnancy at necropsy. All non-pregnant females appeared to be cycling normally and histopathology examination of the selected tissues from these females and their corresponding male partners did not identify any obvious reason for the lack of pregnancy in these females.

At all dose levels, 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) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Gestation Length
Gestation lengths were between 22 and 23½ days and their distribution for the test item-treated females was similar to controls.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOEL

Remarks:

Reproductive toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Litter Responses
In total ten, ten, nine and ten females from the control, 30, 100 or 200 mg/kg bw/day dose groups, respectively, 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 on the mean number of corpora lutea, implantation counts and pre- or post-implantation losses.

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from females treated with the test item at all dose levels was generally comparable with controls. Live birth and survival indices for the 200 mg/kg bw/day dose group appeared to be marginally lower than controls, but this was mainly due to the litter from Female 86 which was observed with 7/11 dead pups following completion of parturition and a further two pups going missing over the subsequent days. In isolation, these findings were considered to be unrelated to treatment with the test item.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated dose groups when compared with controls.

Offspring Growth and Development
Offspring body weights and litter weights on Days 1 and 4 post partum and offspring body weight development over Days 1 to 4 post partum were generally comparable with controls. Any intergroup differences were not dose-related and since the corresponding values from the 200 mg/kg bw/day dose group were similar to controls, these were considered to be incidental.

Surface righting reflex data did not reveal any detrimental effect of treatment with the test item at any dose level in relation to controls.

Female 86 treated with 200 mg/kg bw/day was observed with 7/11 dead pups following completion of parturition whilst the remaining four pups from this litter appeared to be small, cold, weak and with no milk in the stomach on Day 1 post partum with two of these going missing over the subsequent days. Clinical signs detected in pups from the remaining test item-treated dose groups included small size, right hind paw swollen, found dead or missing and, were considered to be low incidence findings observed in offspring in studies of this type and, in isolation, this finding was deemed unlikely to be related to administration of the test item to maternal females.

Key result

Remarks on result:

other: no effects observed

Reproductive effects observed:

not specified

Adult Reponses

Mortality

There were four unscheduled deaths during the study and included two females each from the 100 and 200 mg/kg bw/day dose groups. Clinical signs prior to death and macroscopic necropsy finding for these early decedents are presented below:

 

Dose Level (mg/kg bw/day)	Animal Number	Day of dosing (gestation)	Mode of Death	Clinical Signs (Day of dosing)	Macroscopic Findings at necropsy
100	65	38 (22)	KIE	Noisy respiration (36) Labored respiration (38), Piloerection (38) Lethargy (38) Hunched posture (38) Pale extremities (38) Diarrhoea (38) Chromodacryorrhea (38)	Small spleen Green contents in the stomach Dark brown fluid-filled uterus & cervix
	68	36 (19)	FD	Noisy respiration (35)* Labored respiration (35)* Decreased respiratory rate (35)*	Gaseous distension in duodenum, ileum and jejunum Dark lungs
200	85	3	KIE	Labored respiration (3) Gasping respiration (3) Pale extremities (3) Hypothermia (3)	No abnormalities detected
	94	39 (21)	FD	Noisy respiration (37) Labored respiration (37) Decreased respiratory rate (37)	Dark lungs

FD = Found dead                KIE = Killedin extremis

* = These clinical signs were observed during weekly behavioural assessment

 

These early decedents showed clinical signs of noisy/labored/gasping respiration and/or decreased respiratory rate prior to death but only Females 68 and 94 treated with 100 or 200 mg/kg bw/day, respectively, were observed with dark lungs at necropsy. Histopathological examination of the tissues from Female 65 showed marked inflammatory change in the reproductive tract and secondary lymphoid/cellular depletion and it was deemed likely that these changes were a result of parturition and unrelated to administration of the test item. Histopathology examination of the tissues from the remaining premature decedents revealed inflammatory changes in the trachea with the change considered to have been a major factor in the death of Females 68 and 94. Taking into consideration the overall results from this study, these microscopic changes and, thus the unscheduled deaths, were considered likely to be due to the irritant nature of the test item rather than an indication of its systemic toxicity.

 

There were no further unscheduled deaths during the study.

 

Conclusions:

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item 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…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks to males and up to eight weeks to females including a two week pre-pairing phase, pairing, gestation and early lactation, at dose levels of 30, 100 or 200 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

 

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. Female 85 treated with 200 mg/kg bw/day was killedin extremison Day 3 of dosing and Male 73 was therefore not paired with any female.

 

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 Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post 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 considered to be related to the systemic toxicity of the test item.

 

There were four premature decedents during the course of the study, two each from the 100 or 200 mg/kg bw/day dose groups; one female from either dose group was found dead whilst the other was killedin extremis. Three of these animals showed clinical signs of respiratory distress which correlated with microscopic changes in the trachea with the change considered to have been a major factor in the death of one female from each dose group; these clinical signs and microscopic changes and, thus the deaths, were considered likely to be due to an irritant nature of the test item and not an indication of its systemic toxicity. Microscopic examination of the tissues from the remaining female treated with 200 mg/kg bw/day showed marked inflammatory change in the reproductive tract and secondary lymphoid/cellular depletion and it was deemed likely that these changes were a result of parturition and unrelated to administration of the test item.

 

Clinical Observations

Throughout the dosing period, there were no clinical signs considered to be related to the systemic toxicity of the test item.

 

At 200 mg/kg bw/day, individual animals of either sex surviving to the scheduled necropsy showed a few instances of noisy respiration mainly during the latter half of the treatment period with one of the males also showing noisy/laboured respiration and decreased respiratory rate on Day 35. These were deemed likely to be due to an irritant nature of the test item and not an indication of its systemic toxicity.

 

Behavioral Assessment

At all dose levels, there were no changes in the behavioral parameters measured considered to be related to the systemic toxicity of the test item.

 

Functional Performance Tests

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

 

Sensory Reactivity Assessments

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

 

Body Weight

There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the course of the study.

 

Food Consumption

There was no adverse effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the course of the study.

 

Water Consumption

Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

 

Reproductive Performance

Mating

There was no effect of treatment on mating performance.

 

Fertility

Fertility remained unaffected by treatment with the test item at any dose level.

 

Gestation Lengths

There were no treatment-related 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 corpora lutea count, number of implantations, pre- or post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age at any dose level.

 

Offspring Growth and Development

There was no detrimental effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at any dose level.

 

Laboratory Investigations

Hematology

No treatment-related effects were detected in animals of either sex at any dose level.

 

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex at any dose level.

 

Pathology

Necropsy

Neither the type, incidence or distribution of macroscopic observations in surviving adult animals or offspring indicated any systemic effect of treatment up to a dose level of 200 mg/kg bw/day.

 

Organ Weights

No toxicologically significant effects were detected in animals of either sex at any dose level.

 

Histopathology

No findings were noted in the tissues examined from terminal sacrifice animals which could be unequivocally related to administration of the test item at dose levels up to 200 mg/kg bw/day.

 

Of the four premature decedent animals, three showed inflammatory changes in the trachea with the change considered to have been a major factor in the premature death of two of the animals. These changes are unusual, correlated with the respiratory clinical sign noted and may be indicative of reflux after gavage dosing and thus related to test item irritancy rather than its toxicity.

 

Conclusion

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

 

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day. 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

K1 GLP study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item 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…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks to males and up to eight weeks to females including a two week pre-pairing phase, pairing, gestation and early lactation, at dose levels of 30, 100 or 200 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

 

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. Female 85 treated with 200 mg/kg bw/day was killedin extremison Day 3 of dosing and Male 73 was therefore not paired with any female.

 

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 Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results…….

Reproductive Performance

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on corpora lutea count, number of implantations, pre- or post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age at any dose level.

 

Offspring Growth and Development

There was no detrimental effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at any dose level.

 

Conclusion

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

 

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day. 

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 March 2015 Experimental completion date: 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days before the start of dosing 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 313 to 362g, the females weighed 191 to 232g, and were approximately twelve weeks old.

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, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Appendix 29. 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 late 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 Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a 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:

polyethylene glycol

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 24 days when stored at approximately 4 °C, in the dark. Formulations were therefore prepared approximately fortnightly, aliquoted and stored as above before use.

Samples of the test item formulation were taken and analyzed for concentration of Cyclohexanone peroxide (CAS# 012262-58-7) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were 96 to 108% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consiting of a single peak.

Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 0.2 mg/mL. Standard solutions contained the equivalent amount of vehicle to that of the relevant samples.

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

Analysis of samples
The formulations received were diluted with acetonitrile. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with acetonitrile, which was then shaken to dissolve. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of PEG 400 were accurately fortified with known amounts of test item equivalent to the lowest and hihgest anticipated dose concentrations. These samples were then prepared for analysis.

Preparation of lineraity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1.047 mg/mL by serial dilution covering the conventration range 0 to 0.3141 mg/mL.

Instrumental parameters
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Zorbax Eclipse XDB 5µ C18 (150 x 4.6 mm id)
Mobile phase: Aluent A_Water Eluent B_acetonitrile
Time %B
0 55
4 55
7 100
10 100

Flow-rate: 1 mL/min
UV detector wavelength: 210 nm
Injection volume: 10 µL
Retention time: ~2 mins

Details on mating procedure:

iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days. Female 85 treated with 200 mg/kg bw/day was killed in extremis on Day 3 of dosing and Male 73 was therefore not paired with any female.
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, five selected males per dose group were evaluated for functional/sensory responses to various stimuli.

Duration of treatment / exposure:

Males: approximately six weeks
Females: up to eight weeks

Frequency of treatment:

Daily

Duration of test:

Males: approximately six weeks
Females: up to eight weeks

No. of animals per sex per dose:

12 males, 12 females

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.

Maternal examinations:

OBSERVATIONS
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); see deviations from Study Plan. All observations were recorded.

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 Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin 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

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; see deviations from Study Plan.

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 observed daily by visual inspection of water bottles for any overt changes.

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Female 85 treated with 200 mg/kg bw/day was killed in extremis on Day 3 of dosing and Male 73 was therefore not paired with any female. 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.

Pregnancy and Parturition
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 and public holidays. 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

Laboratory Investigations
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.

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
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids


SACRIFICE AND PATHOLOGY
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 with an asterix (*) were weighed from all remaining animals:

Adrenals
Prostate
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

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 with an asterix (*) 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 (hind limb)
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes
Gross lesions*
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*
Liver
Thymus
Lungs (with bronchi)
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

Tissues were dispatched to the Test Site (Huntingdon Life Sciences Ltd.) for processing. The tissues from five selected control and 200 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to achieve a pregnancy 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 200 mg/kg bw/day animals and any macroscopic abnormalities were also processed. In addition, sections of testes from all control and 200 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.

Microscopic examination was conducted by the Study Pathologist.

Ovaries and uterine content:

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.

Fetal examinations:

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)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Statistics:

Due to the nature and quantity of this data, please see section "Anhy other information on materials and methods"

Indices:

Due to the nature and quantity of this data, please see section "Anhy other information on materials and methods"

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
Please refer to the section "Any other information on results"

Clinical Observations
Of the animals surviving to the scheduled necropsy, 2/12 males and 4/10 females treated with the test item at a dose level of 200 mg/kg bw/day showed sporadic instances of noisy respiration with one of these males also showing clinical signs of noisy/labored respiration and decreased respiratory rate on Day 35 of dosing; the latter clinical signs were observed during behavioral assessment for this male. It is worth noting that the instances of noisy respiration for these animals were generally observed during the latter half of the treatment period although Female 89 showed noisy respiration on Days 2 and 3 of dosing. Additionally, two females each from the 100 or 200 mg/kg bw/day dose groups that were either found dead or killed in extremis due to their deteriorating clinical condition, also showed respiratory signs including noisy/labored/gasping respiration and/or decreased respiratory rate prior to death. For three of these premature decedents, the clinical signs correlated with microscopic changes in the upper respiratory tract which were considered to be likely due to an irritant nature of the test item. The remaining early decedent (Female 65 treated with 100 mg/kg bw/day) also showed clinical signs of labored respiration, piloerection, lethargy, hunched posture, pale extremities, diarrhea and chromodacryorrhea prior to death on Day 22 of gestation and these were deemed to be related to marked inflammatory change in the reproductive tract and thus parturition rather than the administration of the test item.

At 30 or 100 mg/kg bw/day, none of the animals of either sex surviving to the scheduled necropsy were observed with any clinical signs.

At 200 mg/kg bw/day, a few instances of increased post-dose salivation were observed for individual males and one female. Such observations are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are considered to be of no toxicological importance.

Two control males from one cage showed scab formation on some days which is likely to have resulted from fighting amongst cage animals. One control female also showed generalized fur loss during the last week of dosing and this finding was not treatment-related.

Functional Observations
Behavioral Assessments

There were no changes in the behavioral parameters measured that were considered to be related to the toxicity of the test item at any dose level.

Male 73 from the 200 mg/kg bw/day dose group and Female 68, an early decedent from the 100 mg/kg bw/day, were observed with noisy/labored respiration and decreased respiratory rate during behavioral assessment in Week 5 with noisy respiration again being evident for the male during Week 6 assessment. These observations were considered likely to be due to an irritant nature of the test item.

Functional Performance Tests
There were no intergroup differences considered to be related to treatment with the test item.

Grip strength evaluations during the last week of the treatment period revealed statistically significantly higher group mean value for hindlimb strength in males receiving the test item at 200 mg/kg bw/day relative to controls (p<0.01). It is worth noting, however, that this difference was only apparent in 1/3 grip strength tests with no statistically significant intergroup differences during the remaining 2/3 tests and, as such, this observation was considered to be incidental.

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

Body Weight
There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex.

At 200 mg/kg bw/day, group mean body weight gain in males over the first week of dosing was marginally lower than controls albeit without achieving statistical significance. Recovery was evident thereafter such that the weekly group mean weight gains for these males were occasionally slightly higher than controls resulting in a slightly higher overall mean body weight gain for these animals. At 30 or 100 mg/kg bw/day, body weight development in males was generally comparable with controls throughout the treatment period.

During the first week of the pre-pairing phase of the study, group mean body weight gain in females treated with 200 mg/kg bw/day was marginally lower than controls albeit without achieving statistical significance. Thereafter, improvement was apparent and group mean body weight gain over the second week of dosing for these females was comparable with controls. A high degree of inter-individual variation was evident amongst female dose groups, but body weight performance at 30 or 100 mg/kg bw/day during this phase of the study was generally similar to controls. During the gestation and lactation phases of the study, group mean body weight gains for all test item-treated female dose groups were comparable with controls.

Food Consumption
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in Appendix 9. Food efficiency for males and for females during the pre-mating phase is given in Table 10.

There was no adverse effect of treatment with the test item on dietary intake or food conversion efficiency in animals of either sex.

During the second week of dosing, food intake in females given 200 mg/kg bw/day was marginally lower than controls. During gestation and lactation phases of the study, however, food consumption across all test item-treated female dose groups was similar to controls and any initial effect in the 200 mg/kg bw/day dose group was considered not to be of an adverse nature. Food consumption in males from all dose groups remained comparable with controls. Additionally, food conversion efficiency values for animals of either sex were generally comparable with controls with any differences deemed to be reflective of minor intergroup differences in body weight gains.

Water Consumption
Visual inspection of water bottles did not indicate any differences for the animals given the test item in relation to controls.

Reproductive Performance
Mating
There was no effect of treatment with the test item on mating performance with most animals mating within four days after pairing. The only exception was Female 18 from the control group, which showed signs of mating after 12 days of pairing.

Fertility
Fertility as assessed by pregnancy index remained unaffected by treatment with the test item at any dose level.

Of the females reaching the scheduled termination on Day 5 post partum, two females from the control group (Females 22 and 24) and one female each treated with 30 or 100 mg/kg bw/day (Females 48 and 69, respectively) did not achieve pregnancy following evidence of positive mating. Additionally, Female 42 given 30 mg/kg bw/day showed evidence of pregnancy at necropsy but there was no evidence of littering. The three premature decedent females from the 100 or 200 mg/kg bw/day dose groups that were paired with males also showed evidence of pregnancy at necropsy. All non-pregnant females appeared to be cycling normally and histopathology examination of the selected tissues from these females and their corresponding male partners did not identify any obvious reason for the lack of pregnancy in these females.

At all dose levels, 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) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Gestation Length
Gestation lengths were between 22 and 23½ days and their distribution for the test item-treated females was similar to controls.

Laboratory Investigations
Hematology
At the end of the treatment period, there were no intergroup differences considered to be related to treatment with the test item at any dose level in animals of both sexes.

Females receiving 30 mg/kg bw/day showed statistically significantly lower mean corpuscular hemoglobin relative to controls (p<0.05), but there was no dose-relationship and, in isolation, this finding was considered likely to be incidental.

Blood Chemistry
No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

At 200 mg/kg bw/day, males receiving the test item showed statistically significantly lower group mean plasma level of total protein (p<0.05) and alkaline phosphatase activity (p<0.05) in relation to controls. The corresponding values in females from this dose group were similar to control, although these females showed statistically significantly higher level of sodium when compared with the respective controls (p<0.05). With the exception of 2/5 high dose males showing total protein values that were slightly lower than the historical control data ranges, the remaining individual values from these animals were within these ranges. In the absence of any histopathology correlates, these findings were considered to be of no toxicological relevance.

Pathology
Necropsy

Offspring
Macroscopic necropsy findings for control 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 30, 100 or 200 mg/kg bw/day.

Adults
At terminal necropsy, no macroscopic findings were observed which could be related to treatment with the test item. Individual findings across all dose groups included red discolouration of lungs, but the incidence or the distribution of these observations did not indicate any treatment-related effects. At 30 mg/kg bw/day, one female was observed with fluid-filled uterus/cervix & vagina, but this finding was deemed to be unrelated to treatment with the test item.

Organ Weights
No toxicologically significant effects were detected in animals of either sex receiving the test item at any dose level.

At 200 mg/kg bw/day, group mean absolute and body weight-related male spleen weights were statistically significantly higher than controls (p<0.05). A dose-relationship was evident, however, the majority of individual values were within the historical control data ranges. As there were no associated microscopic findings, this observation was considered to be of no toxicological importance.

Histopathology
Histopathological examination of the selected tissues from the 200 mg/kg bw/day animals of either sex reaching scheduled necropsy did not reveal any treatment-related abnormalities.

Premature Decedents
Female 65 treated with 100 mg/kg bw/day was killed in extremis on Day 38 of dosing with the major pathology of marked inflammatory change in the reproductive tract. There was secondary lymphoid/cellular depletion. It is considered likely that these changes were a result of parturition and cannot be related to administration of the test item.

Female 68 receiving 100 mg/kg bw/day was found dead on Day 36. Autolysis was present in most tissues but the major pathological finding was marked necrosis in the trachea. It is considered likely that this was the change likely to have caused the death of this animal and correlates with the clinical sign in the respiratory tract.

Female 85 from 200 mg/kg bw/day dose group was killed in extremis on Day 3 due to respiratory signs. Mild inflammatory change was present in the trachea but no other notable findings were present in the tissues examined to correlate with the clinical findings.

Female 94 given 200 mg/kg bw/day was found dead on Day 39 of dosing. Autolysis was present in most tissues but the major pathological finding was marked necrosis in the trachea. It is considered likely that this was the change likely to have caused the death of this animal and correlates with the clinical sign in the respiratory tract.

Terminal Sacrifice
There was an increase in hyaline droplets in the kidneys of 3/5 males treated with 200 mg/kg bw/day. This was not associated with any damage to the cells or any sign of compromised function. The hyaline droplets within the tubules are consistent with the accumulation of a-2u-globulin, a common finding in untreated male rats (Khan KNM et al., 2002). Hyaline droplet accumulation is specific to the male rat and is not considered to be significant in man and in the absence of any other change in the kidney is considered not to be of toxicological significance and indeed it is unusual not to see this in control animals of this age and strain.

Key result

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Responses
In total ten, ten, nine and ten females from the control, 30, 100 or 200 mg/kg bw/day dose groups, respectively, 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 on the mean number of corpora lutea, implantation counts and pre- or post-implantation losses.

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from females treated with the test item at all dose levels was generally comparable with controls. Live birth and survival indices for the 200 mg/kg bw/day dose group appeared to be marginally lower than controls, but this was mainly due to the litter from Female 86 which was observed with 7/11 dead pups following completion of parturition and a further two pups going missing over the subsequent days. In isolation, these findings were considered to be unrelated to treatment with the test item.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from test item-treated dose groups when compared with controls.

Offspring Growth and Development
Offspring body weights and litter weights on Days 1 and 4 post partum and offspring body weight development over Days 1 to 4 post partum were generally comparable with controls. Any intergroup differences were not dose-related and since the corresponding values from the 200 mg/kg bw/day dose group were similar to controls, these were considered to be incidental.

Surface righting reflex data did not reveal any detrimental effect of treatment with the test item at any dose level in relation to controls.

Female 86 treated with 200 mg/kg bw/day was observed with 7/11 dead pups following completion of parturition whilst the remaining four pups from this litter appeared to be small, cold, weak and with no milk in the stomach on Day 1 post partum with two of these going missing over the subsequent days. Clinical signs detected in pups from the remaining test item-treated dose groups included small size, right hind paw swollen, found dead or missing and, were considered to be low incidence findings observed in offspring in studies of this type and, in isolation, this finding was deemed unlikely to be related to administration of the test item to maternal females.

Key result

Remarks on result:

other: no effects observed

Abnormalities:

not specified

Developmental effects observed:

not specified

1.1.1            Adult Reponses

1.1.1.1      Mortality

There were four unscheduled deaths during the study and included two females each from the 100 and 200 mg/kg bw/day dose groups. Clinical signs prior to death and macroscopic necropsy finding for these early decedents are presented below:

 

Dose Level (mg/kg bw/day)	Animal Number	Day of dosing (gestation)	Mode of Death	Clinical Signs (Day of dosing)	Macroscopic Findings at necropsy
100	65	38 (22)	KIE	Noisy respiration (36) Labored respiration (38), Piloerection (38) Lethargy (38) Hunched posture (38) Pale extremities (38) Diarrhoea (38) Chromodacryorrhea (38)	Small spleen Green contents in the stomach Dark brown fluid-filled uterus & cervix
	68	36 (19)	FD	Noisy respiration (35)* Labored respiration (35)* Decreased respiratory rate (35)*	Gaseous distension in duodenum, ileum and jejunum Dark lungs
200	85	3	KIE	Labored respiration (3) Gasping respiration (3) Pale extremities (3) Hypothermia (3)	No abnormalities detected
	94	39 (21)	FD	Noisy respiration (37) Labored respiration (37) Decreased respiratory rate (37)	Dark lungs

FD = Found dead                KIE = Killedin extremis

* = These clinical signs were observed during weekly behavioural assessment

 

These early decedents showed clinical signs of noisy/labored/gasping respiration and/or decreased respiratory rate prior to death but only Females 68 and 94 treated with 100 or 200 mg/kg bw/day, respectively, were observed with dark lungs at necropsy. Histopathological examination of the tissues from Female 65 showed marked inflammatory change in the reproductive tract and secondary lymphoid/cellular depletion and it was deemed likely that these changes were a result of parturition and unrelated to administration of the test item. Histopathology examination of the tissues from the remaining premature decedents revealed inflammatory changes in the trachea with the change considered to have been a major factor in the death of Females 68 and 94. Taking into consideration the overall results from this study, these microscopic changes and, thus the unscheduled deaths, were considered likely to be due to the irritant nature of the test item rather than an indication of its systemic toxicity.

 

There were no further unscheduled deaths during the study.

Conclusions:

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item 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…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks to males and up to eight weeks to females including a two week pre-pairing phase, pairing, gestation and early lactation, at dose levels of 30, 100 or 200 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

 

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. Female 85 treated with 200 mg/kg bw/day was killedin extremison Day 3 of dosing and Male 73 was therefore not paired with any female.

 

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 Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post 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 considered to be related to the systemic toxicity of the test item.

 

There were four premature decedents during the course of the study, two each from the 100 or 200 mg/kg bw/day dose groups; one female from either dose group was found dead whilst the other was killedin extremis. Three of these animals showed clinical signs of respiratory distress which correlated with microscopic changes in the trachea with the change considered to have been a major factor in the death of one female from each dose group; these clinical signs and microscopic changes and, thus the deaths, were considered likely to be due to an irritant nature of the test item and not an indication of its systemic toxicity. Microscopic examination of the tissues from the remaining female treated with 200 mg/kg bw/day showed marked inflammatory change in the reproductive tract and secondary lymphoid/cellular depletion and it was deemed likely that these changes were a result of parturition and unrelated to administration of the test item.

 

 

Clinical Observations

Throughout the dosing period, there were no clinical signs considered to be related to the systemic toxicity of the test item.

 

At 200 mg/kg bw/day, individual animals of either sex surviving to the scheduled necropsy showed a few instances of noisy respiration mainly during the latter half of the treatment period with one of the males also showing noisy/laboured respiration and decreased respiratory rate on Day 35. These were deemed likely to be due to an irritant nature of the test item and not an indication of its systemic toxicity.

 

 

Behavioral Assessment

At all dose levels, there were no changes in the behavioral parameters measured considered to be related to the systemic toxicity of the test item.

 

 

Functional Performance Tests

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

 

 

Sensory Reactivity Assessments

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

 

 

Body Weight

There was no adverse effect of treatment with the test item on body weight development for animals of either sex during the course of the study.

 

 

Food Consumption

There was no adverse effect of treatment with the test item on food consumption or food conversion efficiency for animals of either sex during the course of the study.

 

 

Water Consumption

Visual inspection of water bottles did not indicate any differences for the animals given the test item in comparison with controls.

 

 

Reproductive Performance

Mating

There was no effect of treatment on mating performance.

 

 

Fertility

Fertility remained unaffected by treatment with the test item at any dose level.

 

 

Gestation Lengths

There were no treatment-related 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 corpora lutea count, number of implantations, pre- or post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age at any dose level.

 

 

Offspring Growth and Development

There was no detrimental effect of treatment indicated by offspring body weight or body weight gain and litter weights, surface righting ability on Day 1 or clinical signs to Day 5 of age at any dose level.

 

 

Laboratory Investigations

Hematology

No treatment-related effects were detected in animals of either sex at any dose level.

 

 

Blood Chemistry

No toxicologically significant effects were detected in animals of either sex at any dose level.

 

 

Pathology

Necropsy

Neither the type, incidence or distribution of macroscopic observations in surviving adult animals or offspring indicated any systemic effect of treatment up to a dose level of 200 mg/kg bw/day.

 

 

Organ Weights

No toxicologically significant effects were detected in animals of either sex at any dose level.

 

 

Histopathology

No findings were noted in the tissues examined from terminal sacrifice animals which could be unequivocally related to administration of the test item at dose levels up to 200 mg/kg bw/day.

 

Of the four premature decedent animals, three showed inflammatory changes in the trachea with the change considered to have been a major factor in the premature death of two of the animals. These changes are unusual, correlated with the respiratory clinical sign noted and may be indicative of reflux after gavage dosing and thus related to test item irritancy rather than its toxicity.

 

 

Conclusion

The oral (gavage) administration of Cyclohexanone peroxide (CAS# 012262-58-7) to Wistar Han™:RccHan™:WIST strain rats, at dose levels of up to 200 mg/kg bw/day resulted in the premature death of two females each from the 100 or 200 mg/kg bw/day dose groups. Prior to death, these early decedents showed clinical signs of respiratory distress which correlated with inflammatory changes observed histopathologically in the trachea in three of these animals. Some surviving animals of either sex receiving 200 mg/kg bw/day also showed a few instances of respiratory signs during dosing, but there were no histopathology correlates. For the surviving animals, there was no adverse effect of treatment with the test item at any dose level on body weight development, dietary intake, hematology or blood chemistry parameters and organ weights. There were no treatment-related macroscopic findings for any of these terminal animals or any histopathology observations that could be unequivocally related to the administration of the test item. Based on the available data, the premature deaths of three females were considered likely due to an irritant nature of the test item rather than an indication of its systemic toxicity and, as such the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity, was considered to be 200 mg/kg bw/day within the confines of this study.

 

There was no effect of treatment on mating performance, fertility, gestation length or any of the maternal and offspring parameters measured and The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, within the confines of this screening study, was considered to be 200 mg/kg bw/day. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

K1 GLP study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information the test substance in not classified for effects on fertility or development.