Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 September 2017 Experimental completion date: 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
As in the study report
Specific details on test material used for the study:
Test item: O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate
Test item identity (including alternative names): tert-Amylperoxy-2-ethylhexylcarbonate
Trigonox 131
CAS number: 70833-40-8
Intended use: Industrial chemical
Appearance: Clear colorless liquid
Storage conditions: Frozen (-10 to -30C) in the dark
Supplier: Sponsor
Batch number: 1702442114
Retest date: 24 March 2019
Purity: 96.5%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Strain/Species RccHan™;WIST rat.
Supplier Envigo RMS (UK) Limited.
Number of animals ordered 44 males and 48 females (92 in total; included 4 spare males and 8 spare females)
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males: six days before commencement of treatment.
Females: 20 days before commencement of treatment.

Age of the animals at the start of treatment Males: 84 to 90 days old.
Females: 98 to 104 days old.

Weight range of the animals at the start of treatment Males: 319 to 358 g.
Females: 199 to 227 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced to ensure variations in body weight of animals did not exceed 20% of the mean for each sex.

Groups were adjusted to reduce inter-/intra-group variation.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Animal Care and Husbandry
Environmental Control

Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing for mating; these were suspended above absorbent paper which was changed daily.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (100g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) and discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: PEG300
Details on exposure:
Correction factor
Used as supplied; no correction factor applied.

Vehicle PEG300.

Method of preparation
O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was prepared for administration as a series of graded concentrations in the vehicle. Starting with the low concentration (50 mg/mL), the formulation was prepared by weighing out the required amount of test item, adding 40 to 50% of the final volume of vehicle and magnetically stirring until all the test item was uniformly mixed. The solution was made up to the required volume with the vehicle and stirred using a magnetic stirrer until homogenous.

The procedure was repeated for the mid and high dose (50 and 200 mg/mL).

Frequency of preparation
Weekly.

Storage of formulation
Refrigerated (2-8ºC).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Details on mating procedure:
Mating Procedure
Paired for mating After two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis

Validated concentration range
20 mg/mL to 200 mg/mL
GLP Study: Harlan Laboratories Study Report: D45837 (homogeneity and stability establish for 4 hours at ambient temperature and 8 days refrigerated storage); 10 mg/mL validated as part of this study.
Stability assessment Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at a concentration of 10 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration during the first and last week of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in acetonitrile (50 mL).

Solutions for instrument calibration were prepared by appropriate dilution of the primarystandard using diluent and contained O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at nominal concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL and 250 μg/mL.

Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurate volume or weight where applicable) was dissolved in a suitable volume of acetonitrile. The extract was diluted using diluent to provide a solution containing O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate at an expected concentration within the range 100 μg/mL to 200 μg/mL.

The concentration of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate in the final solution was quantified by HPLC using UV detection.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (PEG300) with known amounts of O-(2-ethylhexyl)O,O-tert-pentyl peroxycarbonate. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: XBridge Shield RP18, 75 × 4.6 mm, 3.5 μm
Column temperature: 45ºC
Sample temperature: 4ºC
Mobile Phase: ACN/water/ o-H3PO4 65/35/0.1 v/v/v
Flow rate: 1 mL/min
Needle wash: ACN/water 50/50 v/v
Detector wavelength: UV, 210 nm
Injection volume: 20 μL
Run time: 8 minutes
Approximate retention time: 4.3 minutes
Duration of treatment / exposure:
Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation
Frequency of treatment:
Daily
Details on study schedule:
Study initiation (Study Plan signed by Study Director) 21 August 2017

Experimental start date
(Pre-Study chemistry) 20 September 2017
Animal arrival
Males 4 october 2017
Females 20 September 2017

Estrous cycle evaluation commenced 26 September 2017

Treatment commenced 10 October 2017

F0 pairing commenced 24 October 2017

Necropsy
F0 Males 7 November 2017
F0 Females/F1 Offspring 28 November to 2 December 2017

Experimental completion date 22 March 2018
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males
10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
Dose levels of 50, 250 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from an OECD 407 study conducted at 100, 300 and 1000 mg/kg/day (Harlan Laboratories Study Report D45837). In this study findings included:
• Reductions in food consumption at 1000 mg/kg/day
• Slightly high liver weights for females at 300 or 1000 mg/kg/day, with two animals exhibiting minimal centrilobular hepatocellular hypertrophy; considered to be metabolic adaption and therefore not adverse.
• Increased severity of hyaline droplets in the renal proximal tubules of males at 1000 mg/kg/day; this is considered to be an adverse finding in rats but has no toxicological significance in man.
• Forestomach mucosal necrosis for males at 100 mg/kg/day and for both sexes at 300 mg/kg/day or 1000 mg/kg/day; these were considered to be local injuries that resulted in subsequent adverse reactions.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for systemic toxicity was 1000 mg/kg/day. However the no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach.

On this study a high dose of 1000 mg/kg/day was anticipated to elicit parental effects in terms of food consumption, liver pathology and stomach lesions. A low dose of 50 mg/kg/day was selected with the aim to establish a NOEL, with an intermediate dose of 250 mg/kg/day to aid interpretation of any dose response.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males
Week 1 - daily
Week 2 onwards - once each week

F0 females
Week 1 - daily
Week 2 - once
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 12

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose
One to two hours after completion of dosing
As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males
Once each week

F0 females
Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13

Body Weight
The weight of animals was recorded as follows:
F0 males
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females
Weekly during acclimatization.
Before dosing on the day that treatment commenced (Week 0) and weekly before mating.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 animals
Weekly, before pairing.

For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.
Oestrous cyclicity (parental animals):
Estrous Cycles
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:

For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.

After pairing until mating.

For four days before scheduled termination (nominally Days 10-13 of lactation).

Sperm parameters (parental animals):
Testes weight and histopathology
Litter observations:
Records Made During Littering Phase
Clinical observations
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all offspring.
Nipple/areolae count Day 13 of age - male offspring.


Postmortem examinations (parental animals):
Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Tissue and regions examined
Abnormalities
Epididymides (caput, corpus and cauda)
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulation gland)
Stomach
Testes
Thyroid
Uterus with cervix and oviducts

Females
The following were recorded:
Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant females.
For apparently empty uterine horns The absence of uterine implantation sites was confirmed by
staining with ammonium sulphide [modification of the
Salewski staining technique (Salewski, E, 1964)].

Female whose litter died before Day 13 of lactation Mammary tissue appearance.

Light Microscopy
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
Thyroid hormone analysis
- blood samples were collected as follows

Day 4 of age
F1 offspring, two females per litter (where possible).
• one for T4 (serum)#
• one for TSH (plasma)

No females were allocated to these procedures if:
• The resultant live litter size fell below eight offspring.
• The resultant number of live females fell to less than three

If only four female offspring were available in a litter but the overall litter size was more than eight, one female pup was selected with priority given to the serum sample.
# priority was given to serum sample

Day 13 of age
F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample


Premature deaths
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.

F1 offspring on Day 13 of age
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormal offspring were retained in appropriate fixative.
Thyroid glands were preserved from two offspring - one male and one female in each litter, where possible.
Statistics:
Please refer to "Any other information on materials and methods, including tables"
Reproductive indices:
Mating Performance and Fertility

Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:
Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to administration of the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Bodyweight change for males during the four week treatment period was slightly low for males receiving 1000 mg/kg/day at approximately 83% of Controls; this difference did not attain statistical significance. Body weight change at 50 and 250 mg/kg/day was unaffected by treatment.

Body weight change for females during Week 1 of treatment was slightly low at 250 and 1000 mg/kg/day at approximately 57% of Controls; however this difference did not attain statistical significance. The overall weight gain for females for the two weeks before pairing, during gestation and lactation were unaffected by treatment at dose levels up to and including 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption for males for the two weeks before pairing was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.
During the first week of treatment females at 1000 mg/kg/day had slightly but not statistically significant low food consumption, approximately 89% of Controls. At 50 and 250 mg/kg/day food consumption was similar to Controls.

During gestation food consumption was considered unaffected by treatment.

During lactation the overall food consumption for females receiving 1000 mg/kg/day was marginally low when compared with Controls (approximately 93% of Controls), however the mean phase values were not statistically different from Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related findings in the stomach of three males and one female that received 250 mg/kg/day and eight males and one female that received 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The number of implantations and subsequently the live litter size on Days 1 and 4 of age (before selection of offspring for thyroid hormone sampling) were statistically significantly low at 1000 mg/kg/day when compared with concurrent Controls (implantations - p<0.01; litter size - p<0.05) and review of the historical control data showed that two litters on Day 4 were outside the 90 percentile range (7.7-14.4; n=74). Litter size at 50 or 250 mg/kg/day was unaffected by parental treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Implantation count
Key result
Dose descriptor:
NOEL
Remarks:
Parental effects
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Parental effects
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects noted on overall parental effcts

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There no signs at routine physical examination of the offspring that could be attributed to parental treatment
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Offspring survival was unaffected by treatment at dose levels up to and including 1000 mg/kg/day.

At 1000 mg/kg/day one litter comprising of 13 offspring was found dead prior to Day 1 of age (litter no 140); the offspring weighed between 4.4 and 5.3 g. Macroscopic examination of the dam (no. 140) revealed 13 uterine implantation sites and pale/inactive mammary tissue. In the absence of any effect on offspring survival in the remaining litters this isolated litter death is considered to be unrelated to parental treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring bodyweight on Day 1 of age and subsequent weight gain up to Day 4 of age was considered to be unaffected by parental treatment.
At 1000 mg/kg/day body weight gain for male offspring from Day 4 to 7 of age was statistically significantly low when compared with Controls (p<0.05; approximately 83% of Controls). Female offspring at the same dose level showed marginally low weight gain over the same period (91 % of Controls), however this difference did not attain statistical significance. Offspring body weight gain from Day 7 to Day 13 of age and the overall weight gain from Day 1 to Day 13 of age was similar across the groups. Therefore this slight effect on offspring bodyweight gain at 1000 mg/kg/day was not considered to be adverse
Offspring body weight gain at 50 and 250 mg/kg/day was unaffected by parental treatment.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that died prematurely and those at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to parental treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
In general, all samples from adult males at termination and Day 13 of age male and female offspring from the animals in Groups 1 to 4 had concentrations that were comparable with endogenous levels observed in the control matrix used to prepare the QC samples. No further analyses were required.

Litter size, sex ratio and survival indices
At 1000 mg/kg/day one litter comprising of 13 offspring was found dead prior to Day 1 of age (litter no 140); the offspring weighed between 4.4 and 5.3 g. Macroscopic examination of the dam (no. 140) revealed 13 uterine implantation sites and pale/inactive mammary tissue. In the absence of any effect on offspring survival in the remaining litters this isolated litter death is considered to be unrelated to parental treatment.

Sex ratio was unaffected by parental treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Offspring development

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

It was concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. 

Applicant's summary and conclusion

Conclusions:
It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.
Executive summary:

  Summary

This study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with the administration of the test item [O‑(2‑ethylhexyl) O,O-tert-pentyl peroxycarbonate], an industrial chemical, by oral gavage, to Sprague Dawley rats for at least four weeks.

Three groups of ten male and ten female rats received test item at doses of 50, 250 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, PEG300, at the same volume dose as treated groups.

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results and Conclusion

Treatment ofO-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to parental animals at 50, 250 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance or fertility. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

Treatment related findings were restricted to:

·        Low implantations and subsequent live litter size on Days 1 and 4 of age at 1000 mg/kg/day. This effect was slight and in isolation with no effects on any other reproductive parameters or micropathology of the reproductive organs.

·        Macroscopic and microscopic changes in the stomach of parental animals at 250 and 1000 mg/kg/day. The microscopic findings of epithelial hyperplasia and submucosal infiltration of inflammatory cells in the non-glandular region of the stomach were considered to be indicative of a local irritant effect of the test item at 250 and 1000 mg/kg/day, with a dose-relationship in male animals; in males this correlated with gross findings of a thickened forestomach.

It was therefore concluded that the no-observed-adverse-effect-level (NOAEL) for reproductive performance and offspring development based on the effect on implantation count is 250 mg/kg/day. The no-observed effect level (NOEL) for parental effects considering the local irritant effect is 50 mg/kg/day with an overall parental NOAEL of 1000 mg/kg/day.