Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 422 study, the registered substance, had no effects on mating, fertility, reproductive organs or spermatogeneis when tested up to 1000 mg/kg/day.Pre-implantation loss, the implantation rate, the post implantation loss, the number of living pups at first litter check, the post natal loss, and the gestation length, respectively, were considered to be not influenced by treatment with the test item.

Additional tests, for reproductive effects, is not scientifically justified. There were no reproductive or developmental effects in the OECD 422 study. In an OECD 413 study, there were no effects on reproductive organs. An OECD 414 study confirmed the lack of developmental findings in the OECD 422 study.


Short description of key information:
A GLP OECD 422 study, conducted with rats at 0, 100, 300 and 1000 mg/kg/day, did not reveal any adverse effects on reproductive parameters evaluated.

Justification for selection of Effect on fertility via oral route:
Apparently well conducted GLP study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
nine months (approx.)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This is a well documented study conducted according to modern standards of protocol, quality assurance, and good laboratory practices. The test material was 99.2 % pure and was analytically demonstrated to be stable in the test vehicle (corn oil).
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
study plan "amendments": "Male animals aged 8 weeks minimum instead of 10 weeks. Lymph nodes for histopath specified as “one lymph node covering the route of administration and another one distant from the route of admin. to cover systemic effect."
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Species Rat: HanRcc:WIST (SPF)
Rationale: Specified by the international guidelines as the recommended test system
Source: RCC Ltd
Laboratory Animal Services
Wolferstrasse 4
4414 Fullinsdorf / Switzerland

Number of animals: 40 males, 10 per group; 40 females, 10 per group
Age at delivery: 8 weeks (male animals); 10 weeks (female animals)
Body weights: Males: 243 - 291 grams (at first treatment)
Females: 181 - 219 grams (at first treatment)
Acclimatization: Seven days under test conditions with an evaluation of the health status

Identification
Parental animals: Individual animal number tattooed on the pinnae and individual cage card.
F1 pups: Individual intra-litter pup number tattooed with Indian ink on day 1 post partum.

This study was performed under standard laboratory conditions: the animal room was air-conditioned with 10 - 15 air changes per hour; the environment was monitored continuously with recordings of temperature (range 22+/- 3°C) and relative humidity (range 30 - 70%), 12 hours artificial fluorescent light / 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Accommodation: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland). During the pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles. During the pairing period, rats were housed one male / one female in Makrolon pairing cages. After mating, the males and the females were housed individually again. During lactation period (until day 4 of lactation), dams were housed together with their litters. Throughout the study, each cage was identified by a colored label according to the group and recording the study schedule number, animal number(s) and details of treatment.

Diet: Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/ Switzerland) was available ad libitum (Batch No. 67/06). Results of analyses for contaminants are presented in the final report.

Water Tap water from Fullinsdorf in bottles was available ad libitum. Results of the bacteriological, chemical and contaminant analyses scheduled to be conducted at least once yearly by RCC (contaminant analyses only) and by the Official Chemist of the Kanton Basel-Landschaft (bacteriological and chemical analyses) are part of the final report.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Vehicle
Identity – Corn oil
CAS-No. 8001-30-7
EG No. 2322812
Supplier – Carl Roth GmbH & Co., 76185 Karlsruhe / Germany
Batch number – 38679499
Expiry date – 14-NOV-2016
Storage conditions -- Room temperature (20°C +/- 5°C)

Dose formulations were prepared in terms of material as supplied by the Sponsor.
Frequency of dose formulation -- Weekly, based on the results of RCC Study No. A79874
Storage of dose formulations -- Room temperature (20°C +/-5°C)
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle were added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. Having obtained a homogeneous mixture, the vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration. During the daily administration period, homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
Details on mating procedure:
During the pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles.

During the pairing period, rats were housed one male / one female in Makrolon pairing cages.

After mating, the males and the females were housed individually again. During lactation period (until day 4 of lactation), dams were housed together with their litters.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations
Samples for determination of concentration, homogeneity and stability (4 hours and 7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of
the administration period

On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. For determination of stability, samples were taken before dosing from the middle and stored at room temperature for 4 hours and for 7 days in the dark. The samples were stored at approximately 20°C in the dark pending analysis. Samples were shipped to Itingen / Switzerland. Analysis was performed using a method provided by the Sponsor and adapted by the test facility.

After analysis, the analytical results were communicated to the Study Director. Upon receipt and evaluation of these results the Study Director decided about discarding the samples. The report of the Analytical Phase is presented as part of the final report.
Duration of treatment / exposure:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Frequency of treatment:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Details on study schedule:
MALES FEMALES
Acclimatization 7 days (minimum) 7 days (minimum)
Treatment beginning Day 1 of pre-pairing Day 1 of pre-pairing
Pre-pairing 14 days 14 days
Pairing until mating (maximum 14 days) until mating (maximum 14 days)
Gestation N/A* about 21 days
Parturition N/A* expected : on day 21 or 22 post coitum
Lactation N/A* until day 4 post partum
Treatment ending One day prior to the actual day of necropsy on day 4 post partum
(at least 28 days of treatment)
Blood collection On the day of necropsy On the day before necropsy or on the day of necropsy
Termination After the first dams had reached day pups on day 4 post partum
4 post partum dams on day 5 post partum

* Not applicable

Remarks:
Doses / Concentrations:
0; 100; 300; 1000 mg/kg bw
Basis:
other: administered gavage dose
No. of animals per sex per dose:
10 males; 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in agreement with the Sponsor, based on the results of a preliminary dose range-finding study, where 1000 mg/kg/day were used as highest dose level.
Positive control:
None
Parental animals: Observations and examinations:
All animals were checked at least twice daily for any mortalities. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.

All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health. Additionally, the females were observed for signs of difficult or prolonged parturition

Once prior to the first test item administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lcrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also recorded.

Functional observation battery

At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were performed for five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

a) Cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.

b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.

c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.

d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or faeces, soiling, general abnormalities, posture.

e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

A description of all test parameters is listed in this report. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (basing on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Stages of spermatogenesis and histopathology of interstitial testicular cell structure
Litter observations:
Day 0 of lactation was the day on which a female had delivered all her pups. The litters were examined for litter size, live birth, stillbirth and any gross anomalies. The sex ratio of the pups was recorded. The dams were caged together with their litters until day 4 of lactation. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum. The dams and pups were observed daily for survival and behavioural abnormalities in nesting and nursing.
Postmortem examinations (parental animals):
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Examinations were extended to the animals of the other dosage groups, if treatment-related changes were seen in the highest dose group.

All gross lesions were examined. Histological examination of ovaries was carried out on any female that did not give birth. Microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Postmortem examinations (offspring):
Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group 3 and 4 males: mean liver and kidney weight (respectively) slightly increased; maybe due to increased metabolism and excretion of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All male and female animals in groups 2, 3, and 4 were noted without any treatment-related finding at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Clinical signs: all Group 4 males and females noted moving their head through bedding; one to five males and two female animals noted with ruffled fur. No treatment-related signs noted for male or female animals in groups 2 and 3.

Food consumption, males: In group 4, the mean food consumption was statistically significantly decreased during the prepairing period. After the pairing period mean food consumption was not influenced. Mean food consumption in groups 2 and 3 was considered to be not influenced during pre- and after pairing period.

Body weight, males: In group 4, mean body weight started to be statistically significantly decreased on day 11 of the pre-pairing period and continued to be statistically significantly decreased until the end of study in the after pairing period. Mean body weight gain was statistically significantly decreased between day 6 and day 14 of the pre-pairing period and on days 5, 6, and 11 of the after pairing period. In groups 2 and 3, mean body weight was not influenced by treatment with the test item during the whole study. Mean body weight gain was statistically significantly decreased in group 3 between day 3 and 9, and in group 2 on day 3, respectively, during the after pairing period.

Food consumption, females: Mean food consumption in groups 2, 3, and 4 was not influenced during pre-pairing, and gestation period. During lactation period, mean food consumption was statistically not significantly increased in groups 3 and 4.

Body weight, females: In groups 2, 3, and 4, mean body weight and mean body weight gain was considered to be not influenced by treatment with the test item during the whole study.

REPRODUCTION DATA: Except female no. 79 in group 4, all mated females were pregnant. In groups 2, 3, and 4, mating performance was considered to be not influenced by treatment with the test item. The fertility index was 100.0, 100.0, 100.0, and 90.0% in groups 1, 2, 3, and 4, respectively.
In groups 2, 3, and 4, the pre-implantation loss, the implantation rate, the post implantation loss, the number of living pups at first litter check, the post natal loss, and the gestation length, respectively, were considered to be not influenced by treatment with the test item.

HISTOPATHOLOGY

Liver: Minimal centrilobular hepatocellular hypertrophy in 3/5 males and 3/5 females of group 3 and 2/5 males and 2/5 females of group 4. Minimal diffuse hepatocellular hypertrophy in 1/5 males of group 3 and 3/5 males and 3/5 females of group 4. This hypertrophy was associated with consequent increase in diffuse follicular cell hypertrophy in thyroid glands in 4/5 males and 5/5 females.
Kidneys: Moderate diffuse tubular degeneration/regeneration in 5/5 males, in all males associated with minimal to slight multifocal single cell necrosis, and in 3/5 males associated with hyaline casts. Slightly increased severity of hyaline droplets in proximal convoluted tubules in group 4 males.

Forestomach: Minimally increased incidence and severity of diffuse hyperkeratosis in males of group 4 and females of groups 3 and 4.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation not specified (migrated information)
Remarks:
Generation not specified (migrated information)
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation not specified (migrated information)
Remarks:
Generation not specified (migrated information)
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no effects on the reproductive parameters evaluated.
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At scheduled necropsy, no test item-related findings were noted. One animal had a missing tail and anal atresia, one had a missing tip of tail.
Histopathological findings:
not examined
Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 49 / 51, 48 / 52, 43 / 57, and 48 / 52 in groups 1, 2, 3, and 4, respectively, at first litter check.

The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item. Compared with the other groups’ values, slightly higher pup body weights were noted for male and female pups in group 4 that were considered to reflect the normal biological variability.

At scheduled necropsy, no test item-related findings were noted.

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test item-related findings were noted
Reproductive effects observed:
not specified

In groups 2, 3, and 4, the mean number of corpora lutea was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of corpora lutea was 13.8, 13.9, 14.6, and 14.7, respectively.

In groups 2, 3, and 4, the implantation rate and the post implantation loss were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean implantation rate was 12.2, 13.0, 14.1, and 13.7, respectively. In groups 1, 2, 3, and 4, the total post implantation loss was 12, 16, 12, and 10, in 8, 9, 5, and 6 litters, respectively. These data correspond to 9.8%, 12.3%, 8.5%, and 8.1% of implantations.

In groups 2, 3, and 4, the number of living pups at first litter check, were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of living pups was 11.0, 11.4, 12.9, and 12.6, respectively. One, 1, and 2 dead pups were noted in 1, 1, and 2 litters of groups 2, 3, and 4, respectively.

In groups 2, 3, and 4, the postnatal loss was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the postnatal loss was 2, 1, 1, and 3 dead pups noted in 2, 1, 1, and 3 litters, respectively. These data correspond to 1.8%, 0.9%, 0.8%, and 2.7% of dead pups during the postnatal period.

At first litter check, no test item-related findings were noted. Female pup no. 14 in litter 46 (group 1) was noted with a missing tail and an anal atresia. It was killed for ethical reasons on day 1 post partum. Female pup no. 14 in litter 77 (group 4) was noted with a missing tail. It was necropsied as scheduled.

Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 49 / 51, 48 / 52, 43 / 57, and 48 / 52 in groups 1, 2, 3, and 4, respectively, at first litter check.

The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item. Compared with the other groups’ values, slightly higher pup body weights were noted for male and female pups in group 4 that were considered to reflect the normal biological variability.

At scheduled necropsy, no test item-related findings were noted.

Conclusions:
There were no effects on reproductive or developmental parameters up to 1000 mg/kg/day, the highest dose evaluated.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Di-tert-butyl peroxide on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition was provided.

Di-tert-butyl peroxide was administered once daily orally (by gavage) at dosages of 0, 100, 300, and 1000 mg/kg/day, to male rats for 42 days in total and to female rats throughout the prepairing, the pairing, the gestation and the lactation periods until day 4 post partum (last dosing).

Treatment at 1000 mg/kg was associated with body weight effects in male animals. Food consumption was decreased in male animals during the study. A sign of discomfort was noted in all animals at 1000 mg/kg/day in the way that the animals moved their heads through the bedding material after the daily administration of test item. Some male and female animals were noted with ruffled fur.

Treatment at 1000 mg/kg/day and 300 mg/kg/day was associated with increased liver and kidney weights. Histopathological effects were noted in liver (minimal centrilobular and diffuse hepatocellular hypertrophy with association of a consequent increase in diffuse follicular cell hypertrophy in thyroid glands in males and females at 1000 mg/kg/day), kidneys (moderate diffuse tubular degeneration/regeneration with slight multifocal single cell necrosis and hyaline casts as well as hyaline droplets in males at 1000 mg/kg/day), and fore stomach (minimally increased incidence and severity of diffuse hyperkeratosis in males at 1000 mg/kg/day and

females at 1000 mg/kg/day and 300 mg/kg/day).

Behavioral effects were considered to be not influenced by the treatment with the test item. No effects were noted on reproduction data, for the parameters during the clinical laboratory investigations, or for macroscopic findings during necropsy.

The histopathological evaluation of the reproductive organs did not reveal any relevant changes in high-dose animals. Especially the emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high-dose (group 4) males.

Except female no. 79 in group 4, all mated females were pregnant. In groups 2, 3, and 4, mating performance was considered to be not influenced by treatment with the test item. The fertility index was 100.0, 100.0, 100.0, and 90.0% in groups 1, 2, 3, and 4, respectively. In groups 2, 3, and 4, the pre-implantation loss, the implantation rate, the post implantation loss, the number of living pups at first litter check, the post natal loss, and the gestation length, respectively, were considered to be not influenced by treatment with the test item.

Based on these data, it can be concluded that the parental No Observed Effect Level (NOEL) was at 100 mg/kg body weight/day and 1000 mg/kg/day for reproductive and developmental effects.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restrictions.
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
A GLP OECD 414 study, conducted with rats at 0, 100, 300 and 1000 mg/kg/day, did not reveal any adverse effects on developmental parameters evaluated. 
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7th January 2013 to 24 May 2013
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 guidenlines and/or minor methodlogical deficiences, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology Studies, 12 NohSan No 8147, (24 November 2000)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were received in two deliveries each containing two separate batches of females on either Day 0 or Day 1 of presumed gestation. The day that positive evidence of mating was observed at the supplier was designated Day 0 of gestation. On arrival the females weighed 190 to 261g.

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Appendix 17. 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). The diet, drinking water, bedding and environmental enrichment were 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 computerised system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3ºC and 50 ± 20% respectively; there were no deviations from these target ranges.

The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
For purpose of the study, the test item was prepared at the appropriate concentrations as a solution in Corn Oil (Corn oil had been successfully used in previous toxicity work). The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services as part of this study. Results are given in Appendix 14 and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately -20 °C in the dark.

Representative samples were taken of test item formulation used on the study and were analysed for concentration of di-tert butyl peroxide (CAS# 110-05-4) at Harlan Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in Appendix 14. The results indicate that the prepared formulations were within ± 6% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of Di-Tert-butyl peroxide (CAS#110-05-4) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

Samples
The test item formulations were diluted with tetrahydrofuran to give a final, theoretical test item concentration of approximately 0.1 mg/ml.

Standards
Standard solutions of test item were prepared in tetrahydrofuran at a nominal concentration of 0.1 mg/ml.

Procedure
The standard and sample solutions were analysed by GC using the following conditions:

GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-5 (30 m x 0.53 mm id x 5 µm film)
Oven temperature program : initial 40 ºC for 0 mins
rate 10 ºC/min
temp 140 ºC for 0 mins
rate 50 ºC/min
final 260 ºC for 5 mins
Injection temperature : 150 ºC
Flame ionisation detector temperature :250 ºC
Injection volume: 1 µl
Retention time : ~ 4.9 mins


Appendix 14 (continued) Analytical Investigations
1.5 Homogeneity Determinations
The test item formulations were mixed and assessed visually.

Stability Determinations
The test item formulations were sampled, analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within two days of preparation.
Details on mating procedure:
Not described in this study
Duration of treatment / exposure:
16 days (Between days 3 and 19 of gestation)
Frequency of treatment:
Daily
Duration of test:
20 days
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.


Justification
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. An additional observation was also performed five hours after dosing during the normal working week (see Deviations from Study Plan). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

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


All implantations and viable foetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn

L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16

V = viable foetus

Organ Weights
The liver and kidneys were removed from all animals that were killed at the end of the study and were dissected free from fat and weighed before fixation. These tissues were not processed any further and are retained with the raw data for the study.
Ovaries and uterine content:
Post Mortem
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium poly sulphide to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue

Late Death: Separate embryonic/foetal and placental tissue visible

Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Fetal examinations:
The foetuses were killed by subcutaneous injection of sodium pentobarbitone. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red S. The foetuses were examined for skeletal development and anomalies. Following examination foetuses that were examined for skeletal development were placed in 100% glycerol.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, absolute and body weight relative organ weights, litter data and foetal litter and placental weights: Bartlett’s test for homogeneity of variance. Where the data were shown to be homogeneous one way analysis of variance and, if significant, Dunnett’s multiple comparison test was employed; where the data were found to nonhomogeneous Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was employed.

Foetal evaluation parameters, including skeletal or visceral findings were analyzed by Kruskal-Wallis and, if significant, Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality
There were no unscheduled deaths.


Clinical Observations
A summary incidence of daily clinical observations is given in Table 2. Individual data is presented in Appendix 1.

At 1000 mg/kg bw/day, the majority of females showed isolated incidences of increased salivation between Day 13 and Day 17 of gestation. One female at this dosage also showed noisy respiration on Days 8 to 10 and Day 14. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and, in isolation, are considered to be of no toxicological importance.

There were no other signs considered to indicate an effect of treatment.

At 300 mg/kg bw/day, one female treated showed chromodacryorrhoea on Days 4 to 5 and another female at 100 mg/kg bw/day showed this clinical sign on Day 10. Additionally at 100 mg/kg bw/day, a further female showed increased salivation and decreased respiratory rate on Day 3 with noisy respiration on Days 3 and 5 and one female had fur loss from Day 14 onwards. In isolation, these low incidence findings were considered to incidental and unrelated to treatment.


Body Weight
Group mean body weights and standard deviations are given in Table 3. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5 and cumulative body weight change is presented graphically in Figure 1. Individual data are given in Appendix 2 to Appendix 4.

Body weights and body weight gain, including values adjusted for the contribution of the gravid uterus, were unaffected by treatment at 100, 300 and 1000 mg/kg bw/day.


Food Consumption
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

There were no effects of treatment on food intake during gestation at 100, 300 or 1000 mg/kg bw/day.


Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.


Post Mortem Studies
A summary incidence of female necropsy findings is given in Table 7. Individual data are given in Appendix 6.

No macroscopic abnormalities were detected for adult animals at Day 20 of gestation.


Organ Weights
Group mean absolute and body weight-relative values and standard deviations for test and control animals are presented in Table 8. Individual data are given in Appendix 7.

At 1000 mg/kg bw/day mean absolute and body weight-relative liver weight were higher than control with differences for relative weights attaining statistical significance. Absolute and body weight-relative kidney weights at this dosage were considered to be unaffected by treatment and there were no statistically significant differences in kidney weights compared with control.

At 100 and 300 mg/kg bw/day there were no statistically significant differences in absolute and body weight-relative liver and kidney weight compared with control.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
300 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 Data and Litter Placental and Foetal Weights
Group Mean Litter data is given in Table 9. Individual data are given in Appendix 8 and Appendix 9.

There was no effect of treatment on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and pre and post-implantation losses at 100, 300 and 1000 mg/kg bw/day.

There were no effects of treatment on mean foetal, litter or placental weights at 100, 300 or 1000 mg/kg bw/day.

At 300 mg/kg bw/day, one female showed total resorption (total litter loss in utero). In the absence of any observed increase in mean post-implantation loss for other litters at this dosage, or at 1000 mg/kg bw/day, this finding was considered incidental and unrelated to treatment.


Foetal Examination
Summary foetal external findings, visceral findings, skeletal findings and skeletal development are given in Table 10 to Table 13. Individual data are given in Appendix 10 to Appendix 13.

The type, incidence or distribution of findings observed externally at necropsy examination and subsequently during detailed visceral and skeletal assessment did not indicated any effect of treatment on foetal growth or development.

Females from all treatment groups showed a statistically significant increase in the number of foetuses presenting with one thoracic vertebral centra semi-bipartite. Additionally females treated with 1000 mg/kg bw/day showed a statistically significant reduction in the number of foetuses presenting with incomplete ossification of more than one cranial bone variant. The observation of one variant at a higher or lower incidence when compared to controls is not significant when evaluated in isolation and, due to the number of parameters evaluated, it is highly likely that at least one finding will be more/less frequently observed in dosed groups in comparison to controls. A true developmental effect is represented by effects observed in a number of variants or a syndrome of variance and therefore the intergroup differences observed in this study were considered incidental and unrelated to treatment.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
A dosage of 1000 mg/kg bw/day was considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female, with the No Observed Effect Level (NOEL) being 300 mg/kg bw/day. The NOEL for the survival, growth and morphological development of the conceptus was considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was performed according to the study plan presented in Appendix15and was designed to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female (from Day 3 to Day 19 of gestation) (and including period of organogenesis). The results of the study are believed to be of value in predicting the toxicity of the test item during pregnancy, and the estimation of both a maternal and embryofoetal ‘No Observed Effect Level’ (NOEL).

 

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     US EPA Health Effects Test Guideline OPPTS 870.3700, “Prenatal Developmental Toxicity Study” (August 1998)

·     Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

·     OECD Guidelines for Testing of Chemicals, No 414 “Prenatal Developmental Toxicity Study” (adopted 22 January 2001)

·     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 twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Corn Oil) to serve as a control.

Clinical signs, body weight change and food and water consumptions were monitored during the study. Liver and kidney weights were recorded for all females at termination.

 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in 70% Industrial Methylated Spirit (IMS) in distilled water and then following examination for skeletal development transferred into 100% glycerol. The remaining half were preserved in Bouin’s solution and transferred to 90% IMS in distilled water and the viscera were examined.

 

Results…….

Mortality

There were no unscheduled deaths.

 

Clinical Observations

There were no toxicological significant clinical signs observed at 100, 300 or 1000 mg/kg bw/day.

 

Body Weight

There were no effects of treatment on body weight or body weight gain during gestation at 100, 300 or 1000 mg/kg bw/day.

 

Food Consumption

There were no effects of treatment on food intake during gestation at 100, 300 or 1000 mg/kg bw/day.

 

Water Consumption

No effect on water consumption during gestation was detected at 100, 300 or 1000 mg/kg bw/day.

 

Post Mortem Studies

No macroscopic abnormalities were detected for adult animals.

 

Organ Weights

At 1000 mg/kg bw/day, liver weights were considered to be increased in comparison to control.

 

Litter Data and Litter Placental and Foetal Weights

There were no effects of maternal treatment onin uterolitter data or on litter, placental or foetal weights at 100, 300 or 1000 mg/kg bw/day.

 

Foetal Examination

Detailed examination of foetal morphology did not indicate any effect of maternal treatment on embryofoetal development at 100, 300 or 1000 mg/kg bw/day.

 

Conclusion

A dosage of 1000 mg/kg bw/day was considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female, with the No Observed Effect Level (NOEL) being 300 mg/kg bw/day. The NOEL for the survival, growth and morphological development of the conceptus was considered to be 1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Species:
rabbit
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This is a well documented study conducted according to modern standards of protocol, quality assurance, and good laboratory practices. The test material was 99.2 % pure and was analytically demonstrated to be stable in the test vehicle (corn oil).
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
yes
Remarks:
study plan "amendments": "Male animals aged 8 weeks minimum instead of 10 weeks. Lymph nodes for histopath specified as “one lymph node covering the route of administration and another one distant from the route of admin. to cover systemic effect."
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Details on test animals and environmental conditions:
Species Rat: HanRcc:WIST (SPF)
Rationale: Specified by the international guidelines as the recommended test system
Source: RCC Ltd
Laboratory Animal Services
Wolferstrasse 4
4414 Fullinsdorf / Switzerland

Number of animals: 40 males, 10 per group; 40 females, 10 per group
Age at delivery: 8 weeks (male animals); 10 weeks (female animals)
Body weights: Males: 243 - 291 grams (at first treatment)
Females: 181 - 219 grams (at first treatment)
Acclimatization: Seven days under test conditions with an evaluation of the health status

Identification
Parental animals: Individual animal number tattooed on the pinnae and individual cage card.
F1 pups: Individual intra-litter pup number tattooed with Indian ink on day 1 post partum.

This study was performed under standard laboratory conditions: the animal room was air-conditioned with 10 - 15 air changes per hour; the environment was monitored continuously with recordings of temperature (range 22+/- 3°C) and relative humidity (range 30 - 70%), 12 hours artificial fluorescent light / 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Accommodation: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland). During the pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles. During the pairing period, rats were housed one male / one female in Makrolon pairing cages. After mating, the males and the females were housed individually again. During lactation period (until day 4 of lactation), dams were housed together with their litters. Throughout the study, each cage was identified by a colored label according to the group and recording the study schedule number, animal number(s) and details of treatment.

Diet: Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/ Switzerland) was available ad libitum (Batch No. 67/06). Results of analyses for contaminants are presented in the final report.

Water Tap water from Fullinsdorf in bottles was available ad libitum. Results of the bacteriological, chemical and contaminant analyses scheduled to be conducted at least once yearly by RCC (contaminant analyses only) and by the Official Chemist of the Kanton Basel-Landschaft (bacteriological and chemical analyses) are part of the final report.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Vehicle
Identity – Corn oil
CAS-No. 8001-30-7
EG No. 2322812
Supplier – Carl Roth GmbH & Co., 76185 Karlsruhe / Germany
Batch number – 38679499
Expiry date – 14-NOV-2016
Storage conditions -- Room temperature (20°C +/- 5°C)

Dose formulations were prepared in terms of material as supplied by the Sponsor.
Frequency of dose formulation -- Weekly, based on the results of RCC Study No. A79874
Storage of dose formulations -- Room temperature (20°C +/-5°C)
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle were added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. Having obtained a homogeneous mixture, the vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration. During the daily administration period, homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations
Samples for determination of concentration, homogeneity and stability (4 hours and 7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of
the administration period

On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. For determination of stability, samples were taken before dosing from the middle and stored at room temperature for 4 hours and for 7 days in the dark. The samples were stored at approximately 20°C in the dark pending analysis. Samples were shipped to Itingen / Switzerland. Analysis was performed using a method provided by the Sponsor and adapted by the test facility.

After analysis, the analytical results were communicated to the Study Director. Upon receipt and evaluation of these results the Study Director decided about discarding the samples. The report of the Analytical Phase is presented as part of the final report.
Details on mating procedure:
During the pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles.

During the pairing period, rats were housed one male / one female in Makrolon pairing cages.

After mating, the males and the females were housed individually again. During lactation period (until day 4 of lactation), dams were housed together with their litters.
Duration of treatment / exposure:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Frequency of treatment:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Duration of test:
MALES FEMALES
Acclimatization 7 days (minimum) 7 days (minimum)
Treatment beginning Day 1 of pre-pairing Day 1 of pre-pairing
Pre-pairing 14 days 14 days
Pairing until mating (maximum 14 days) until mating (maximum 14 days)
Gestation N/A* about 21 days
Parturition N/A* expected : on day 21 or 22 post coitum
Lactation N/A* until day 4 post partum
Treatment ending One day prior to the actual day of necropsy on day 4 post partum
(at least 28 days of treatment)
Blood collection On the day of necropsy On the day before necropsy or on the day of necropsy
Termination After the first dams had reached day pups on day 4 post partum
4 post partum dams on day 5 post partum

* Not applicable
No. of animals per sex per dose:
10 males; 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in agreement with the Sponsor, based on the results of a preliminary dose range-finding study, where 1000 mg/kg/day were used as highest dose level.
Maternal examinations:
All animals were checked at least twice daily for any mortalities. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.

All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health. Additionally, the females were observed for signs of difficult or prolonged parturition

Once prior to the first test item administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also recorded.

Functional observation battery

At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were performed for five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

a) Cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well as resistance to removal.

b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.

c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.

d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or faeces, soiling, general abnormalities, posture.

e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

A description of all test parameters is listed in this report. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (basing on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
Ovaries and uterine content:
The number of implantation sites and corpora
lutea was recorded for all dams with litters. The uteri of
non-pregnant females were placed in a solution of
ammonium sulfide to visualize possible hemorrhagic
areas of implantation sites.
Fetal examinations:
Day 0 of lactation was the day on which a female had
delivered all her pups. The litters were examined for litter
size, live birth, stillbirth and any gross anomalies. The sex
ratio of the pups was recorded. The dams were caged
together with their litters until day 4 of lactation. Pups
were weighed individually (without identification) on days
0 (if possible), 1 and 4 post partum.
The dams and pups were observed daily for survival and
behavioural abnormalities in nesting and nursing.

Dead pups (except if excessively cannibalized) and pups
killed at day 4 of lactation were examined macroscopically.
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group).
• The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
The calculations for evaluation of the reproduction data were performed by a computer program
based on on-line recorded data.
Historical control data:
Included in report.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment at 1000 mg/kg/day and 300 mg/kg/day was associated with increased liver and kidney weights. Histopathological effects were noted in liver (minimal cetrilobular and diffuse hepatocellular hypertrophy with association of a consequent increase in diffuse follicular sell hypertrophy in thyroid glands in males and females at 1000 mg/kg/day), kidneys (moderate diffuse tubular degeneration/regeneration with slight multifocal single cell necrosis and hyaline casts as well as hyaline droplets in males at 1000 mg/kg/day), and forestomach (minimally increased incidence adn severity of diffuse hyperkeratosis in males at 1000 mg/kg/day and females at 1000 mg/kg/day and 300 mg/kg/day).
Dose descriptor:
NOAEL
Remarks:
100 mg/kg/day
Effect level:
ca. 300 mg/kg bw/day (nominal)
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:
There were no effects on viability, clinical signs, body weight or gross pathology.

Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 49 / 51, 48 / 52, 43 / 57, and 48 / 52 in groups 1, 2, 3, and 4, respectively, at first litter check.

The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item. Compared with the other groups’ values, slightly higher pup body weights were noted for male and female pups in group 4 that were considered to reflect the normal biological variability.

At scheduled necropsy, no test item-related findings were noted.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

In groups 2, 3, and 4, the mean number of corpora lutea was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of corpora lutea was 13.8, 13.9, 14.6, and 14.7, respectively.

In groups 2, 3, and 4, the implantation rate and the post implantation loss were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean implantation rate was 12.2, 13.0, 14.1, and 13.7, respectively. In groups 1, 2, 3, and 4, the total post implantation loss was 12, 16, 12, and 10, in 8, 9, 5, and 6 litters, respectively. These data correspond to 9.8%, 12.3%, 8.5%, and 8.1% of implantations.

In groups 2, 3, and 4, the number of living pups at first litter check, were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of living pups was 11.0, 11.4, 12.9, and 12.6, respectively. One, 1, and 2 dead pups were noted in 1, 1, and 2 litters of groups 2, 3, and 4, respectively.

In groups 2, 3, and 4, the postnatal loss was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the postnatal loss was 2, 1, 1, and 3 dead pups noted in 2, 1, 1, and 3 litters, respectively. These data correspond to 1.8%, 0.9%, 0.8%, and 2.7% of dead pups during the postnatal period.

At first litter check, no test item-related findings were noted. Female pup no. 14 in litter 46 (group 1) was noted with a missing tail and an anal atresia. It was killed for ethical reasons on day 1 post partum. Female pup no. 14 in litter 77 (group 4) was noted with a missing tail. It was necropsied as scheduled.

Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 49 / 51, 48 / 52, 43 / 57, and 48 / 52 in groups 1, 2, 3, and 4, respectively, at first litter check.

The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item. Compared with the other groups’ values, slightly higher pup body weights were noted for male and female pups in group 4 that were considered to reflect the normal biological variability.

At scheduled necropsy, no test item-related findings were noted.

Conclusions:
Based on these data, it can be concluded that the maternal No Observed Effect Level (NOEL) was at 100 mg/kg body weight/day. The NOEL for developmental toxicity was 1000 mg/kg/day, the highest dose administered to dams.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Di-tert-butyl peroxide on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition was provided.

Di-tert-butyl peroxide was administered once daily orally (by gavage) at dosages of 0, 100, 300, and 1000 mg/kg/day, to male rats for 42 days in total and to female rats throughout the prepairing, the pairing, the gestation and the lactation periods until day 4 post partum (last dosing).

Treatment at 1000 mg/kg was associated with body weight effects in male animals. Food consumption was decreased in male animals during the study. A sign of discomfort was noted in all animals at 1000 mg/kg/day in the way that the animals moved their heads through the bedding material after the daily administration of test item. Some male and female animals were noted with ruffled fur.

Treatment at 1000 mg/kg/day and 300 mg/kg/day was associated with increased liver and kidney weights. Histopathological effects were noted in liver (minimal centrilobular and diffuse hepatocellular hypertrophy with association of a consequent increase in diffuse follicular cell hypertrophy in thyroid glands in males and females at 1000 mg/kg/day), kidneys (moderate diffuse tubular degeneration/regeneration with slight multifocal single cell necrosis and hyaline casts as well as hyaline droplets in males at 1000 mg/kg/day), and fore stomach (minimally increased incidence and severity of diffuse hyperkeratosis in males at 1000 mg/kg/day and

females at 1000 mg/kg/day and 300 mg/kg/day).

Behavioral effects were considered to be not influenced by the treatment with the test item. No effects were noted on reproduction data, for the parameters during the clinical laboratory investigations, or for macroscopic findings during necropsy.

The histopathological evaluation of the reproductive organs did not reveal any relevant changes in high-dose animals. Especially the emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high-dose (group 4) males.

Except female no. 79 in group 4, all mated females were pregnant. In groups 2, 3, and 4, mating performance was considered to be not influenced by treatment with the test item. The fertility index was 100.0, 100.0, 100.0, and 90.0% in groups 1, 2, 3, and 4, respectively. In groups 2, 3, and 4, the pre-implantation loss, the implantation rate, the post implantation loss, the number of living pups at first litter check, the post natal loss, and the gestation length, respectively, were considered to be not influenced by treatment with the test item.

Based on these data, it can be concluded that the maternal No Observed Effect Level (NOEL) was at 100 mg/kg body weight/day and 1000 mg/kg/day for reproductive and developmental effects.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restrictions.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 414 study, a dosage of 1000 mg/kg bw/day was considered to represent a No Observed Adverse Effect Level (NOAEL) for the pregnant female, with the No Observed Effect Level (NOEL) being 300 mg/kg bw/day. The NOEL for the survival, growth and morphological development of the conceptus was considered to be 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Apparently well conducted GLP study.

Justification for classification or non-classification

Does not meet the criteria for classification due to lack of reproductive or developmental effects.