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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:
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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identity Di-tert-butyl peroxide
Batch number 11648 995507-109
Description liquid
Purity 99.2%
CAS-No. 110-05-4
Expiry date 12-APR-2007
Storage Room temperature (20°C ± 5°C)
Stability in the vehicle at least 4 hours demonstrated analytically

Test animals

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.

Administration / exposure

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

Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.