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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 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
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 oral 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.
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.
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 preliminarydose range-finding study, where 1000 mg/kg/day were used as highest dose level.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
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 pups on day 4 post partum
day 4 post partum dams on day 5 post partum


++++++++++++++++++++++++++++++++++++++

Mortality rate -- 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.

Signs and/or symptoms -- 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.

Detailed clinical observations -- Once prior to the first test item administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for thefollowing: 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 asresistance 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 the final 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.

Body weight--The animals were weighed daily during the entire study.

Food consumption
Males: Food consumption was recorded weekly during the prepairing and post-pairing period.
Females: Food consumption was recorded for the following periods: days 1-8 and 8-14 of the pre-pairing period; days 0-7, 7- 14 and 14-21 post coitum and days 1-4 post partum.
Food consumption was not recorded during the pairing period (mixed values of males and females).

Lactation and litter data -- 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.

Sacrifice and pathology:
PATHOLOGY
Males were sacrificed after the first dams had reached postmortem examination day 4 post partum (42 days of administration). Females were sacrificed on day 5 post partum. Pups were sacrificed on day 4 post partum. Males and females were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. Pups were killed by an intraperitoneal injection of sodium pentobarbital (EuthaR 77). The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. 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. Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically. If birth did not occur on the expected date (day 21 post coitum), the female was treated until day 24 post coitum, sacrificed on day 25 post coitum and examined as described.

ORGAN WEIGHTS
The testes* and epididymides* of all parental males were weighed. In addition for five adult males and females, randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:• liver • spleeen • adrenals* • brain • thymus • heart • kidneys* [ * weighed as pairs]


TISSUE PRESERVATION
Of all parental males the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • prostate • testes (in Bouin's fixative) • seminal vesicles with coagulation gland • epididymides (in Bouin's fixative)

Of all parental females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • ovaries

In addition, of the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • gross lesions • heart • brain • thymus • spinal cord • thyroid • small and large intestines (incl. Peyer's patches) • trachea and lungs (preserved by inflation with fixative and then immersion) • stomach • uterus (with vagina) • liver • urinary bladder • kidneys • lymph nodes (mandibular, mesenteric) • adrenals • peripheral nerve • spleen • bone marrow


HISTOPATHOLOGY
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, ifnecessary.
Other examinations:
CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day before or on the day of scheduled necropsy from all P generation males after they had been fasted overnight. Blood samples of P generation females were obtained on day 5 post partum after the females had been fasted overnight. Blood samples were collected sublingually with the animal under light isoflurane anaesthesia. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HAEMATOLOGY
The following haematology parameters were determined:
Erythrocyte count Reticulocyte count
Haemoglobin Reticulocyte maturity index
Haematocrit Total leukocyte count
Mean corpuscular volume Differential leukocyte count
Red cell volume distribution width Platelet count
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration Thromboplastin time
Haemoglobin concentration distribution width Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
The following parameters were determined:
Glucose Sodium
Urea Potassium
Creatinine Chloride
Bilirubin, total Calcium
Cholesterol, total Phosphorus inorganic
Aspartate aminotransferase Protein, total
Alanine aminotransferase Albumin
Bile acids Globulin
Alkaline phosphatase Albumin/Globulin ratio
Gamma-glutamyl-transferase
Statistics:
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
PARENTAL ANIMALS
MORTALITY
All male and female animals survived until scheduled necropsy.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
In group 4, all male and female animals were noted with moving their head through the bedding material starting on days 11 and 12, respectively, until the last administration. About one to 5 male and 2 female animals were noted with ruffled fur starting on day 12 of the study. It continued until day 19 of the after pairing period for one male (no. 31). In two female animals (nos. 79 and 80), ruffled fur disappeared on days 6 and 7, respectively, of the gestation period. No treatment related clinical signs were noted for male and female animals in groups 2 and 3. Male no. 14 in group 2 was noted with a black tip of tail starting on day 8 until day 13, with body weight loss on day 8. On day 14, the tip of tail fell off. No survival and behavioural abnormalities were noted in the female animals and pups during nesting and nursing.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
Four male animals (nos. 31, 33, 36, and 39) and two female animals (nos. 79 and 80) in group 4 were noted with ruffled fur on day 26 of study, respectively (after pairing period / gestation period). One male animal (no. 31) was again noted with ruffled fur on day 40 of study (after pairing period). No other findings were noted during the detailed clinical observations in any other animal in any group.

FOOD CONSUMPTION - MALES
Pre-pairing and After Pairing Period
In group 4, the mean food consumption was statistically significantly decreased during the prepairing period. During after 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 WEIGHTS - MALES
Pre-pairing, Pairing and After Pairing Period
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, andin group 2 on day 3, respectively, during the after pairing period.

FOOD CONSUMPTION - FEMALES
Pre-pairing, Gestation and Lactation Period
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 WEIGHTS - FEMALES
Pre-pairing, Pairing, Gestation and Lactation Period
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.

FUNCTIONAL OBSERVATIONAL BATTERY
None of the parameters under investigation was considered to be affected by exposure to the test item during the functional observational battery. One male animal was noted with reduced activity in group 4. Three males in group 1, one male in group 3 and two males in group 4 were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 3 males each in groups 1 and 2. Spontaneous vocalization, when removed was noted in two females in group 1 and one female in group 2. One female animal was noted with reduced activity in group 1. One female in group 1 and one female in group 2 were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2 females in groups 1 and one female in group 2.

Grip Strength, Landing Foot Splay and Body Temperature
In groups 2, 3, and 4, mean values for grip strength (fore- and hind paws), landing foot splay, and body temperature (rectal) were considered not influenced by treatment with the test item.

Locomotor Activity In groups 2, 3, and 4, locomotor activity, as assessed in terms of low beam counts in an activity monitor, was considered to be notinfluenced by treatment with the test item.

REPRODUCTION DATA
FERTILITY AND MATING PERFORMANCE
All mated females except one (no. 79 in group 4) were pregnant. In groups 2, 3, and 4, mating performance was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the median pre-coital time was 3, 3, 2, and 3 days, and the mean precoital time was 2.4, 2.7, 2.3, and 3.0 days, respectively. The fertility index was 100.0, 100.0, 100.0, and 90.0% in groups 1, 2, 3, and 4, respectively.
DURATION OF GESTATION
In groups 2, 3, and 4, the gestation length was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean duration of gestation was 21.6, 21.5, 21.7, and 21.8 days, respectively.
CORPORA LUTEA COUNT
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.
IMPLANTATION RATE AND POST-IMPLANTATION LOSS
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.
LITTER SIZE AT FIRST LITTER CHECK
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.
POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
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.

LITTER DATA - F1 PUPS
ABNORMAL FINDINGS AT FIRST LITTER CHECK UNTIL DAY 4 POST PARTUM
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
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.
PUP WEIGHTS TO DAY 4 POST PARTUM
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.

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

CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
None of the parameters under investigation for hematology was considered to be affected by exposure to the test item. In males, the mean hemoglobin concentration distribution width and the mean relative reticulocyte count were noted with statistically significantly increased values in group 4. Mean prothombin time was statistically significantly increased in groups 3 and 4. The mean activated partial thromboplastin time was statistically significantly decreased in group 4. These findings were considered incidental as these statistically significant values were within the range of reference values. In females, the mean relative reticulocyte count was noted with increased values in group 4. This finding was considered incidental as this parameter exceeded the reference values in all groups. The parameters mean absolute reticulocyte count and mean total leukocytes also exceeded the referencevalues in all groups. Therefore, these findings were also considered incidental.
CLINICAL BIOCHEMISTRY
None of the parameters under investigation for clinical biochemistry was considered to be affected by exposure to the test item. A few parameters, like creatinine, cholesterol, sodium, and chloride in males, were noted with statistically significantly values in groups 3 and 4. Yet, these were considered incidental as the statistically significant values were within the range of reference values. In females in group 4, statistically significant values were noted at the parameters of glucose and potassium. The value for potassium exceeded the reference values for females from 13 to 18 weeks of age. As the parameter of phosphorus in all groups also exceeded the reference values, these incidences were considered incidental.

TERMINAL FINDINGS
NECROPSY
All male and female animals in groups 2, 3, and 4 were noted without any treatment-related finding at necropsy.
ORGAN WEIGHTS
Males
In male animals of groups 3 and 4, respectively, mean organ weights of liver and kidneys were slightly increased. For organ / body weight ratio these organs weights were statistically significantly increased in groups 3 and 4, respectively. These findings may be due to an increased metabolism and excretion of the test item and was therefore considered test item related. All other mean organ weights were considered to be not influenced by the treatment with the test item. For organ / body weight ratio the weight of the testes was statistically significantly increased in group 4. As the mean body weight in group 4 was statistically significantly decreased at the end of study, this relative mean organ weight increase of the testes was considered incidental.

Females
In female animals of group 4, the mean liver weight was statistically significantly increased. The mean kidney weights were slightly increased in groups 3 and 4. For organ / body weight ratio, the relative liver weight was statistically significantly increased in group 4, and the relative kidney weights were statistically significantly increased in groups 3 and 4. These findings may be due to an increased metabolism and excretion of the test item and was therefore considered test item related. All other mean organ weights were considered to be not influenced by the treatment with the test item. For organ / body weight ratio the heart was statistically significantly increased in group 4. This relative mean organ weight increase of the heart was considered incidental.

HISTOPATHOLOGY
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.

The oral application of Di-tert-butyl peroxide at the dose levels of 300 and 1000 mg/kg/day (groups 3 and 4) induced the following findings likely related to an effect of the test item in organs other than reproductive:

Liver: Minimal centrilobular hepatocellular hypertrophy was present in 3/5 males and 3/5 females of group 3 and 2/5 males and 2/5 females of group 4. Minimal to slight diffuse hepatocellular hypertrophy occurred in 1/5 males of group 3 and 3/5 males and 3/5 females of group 4. In one male, the hypertrophy was associated with focal single cell necrosis.

Thyroid gland: Minimally to moderately increased incidence and minimally increased severity of diffuse follicular cell hypertrophy was noted in 4/5 males and 5/5 females and was likely consequent to an enhanced liver cell metabolism due to the hepatocellular hypertrophy.

Kidneys: Moderate diffuse tubular degeneration/regeneration was found in 5/5 males, in all males associated with minimal to slight multifocal single cell necrosis, and in 3/5 males associated with minimal to moderate hyaline casts. In addition, slightly increased severity of hyaline droplets occurred in proximal convoluted tubules of group 4 males, representing alpha-2μ-globulin in the cytoplasm (phagolysosomes) of proximal convoluted tubules of sexually mature males.

Forestomach: Minimally increased incidence and severity of diffuse hyperkeratosis was diagnosed in group 4 males and in group 3 and 4 females.


These microscopic findings occurring in treated group 4 animals or being increased in group 4 rats, were considered to be likely related to an effect of the test item. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence were indistinguishable from controls.

Effect levels

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'
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'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

SUMMARY OF PERFORMANCE                      F0 ANIMALS BREEDING FOR F1 LITTERS

Group

1

2

3

4

(mg/kg/day)

0

100

300

1000

Female numbers

41-50

 

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of non pregnant females(A)

0

0

0

1

Number of females, which lost their

litters

0

0

0

0

Number of females which reared

their pups until day 4 post partum

10

10

10

9

(A) = Female No. 79 was not pregnant

 

 

 

Applicant's summary and conclusion

Conclusions:
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.

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

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

The microscopic findings occurring in the high dose group animals or being increased in the high dose animals, were considered to be likely related to an effect of the test item. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence were indistinguishable from controls. Therefore, 300 mg/kg/day will be used as the NOAEL for DNEL calculation.