Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
26 feb 2007 to 30 april 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: Guideline study, GLP

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)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 2,2-Bis(t-butylperoxyisopropyl)benzene (CAS 25155-25-3)
- Physical state: pale yellow solid, acrid odour
- Analytical purity: 96.5 %
- Stability under test conditions: at least 4 hours in the vehicle
- Storage condition of test material: room temperature (15-25 °C), dark
- Frequence of dose formulation: weekly, based on a previous results

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd
- Age at study initiation: 8 weeks (males) and 10 weeks (female)
- Weight at study initiation: 243-291 g (males) and 190-217 g (females)
- Fasting period before study:overnight
- Housing:in Marolon cages ( individually during the pre-pairing period et after the mating , and one male/one female during the paring period)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
in corn oil, the dosing solutionsn were made weekly

VEHICLE
- Justification for use and choice of vehicle: justified by analytical considerations
- Concentration in vehicle: 0, 25, 75 or 250 mg/ml
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. : 38679499
Details on mating procedure:
- M/F ratio per cage: one/one
- Length of cohabitation: during the mating
- Proof of pregnancy: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive.
- If a female was not mated during the 14-day pairing period, the female was paired with a male of the same group which already mated successfully.
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dose formulations were taken during the first and the last week of the administration period. Three samples were taken from the top, middle and bottom of each formulation, dilution were performed then the samples were analysed with HPLC. Furthemore samples were taken for analysing the stability after 2 and 16 days. The application formulations investigated during the study were found to comprise 2,2-Bis(t-butylperoxy isopropyl)benzene in the range from 81.8% to 118.3% and, thus, the required content limit of ±20% with reference to the nominal concentration was met.
Duration of treatment / exposure:
The test item was administered for at least 44 days.
Females: The test item was administered throughout the pre-pairing (14 days), pairing , gestation (21 days) and lactation periods (until day 4 of post partum)
Males: dosing of males was continued until the first dams had reached day 4 post partum
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
actual ingested
100, 300, 1000 mg/kg
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in agreement with the Sponsor, based on the results of a preliminary dose range-finding study (RCC Study No. A77242)
- Prior to start of treatment, parental animals were assigned to the different groups using a computer-generated random algorithm. In addition, body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Examinations

Parental animals: Observations and examinations:
MORTALITY:
- All animals were checked at least twice daily for mortality

SIGNS AND/OR SYMPOTMS:
- 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.

FUNCTIONNAL OBSERVATION BATTERY (FOB):
- Time schedule: at one time during the stuy (males: shortly before sacrifice; females: on day 3 or 4 post-partum)
- Cage side observations were included: unusual body movements (tremor, convulsions), abnormal behavior (circling, stereotypy), posture, resistance to removal
- Hand-held observations: palpebral closure, pinna, reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
- 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.
- Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

MORTALITY:
- All animals were checked at least twice daily for mortality

SIGNS AND/OR SYMPOTMS:
- 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.

FUNCTIONNAL OBSERVATION BATTERY (FOB):
- Time schedule: at one time during the stuy (males: shortly before sacrifice; females: on day 3 or 4 post-partum)
- Cage side observations were included: unusual body movements (tremor, convulsions), abnormal behavior (circling, stereotypy), posture, resistance to removal
- Hand-held observations: palpebral closure, pinna, reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
- 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.
- Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

DETAILED CLINICAL OBSERVATIONS:
- Following observations were included: changes in skin, fur eyes, mucous membranes, occurence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern)
-Time schedule: once prior to the first item administration and weekly thereafter

BODY WEIGHT
- The animals were weighed daily during the entire study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 durin the pairing period

MORTALITY:
- All animals were checked at least twice daily for mortality

SIGNS AND/OR SYMPOTMS:
- 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.

FUNCTIONNAL OBSERVATION BATTERY (FOB):
- Time schedule: at one time during the stuy (males: shortly before sacrifice; females: on day 3 or 4 post-partum)
- Cage side observations were included: unusual body movements (tremor, convulsions), abnormal behavior (circling, stereotypy), posture, resistance to removal
- Hand-held observations: palpebral closure, pinna, reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
- 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.
- Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

DETAILED CLINICAL OBSERVATIONS:
- Following observations were included: changes in skin, fur eyes, mucous membranes, occurence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern)
-Time schedule: once prior to the first item administration and weekly thereafter


BODY WEIGHT
- The animals were weighed daily during the entire study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 durin the pairing period

OPHTHALMOSCOPIC EXAMINATION: Not analysed

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Time schedule for collection of blood: early in the morningn on the day before or on the day of scheduled necropsy for malesn abd on day 5 of postpartum for female
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: from all P generation males and females
- Following parameters were examined for hameatology
1/Complete blood cell count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width
Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index, Leukocyte count total, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells) Platelet count
2/ Coagulation: Prothrombin time (=Thromboplastin time), Activated partial thromboplastin time
- Following parameters were examined for clinical chemistry : Glucose, Urea, Creatinine, total Bilirubin, Bile acids, total Cholesterol, ALAT, ASAT, PAL, Gamma-GT, Sodium, Potassium, Chloride, Calcium, Phosphorous, total Protein, albumine, Globulin

URINALYSIS: only discolorations were reported

OTHER: The females were observed twice daily for signs of difficult or prolonged parturition
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]
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.


STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]


GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
Males were sacrificed after the first dams had reached postmortem examination day 4 post partum. Females were sacrificed on day 5 post partum
GROSS PATHOLOGY: The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system

ORGAN WEIGHT
The testes and epididymes of all parental males were weighed.
In addition for five adult males (all groups) and for five females (groups 1 and 2), six females (group 3) and all females (group 4), randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: live, spleen, adrenals, brain, thymus, heart, kidneys

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, if necessary.
For all animals: prostate, testes, seminal vesicles with coagulation gland, epididymides, ovaries were analysed

In addition, of the five males (all groups) and of five females (groups 1 and 2), six females (group 3) and all females (group 4), selected for organ weights, the following tissues were preserved: gross lesions, heart, brain, thymus, spinal cord, thyroid, small and large intestines, trachea and lungs, stomach, uterus (with vagina), liver, urinary bladder, kidneys, lymph nodes (mandibular, mesentericl), adrenals, peripheral nerve, spleen, bone marrow

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.

All gros lesions were examined.
Postmortem examinations (offspring):
SACRIFICE
- Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
For 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; The Steel test (rank test) was applied when the data could not be assumed tofollow a normal distribution, Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
The testes and epididymides of all parental males were weighed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

FOOD CONSUMPTION:
- slight difference (increase or decrease). The food consumption was not the same il all the groups and within all the period (pre-pairing period, pairing period)

BODY WEIGHT:
- statistically decreased only for males in the group 4 (from the day 3 until necropsy), in the group 3 (from day 12 until necropsy) and in the group 2 (from day 5 until necropsy)
- only statistically decreased for females in the groupe 4, during the gestation and lactation periods (not clear in the pre-pairing and pairing periods)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): the emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between controls (group 1) and high-dose (group 4) males.

ORGAN WEIGHTS (PARENTAL ANIMALS):
- Kidney were statistically significantly increased in group 3 and 4; it may be due to an increased metabolism and excretion of test item, and was therefore considered test item-related

HISTOPATHOLOGY:
- No changes in the reproductive organs
- Centrilobular and/or diffuse hepatocellular hypertrophy in groups 3 and 4, considered as being adaptative changes and not adverse effect of the test item.
- Kidney: slight to moderate diffuse tubulare degeneration/regeneration in all 5 males of group 4, minimal to slight multifocal tubular degeneration/regeneration in 3/5 males of group 3 and 5/10 females of group 4. In addition, slightly increased incidence of focal tubular degeneration/regeneration in females of group 4. Slightly increased incidence and severity of hyaline droplets in proximal convoluted tubules in males of groups 3 and 4.

OTHER FINDINGS (PARENTAL ANIMALS):
- Duration of gestation: not influenced by test item treatment
- Corpus lutea count: reduced only at 1000 mg/kg
- Implantation rate : reduced onlyat 1000 mg/kg
- Post-implantation loss: reduced only at 1000 mg/kg
- Litter size at first litter check: reduced only at 1000 mg/kg
- Postnatal loss day 0-4 post-partum: increased at 1000 mg/kg
- Hematology and clinical biochemistry: no effect

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Kidney: multifocal tubular degeneration / regeneration
Dose descriptor:
dose level:
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Increase in kidney/ body weight ratio

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

BODY WEIGHT (OFFSPRING): The mean body weight of pups per group was statistically reduced on day 4 post-partum in groups 3 and 4.

SEX RATIO (OFFSPRING) : not influenced by treatment

NECROPSY FINDINGS: no test item related findings were noted

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: In the living pups at 1000 mg/kg and at 300 mg/kg, body weight gain was decreased until day 4 post-partum

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Fertility performance: the fertility index was 100 %, 100 %, 100 % and 70 % in groups 1, 2, 3, and 4 respectively

Tabular summary report of effects on reproduction/developpemnt

Observations  
Dosage(mg/kgbody weight) 0 100 300 1000
Pairs started (N) 10 10 10 10
Females showing evidence of copulation (N) 10 10 10 10
Females achieving pregnancy (N) 10 10 10 7
Conceiving days1 –5 (N) 9 8 8 9
Conceiving days6 –16* (N) 1 2 2 1
Pregnancy = 21 days (N) 2 3 5 2
Pregnancy = 22 days (N) 7 7 5 5
Pregnancy = 23 days (N) 1 - - -
Dams with live young born (N) 10 10 10 7
Dams with live young at day 4 pp (N) 10 10 10 6
Corpora lutea / dam (mean) 15.2 15.5 15.4 12.4
Implantations / dam (mean) 13.8 15.2 14.4 11.0
Live pups / dam at birth (mean) 10.5 14.4 13.8 9.1
Live pups / dam at day 4 (mean) 10.3 14.0 13.2 7.9
Sex ratio (m/f) at birth (mean) 49 / 51 45 / 55 49 / 51 45 / 55
Sex ratio (m/f) at day 4 (mean) 50 / 50 46 / 54 48 / 52 47 / 53
Litter weight at birth, day 1 (mean) 66.8 84.4 79.0 55.0
Litter weight at day 4 (mean) 87.4 116.9 100.2 71.4
Pup weight at birth, day 1 (mean) 6.3 5.9 5.7 6.1
Pup weight at day 4 (mean) 9.2 8.4 7.6 7.8
ABNORMAL PUPS
Dams with 0 10 10 10 7
LOSS OF OFFSPRING
Pre-implantation(corpora lutea minus implantations)
Females with 0 3 8 4 3
Females with 1 3 1 4 1
Females with 2 2 1 1 2
Females with 3 1 - - -
Females with4 /5 1 - 1 1
Pre-natal(implantations minus live births)
Females with 0 - 3 7 1
Females with 1 4 6 1 1
Females with 2 1 1 1 4
Females with 3 3 - 1 -
Females with 4 - - - 1
Females with 8 / 10 2 - - -
Post-natal(live births minus alive at day 4 pp)
Females with 0 8 6 7 5
Females with 1 2 4 1 1
Females with 2 / 3 - - 2 -
Females with 8 - - - 1

Selected Microscopic Findings for 2,2-Bis(t-butylperoxy isopropyl)benzene in Males:
Groups 1 2 3 4
Dose Levels of 2,2-Bis 0 100 300 1000
(t-butylperoxy isopropyl)benzene mg/kg/day mg/kg/day mg/kg/day mg/kg/day
Liver(nos. examined) 5 5 5 5
- Centr. hep. hypertrophy Grade 1 0 3 1 0
Grade 2 0 0 3 0
Grade 3 0 0 0 3
Mean Severity 0 1.0 1.8 3.0
- Diff. hep. hypertrophy Grade 1 0 0 1 0
Grade 2 0 0 0 2
Mean Severity 0 0 1.0 2.0
Thyroid gland(nos. examined) 5 0 0 5
- Follic.cell hypertrophy Grade 1 4 0 0 0
Grade 2 0 0 0 4
Grade 3 0 0 0 1
Mean Severity 1.0 0 0 2.2
Kidneys(nos. examined) 5 5 5 5
- Multifocal tubular
degener./regeneration Grade 1
0 0 2 0
Grade 2 0 0 1 0
Mean Severity 0 0 1.3 0
- Diffuse tubular
degener./regeneration Grade 2
0 0 0 2
Grade 3 0 0 0 3
Mean Severity 0 0 0 2.6
- Hyaline droplets Grade 1 2 2 0 0
Grade 2 0 1 4 3
Grade 3 0 0 1 2
Mean Severity 1.0 1.3 2.2 2.4

Selected Microscopic Findings for 2,2-Bis(t-butylperoxy isopropyl)benzene in Females:
Groups 1 2 3 4
Dose Levels of 2,2-Bis 0 100 300 1000
(t-butylperoxy isopropyl)benzene mg/kg/day mg/kg/day mg/kg/day mg/kg/day
Liver(nos. examined) 5 5 6 10
- Centr. hep. hypertrophy Grade 1 0 1 2 0
Grade 2 0 0 2 0
Grade 3 0 0 0 5
Mean Severity 0 1.0 1.5 3.0
- Diff. hep. Hypertrophy Grade 1 0 0 2 0
Grade 2 0 0 0 5
Mean Severity 0 0 1.0 2.0
Thyroid gland(nos. examined) 5 0 0 5
- Follic. cell hypertrophy Grade 1 2 0 0 0
Grade 2 0 0 0 1
Grade 3 0 0 0 4
Mean Severity 1,0 0 0 2.8
Kidneys(nos. examined) 5 5 6 10
- Focal tubul. deg./regen. Grade 1 1 2 1 5
Mean Severity 1.0 1.0 1.0 1.0
- Multifocal tubular
degener./regeneration Grade 1
0 0 0 3
Grade 2 0 0 0 2
Mean Severity 0 0 0 1.4
- Hyaline casts Grade 1 0 0 0 1
Grade 2 0 0 0 1
Mean severity 0 0 0 1.5
- Pelvic dilation Grade 1 0 0 0 1
Mean Severity 0 0 0 1.0

Applicant's summary and conclusion

Conclusions:
The NOAEL is 100 mg/kg bw/day based on kidney multifocal tubular degeneration / regeneration in males at 300 mg/kg, increase in kidney/body weight ratio in males and females at 300 mg/kg , and body weight gain decreased in offspring (until day 4 post-partum) at 300 mg/kg
Executive summary:

The potential toxic effects of 2,2-Bis(t-butylperoxy isopropyl) benzene on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition was evaluated in a combined repeated dose and reproduction/developmental screening study according to the OECD Guideline No. 422.

Four groups of Sprague Dawley rats (10 males and 10 females) were treated orally with 2,2-Bis(t-butylperoxy isopropyl)benzene at 0, 100, 300, 1000 mg/kg/d by gavage.

Mortality and clinical signs were checked daily. Body weight and food consumption were recorded at regular intervals. Females were paired with males from the same dose-level group until mating occurred. Gestation was monitored. Females were allowed to deliver normally and to rear their progeny until day 4 post-partum. During the lactation period, the pups were examined daily for survival, external abnormalities and clinical signs. Their body weights were recorded on days 1 and 4 post-partum. At final sacrifice, the pups were examined for gross external abnormalities and then discarded. At final sacrifice of the parent, the testis and epididymides were weighed for the males and a complete macroscopic post-mortem examination was performed for both gender. A microscopic examination was performed on the ovaries, testes and epididymides of females and males, respectively.

At any dose level, no test item-related effect was observed on the mortality rate, the clinical signs and behavior in parental generation, the body weight gain and the food consumption. . All male and female animals in groups 2, 3, and 4 were noted without any test item related finding at necropsy.

The mean body weight of pups per group was statistically significantly reduced on day 4 post

partum in groups 3 and 4.

Mean kidneys weights were statistically significantly increased at 1000 mg/kg for male and female. Kidney / body weight ratio were increased at 300 and 1000 mg/kg. These findings may be due to an increased metabolism and excretion of the test item and were therefore considered test item related.

Treatment at 1000 mg/kg was associated with continuing body weight decrease during the study.

Treatment with 1000 mg/kg was associated with a smaller number of pregnant dams that were noted with less corpora lutea, less implantation sites, less live embryos at first litter check, and a higher postnatal loss. The living pups at 1000 mg/kg and at 300 mg/kg were noted with less body weight gain until day 4 post partum. The litter weight gain at day 4 post partum was reduced.

Moreover treatment at 1000 mg/kg/day was associated in kidneys with a diffuse tubular degeneration / regeneration in all males. A multifocal tubular degeneration / regeneration was observed in some males at 300 mg/kg and some females at 1000 mg/kg. A slightly increased incidence of focal tubular degeneration / regeneration was noted in the females at 1000 mg/kg. A slightly increased incidence and severity of hyaline droplets in proximal convoluted tubules was noted in males at 1000 and 300 mg/kg.

No effects were noted for the parameters on clinical laboratory investigations, or for other macroscopic findings during scheduled necropsy.

Based on these data, it can be concluded that the No Observed Adverse Effect Level (NOAEL) was 100 mg/kg body weight/day (based both on parental and foetal toxicity).