Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents.
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the vehicle: Stability for at least 5 hours at room temperature, 13 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males between 275 and 319 g; females between 187 and 230 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon
plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pre-test period (females) or 6 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 47 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Sept 2017 To: 15 Nov 2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 9 days (Groups 1-3 as a clear solution) or every 1-2 days (Group 4 as an opaque suspension), formulated in daily portions. Formulations were heated to a
maximum temperature of 50±5°C for maximally 10 minutes to obtain visual homogeneity. Formulations were stored in the refrigerator. When formulations were used on the day of preparation, formulations were released for dosing when they had reached a temperature of 40°C or lower. When
dosing formulations were prepared and stored in the refrigerator, formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations and vehicle were continuously stirred until and during dosing.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in Week 1 for concentration anaysis in all groups and for homogeity in groups 2 and 4 (low and high dose groups).
All samples were stored on dry ice immediately after sampling.
Concentration analysis was performed for duplicate sets of samples (approximately 500 mg). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions (Group 1-3) and ± 15% for suspensions (Group 4) of target concentration.
Homogeneity analysis was performed on duplicate sets of samples (approximately 500 mg).
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was # 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Details on mating procedure:
- M/F ratio per cage: 1/1
Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were
separated.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver were treated for 41 days.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 17-day dose range finder with oral administration of 1,3-bis(tert-butylperoxyisopropyl)benzene in rats, and in an attempt to produce graded responses to the test item.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were at least conducted immediately after dosing and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND
1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.

IMMUNOLOGY: No

other: organ weights F0 generation according to Guidelines

Sacrifice and pathology
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations
F0 generation: Estrous cycle determination, cohabitating/mating procedure, general reproduction data, TSH and total T4 measurement
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: No
Fetal examinations:
Post implantation survival index, litter size, live birth index, viability index, lactation index, clinical signs, body weights, sex ratio, AGD, areola/nipple retention, serum T4 levels, macroscopy
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Paremetric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidene: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in about half of the animals treated at 1000 mg/kg bw/day, starting after about one (males) or two (females) weeks of treatment. In females piloerection occurred mostly in the post-coitum period. Salivation seen among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its modest severity (slight or moderate) and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated
to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
The premature death of one female of the 1000 mg/kg bw/day group was considered to be related to treatment. All other animals survived until scheduled sacrifice.
The high dose female was found dead, while in labour, on Day 24 of the post-coitum period. On the day prior to death, she showed piloerection. Her body weight development and food consumption were unremarkable. The main macroscopic finding was the presence of dead fetuses in the uterus (10 in total). Main microscopic findings were moderate centrilobular hepatocellular necrosis in the liver and slight bone marrow atrophy. The hepatocellular necrosis was considered to be the main cause of death. As test item-related hepatocellular necrosis was also noted in a surviving male of the 1000 mg/kg bw/day group, the death of the high dose female was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males treated at 1000 mg/kg bw/day was reduced throughout the treatment period (statistically significant), with slight weight loss in several animals in the last week of this per iod. The resulting reductions in mean body weights were statistically significant at Days 8 and 15 of the mating period (8% difference from controls at the end of treatment). To a lesser extent, reduced weight gain was noted in males treated at 300 mg/kg bw/day (not statistically significant). Mean terminal body weight of 300 mg/kg bw/day males was 5% lower than that of controls.
Body weights and body weight gain of females were considered not to be affected by treatment up to and including 1000 mg/kg bw/day. The statistically significantly higher mean body weight gain noted in 1000 mg/kg bw/day females at Day 7 of the lactation period, mainly due to higher weight gain of one female, was regarded as a chance finding.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw/day consumed less food than controls in treatment weeks 1-2 and, to a lesser extent, weeks 3-4. Their food consumption after correction for body weight was reduced only in weeks 1-2.
Food consumption of females (before and after correction for body weight) was considered not to be affected by treatment. Occasional, slightly higher values noted at 1000 mg/kg bw/day (between Days 11-17 of the post-coitum period and Days 1-4 of the lactation period) were regarded as unrelated to treatment as mean food consumption of 1000 mg/kg bw/day females remained close to that of controls at the other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters showed the following statistically significant differences between treated rats and controls (percentage differences from the control group mean are indicated between parentheses):
- Lower haemoglobin concentration in males at 1000 mg/kg bw/day (9%).
- Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg bw/day (5 and 6%, respectively).
There were no treatment-related changes in white blood cell parameters or number of platelets. There were no test item-related differences in coagulation parameters. The lower mean activated partial
thromboplastin time (APTT) values noted at 1000 mg/kg bw/day (statistically significant in males only) were considered to have arisen as a result of slightly high concurrent control values and, therefore, regarded as unrelated to treatment. Mean APTT values at 1000 mg/kg bw/day remained in the normal ranges.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameter, mostly in 1000 mg/kg bw/day males, distinguished treated animals from control animals (percentage differences from the control group mean are indicated between parentheses). Mean values for these parameters in the affected group(s) of males were (slightly) out of the normal ranges.
- Higher alanine aminotransferase (ALAT) in males at 1000 mg/kg bw/day (48%).
- Higher total protein in males at 300 and 1000 mg/kg bw/day (5 and 10%, respectively).
- Higher albumin in males at 1000 mg/kg bw/day (10%).
- Higher creatinine in males at 1000 mg/kg bw/day (23%).
- Higher potassium in males at 1000 mg/kg bw/day (14%). Potassium was also higher (10%) in males at 300 mg/kg bw/day but the difference from controls was not statistically significant.
- Lower cholesterol in males at 1000 mg/kg bw/day (22%).
The other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response, slightly high control value and/or a high value in a single animal.
Thyroid hormone analyses:
Mean serum T4 levels were statistically significantly decreased in F0 males at 300 and 1000 mg/kg bw/day (relative differences from controls: 32 and 51%, respectively), whereas mean serum TSH levels in these males were increased (4.3 and 9.7-fold, respectively; statistically significant at 1000 mg/kg bw/day only). The mean values in these groups of males were out of the historical control ranges.
Serum levels of T4 and TSH in F0 females were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females of the 1000 mg/kg bw/day group had lower mean numbers of total movements and ambulations compared to those of the control group (nearly 60% difference, statistically significant for total movements only). Motor activity was particularly low in 3 females which had values below the historical control ranges (2). The other two 1000 mg/kg bw/day females tested had activity values at or just below the concurrent control group ranges.
Activity habituation profiles were similar between the treated and control groups with a decreasing trend in activity over the duration of the test period Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
(2) Historical control data for motor activity in female Wistar Han rats (period 2015-June 2017):
Total movements: mean = 3502, P5 - P95 = 1478 - 5683 (n=195).
Ambulations: mean = 855, P5 - P95 = 301 - 1467 (n=195).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the weights of the thymus in males and of the thyroid gland, liver and kidneys in both sexes, as described below. Relative differences from control values and statistical significances are shown in the Text Table 1 (see any other information).
- Thymus (absolute and relative to body weight): lower (not statistically significant) in males at 300 and 1000 mg/kg bw/day.
- Thyroid gland (absolute and relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Liver (absolute and relative to body weight): higher (statistically significant) in males at 100, 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Kidneys (relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
There were no other test item-related organ weight changes. A few statistically significant organ weight differences noted at 1000 mg/kg bw/day were regarded as secondary to differences in terminal body weight rather than as primary effects of the test item (lower absolute and relative prostate gland weights, higher relative brain weight in females).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings consisted of:
- Liver: Enlarged at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (9/10 males, 2/10 females).
- Liver: Black-brown discoloration at 300 mg/kg bw/day (2/10 males) and 1000 mg/kg bw/day (10/10 males, 3/10 females).
- Thyroid gland: Enlarged at 1000 mg/kg bw/day (3/10 males).
- General: Emaciated appearance at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (5/10 females). However, as terminal body weights were within normal limits, this finding was not considered toxicologically significant.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were herefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thymus of males and the thyroid (see also 'any other information').
Kidneys:
- Tubular degeneration was present in males and females at 1000 mg/kg bw/day up to moderate degree.
- Tubular basophilia was present at increased incidence and/or severity in males starting at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree.
- Hyaline droplet accumulation was present at increased severity in males starting at 300 mg/kg bw/ day up to moderate degree.
- Tubular mineralization was present at increased incidence and/or severity in females starting at 300 mg/kg bw/day up to moderate degree.
Liver:
- Hepatocellular hypertrophy was present in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Pigment deposition was present in males at 300 and 1000 mg/kg bw/day up to slight degree. This sometimes correlated with the macroscopic black-brown discoloration.
- Hepatocellular necrosis was present in a single male at 1000 mg/kg bw/day at slight degree.
Thyroid gland:
- Follicular cell hypertrophy was present at increased incidence and/or severity in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day up to moderate degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Colloid alteration was present in males and females at 1000 mg/kg bw/day up to minimal degree.
Thymus:
- Lymphoid atrophy was present in males at 1000 mg/kg bw/day at minimal degree. This correlated with the decreased organ weight.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the other pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment. The survival indices were 81, 94, 88 and 81% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
It was noted that the post-implantation survival indices for the control and 1000 mg/kg bw/day groups were lower than normal. Two control females had more unaccounted for sites (i.e. difference between number of implantation sites and total number of pups born) than observed normally (2 females with 6 and 7 unaccounted for sites, respectively). The low post-implantation survival index for the 1000 mg/kg bw/day group was due to one female which died while in labour. In her uterus 13 implantation sites, 10 dead fetuses and 3 early resorptions were noted. As the death of that females was related to liver toxicity and the other 1000 mg/kg bw/day females had zero (4 females) or only 1-2 (4 females) unaccounted for sites, postimplantation survival at 1000 mg/kg bw/day was judged to be unaffected by treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In the uterus of one high dose female 3 early resorptions were noted, which is considered not relatd to treatment, and is within normal limits.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99-100%. The numbers of dead pups at first litter check in the control and 100 mg/kg bw/day groups were within normal limits (one dead pup in two liters) and the incidental pup mortality in the lowest dose group (100 mg/kg bw/day) was regarded as unrelated to treatment.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Fertility index was considered not to be affected by treatment.
Except for one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day, all mated females were pregnant. The fertility indices were 100% for the control and 100 mg/kg bw/day groups and 90% for the 300 and 1000 mg/kg bw/day groups.
The non-pregnancy of one female was due to infertility of the male she was paired with (see histopathology changes in the testes and epididymides of this male). The non-pregnancy of another female was not associated with related histopathology changes in reproductive organs. These incidental cases of male infertility/non-pregnancy were regarded as unrelated to treatment as the incidence showed no dose-related trend and was within normal limits.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, treatment related lower body weights of pups were noted on PND 13. This was statistically significant for male pups, female pups and sexes combined, and resulted in a 20% lower mean body weight when compared to concurrent controls.
The remaining statistical significant changes (on PND 7 at 100 and 1000 mg/kg bw/day and on PND 13 at 100 and 300 mg/kg bw/day) were not considered toxicologically relevant as no dose response was apparent, all values were within normal limits7 of which the control group values were at the higher end.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99-100%. The numbers of dead pups at first litter check in the control and 100 mg/kg bw/day groups were within normal limits (one dead pup in two litters nos. 47 and 51) and the incidental pup mortality in the lowest dose group (100 mg/kg bw/day) was regarded as unrelated to treatment.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 300 mg/kg bw/day group and 99% for the other groups. One pup of the control group died spontaneously at PND 1, one pup of the 100 mg/kg bw/day group was euthanized for humane reasons at PND 2 and one pup of the 1000 mg/kg bw/day group went missing (presumably cannibalized) at PND 2. This incidental pup mortality was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
see reduction in number of live offspring
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 14-16 pups were judged to be unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 96.2% and 98.7%). No test item was detected in the Group

1 formulation.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 2.8% and 2.1% in the low and high dose goups, respectively).

Text Table 1 Mean Percent Organ Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

              Absolute

5

-27

-41

-

-

-

              Relative to body weight

3

-19

-35

-

-

-

 

 

 

 

 

 

 

THYROID GLAND

 

 

 

 

 

 

              Absolute

13

38**

38**

6

13

19*

              Relative to body weight

0

40**

40**

0

17

17**

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

              Absolute

28*

26*

63**

-2

15

25**

              Relative to body weight

25**

38**

81**

0

11

39**

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

              Absolute

12

16

16

2

4

8

              Relative to body weight

9

27**

28**

3

0

19**

*: P<0.05, **: P<0.01. - no remarkable findings

Text Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Tubular degeneration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

-

-

-

2/5

        Slight

-

-

-

1/5

-

-

-

-

        Moderate

-

-

-

2/5

-

-

-

-

    Tubular basophilia

 

 

 

 

 

 

 

 

        Minimal

2/5

3/5

2/5

-

-

1/5

-

2/5

        Slight

-

-

2/5

3/5

-

-

-

1/5

a = Number of tissues examined from each group.

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Hyaline droplet accumulation

 

 

 

 

 

 

 

 

        Minimal

3/5

3/5

-

-

-

-

-

-

        Slight

2/5

1/5

1/5

3/5

-

-

-

-

        Moderate

-

1/5

4/5

2/5

-

-

-

-

    Tubular mineralization

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

2/5

3/5

2/5

-

        Slight

-

-

-

-

-

-

1/5

2/5

        Moderate

-

-

-

-

-

-

1/5

1/5

LIVERa

5

5

7

10

5

5

5

5

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

3/5

4/7

1/10

-

-

-

2/5

        Slight

-

1/5

1/7

8/10

-

-

-

3/5

    Pigment deposition

 

 

 

 

 

 

 

 

        Minimal

-

-

1/7

2/10

-

-

-

-

        Slight

-

-

1/7

-

-

-

-

-

    Hepatocellular necrosis

 

 

 

 

 

 

 

 

        Slight

-

-

-

1/10

-

-

-

-

THYROID GLANDa

5

5

5

6

5

5

5

5

    Follicular cell hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

1/5

2/5

1/6

3/5

3/5

1/5

-

        Slight

-

3/5

3/5

5/6

1/5

1/5

4/5

2/5

        Moderate

-

-

-

-

-

-

-

3/5

    Colloid alteration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

1/6

-

-

-

4/5

THYMUSa

5

5

6

5

5

1

-

5

    Lymphoid atrophy

 

 

 

 

 

 

 

 

        Minimal

-

-

-

3/5

-

-

-

-

a = Number of tissues examined from each group.

Conclusions:
The NOAEL for developmental toxicity is 300 mg/kg bw/day, based on reduced body weight gain of male and female pups at 1000 mg/kg bw/day.
Executive summary:

In an OECD 422 study, Wistar Han rats were treated with 1,3-bis(tert-butylperoxyisopropyl)benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14 or16 days of lactation (for 50-56 days). Females without offspring were treated for 41 days (two non-pregnant females). Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Developmental results

Developmental toxicity occurred at 1000 mg/kg bw/day and was characterized by reduced body weights gain of male and female pups. This resulted in about 20% lower mean body weights at PND 13. Birth weight of 1000 mg/kg bw/day pups was not affected by treatment. No treatment-related changes were noted in the other developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, anogenital distance, areola/nipple retention, serum T4 thyroid hormone levels and macroscopic examination).

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAELs) of 1,3-bis(tert-butylperoxyisopropyl)benzene was established: Developmental NOAEL: 300 mg/kg bw/day, based on reduced body weight gain of male and female pups at 1000 mg/kg bw/day.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the vehicle: Stability for at least 5 hours at room temperature, 13 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males between 275 and 319 g; females between 187 and 230 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon
plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pre-test period (females) or 6 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 47 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Sept 2017 To: 15 Nov 2017
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 9 days (Groups 1-3 as a clear solution) or every 1-2 days (Group 4 as an opaque suspension), formulated in daily portions. Formulations were heated to a
maximum temperature of 50±5°C for maximally 10 minutes to obtain visual homogeneity. Formulations were stored in the refrigerator. When formulations were used on the day of preparation, formulations were released for dosing when they had reached a temperature of 40°C or lower. When dosing formulations were prepared and stored in the refrigerator, formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations and vehicle were continuously stirred until and during dosing.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in Week 1 for concentration anaysis in all groups and for homogeity in groups 2 and 4 (low and high dose groups).
All samples were stored on dry ice immediately after sampling.
Concentration analysis was performed for duplicate sets of samples (approximately 500 mg). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions (Group 1-3) and ± 15% for suspensions (Group 4) of target concentration.
Homogeneity analysis was performed on duplicate sets of samples (approximately 500 mg). Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41 days.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 17-day dose range finder with oral administration of 1,3-bis(tert-butylperoxyisopropyl)benzene in rats, and in an attempt to produce graded responses to the test item.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were at least conducted immediately after dosing and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.

IMMUNOLOGY: No

other: organ weights F0 generation according to Guidelines
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to Guidelines

HISTOPATHOLOGY: Yes, according to Guidelines
Other examinations:
F0 generation: Estrous cycle determination, cohabitating/mating procedure, general reproduction data, TSH and total T4 measurement
F1 generation: mortality, clinical observations, BW, sex, AGD, areola/nipple retention, total T4 measurement

Reproduction and Developmental parameters will be discussed in sections 7.8.1 and 7.8.2.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Paremetric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidene: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was observed in about half of the animals treated at 1000 mg/kg bw/day, starting after about one (males) or two (females) weeks of treatment. In females piloerection occurred mostly in the post-coitum period. Salivation seen among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its modest severity (slight or moderate) and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
The premature death of one female of the 1000 mg/kg bw/day group was considered to be related to treatment. All other animals survived until scheduled sacrifice.
The high dose female was found dead, while in labour, on Day 24 of the post-coitum period. On the day prior to death, she showed piloerection. Her body weight development and food consumption were unremarkable. The main macroscopic finding was the presence of dead fetuses in the uterus (10 in total). Main microscopic findings were moderate centrilobular hepatocellular necrosis in the liver and slight bone marrow atrophy. The hepatocellular necrosis was considered to be the main cause of death. As test item-related hepatocellular necrosis was also noted in a surviving male of the 1000 mg/kg bw/day group, the death of the high dose female was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males treated at 1000 mg/kg bw/day was reduced throughout the treatment period (statistically significant), with slight weight loss in several animals in the last week of this period. The resulting reductions in mean body weights were statistically significant at Days 8 and 15 of the mating period (8% difference from controls at the end of treatment). To a lesser extent, reduced weight gain was noted in males treated at 300 mg/kg bw/day (not statistically significant). Mean terminal body weight of 300 mg/kg bw/day males was 5% lower than that of controls.
Body weights and body weight gain of females were considered not to be affected by treatment up to and including 1000 mg/kg bw/day. The statistically significantly higher mean body weight gain noted in 1000 mg/kg bw/day females at Day 7 of the lactation period, mainly due to higher weight gain of one female, was regarded as a chance finding.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw/day consumed less food than controls in treatment weeks 1-2 and, to a lesser extent, weeks 3-4. Their food consumption after correction for body weight was reduced only in weeks 1-2.
Food consumption of females (before and after correction for body weight) was considered not to be affected by treatment. Occasional, slightly higher values noted at 1000 mg/kg bw/day (between Days 11-17 of the post-coitum period and Days 1-4 of the lactation period) were regarded as unrelated to treatment as mean food consumption of 1000 mg/kg bw/day females remained close to that of controls at the other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters showed the following statistically significant differences between treated rats and controls (percentage differences from the control group mean are indicated between parentheses):
- Lower haemoglobin concentration in males at 1000 mg/kg bw/day (9%).
- Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg bw/day (5 and 6%, respectively).
There were no treatment-related changes in white blood cell parameters or number of platelets. There were no test item-related differences in coagulation parameters. The lower mean activated partial thromboplastin time (APTT) values noted at 1000 mg/kg bw/day (statistically significant in males only) were considered to have arisen as a result of slightly high concurrent control values and, therefore, regarded as unrelated to treatment. Mean APTT values at 1000 mg/kg bw/day remained in the normal ranges.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameter, mostly in 1000 mg/kg bw/day males, distinguished treated animals from control animals (percentage differences from the control group mean are indicated between parentheses). Mean values for these parameters in the affected group(s) of males were (slightly) out of the normal ranges.
- Higher alanine aminotransferase (ALAT) in males at 1000 mg/kg bw/day (48%).
- Higher total protein in males at 300 and 1000 mg/kg bw/day (5 and 10%, respectively).
- Higher albumin in males at 1000 mg/kg bw/day (10%).
- Higher creatinine in males at 1000 mg/kg bw/day (23%).
- Higher potassium in males at 1000 mg/kg bw/day (14%). Potassium was also higher (10%) in males at 300 mg/kg bw/day but the difference from controls was not statistically significant.
- Lower cholesterol in males at 1000 mg/kg bw/day (22%).
The other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response, slightly high control value and/or a high value in a single animal.

Thyroid hormone analyses:
Mean serum T4 levels were statistically significantly decreased in F0 males at 300 and 1000 mg/kg bw/day (relative differences from controls: 32 and 51%, respectively), whereas mean serum TSH levels in these males were increased (4.3 and 9.7-fold, respectively; statistically significant at 1000 mg/kg bw/day only). The mean values in these groups of males were out of the historical control ranges.
Serum levels of T4 and TSH in F0 females were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females of the 1000 mg/kg bw/day group had lower mean numbers of total movements and ambulations compared to those of the control group (nearly 60% difference, statistically significant for total movements only). Motor activity was particularly low in 3 females which had values below the historical control ranges (2). The other two 1000 mg/kg bw/day females tested had activity values at or just below the concurrent control group ranges.
Activity habituation profiles were similar between the treated and control groups with a decreasing trend in activity over the duration of the test period Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.

(2) Historical control data for motor activity in female Wistar Han rats (period 2015-June 2017):
Total movements: mean = 3502, P5 - P95 = 1478 - 5683 (n=195).
Ambulations: mean = 855, P5 - P95 = 301 - 1467 (n=195).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the weights of the thymus in males and of the thyroid gland, liver and kidneys in both sexes, as described below. Relative differences from control values and statistical significances are shown in the Text Table 1 (see any other information).
- Thymus (absolute and relative to body weight): lower (not statistically significant) in males at 300 and 1000 mg/kg bw/day.
- Thyroid gland (absolute and relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Liver (absolute and relative to body weight): higher (statistically significant) in males at 100, 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.
- Kidneys (relative to body weight): higher (statistically significant) in males at 300 and 1000 mg/kg bw/day and females at 1000 mg/kg bw/day.

There were no other test item-related organ weight changes. A few statistically significant organ weight differences noted at 1000 mg/kg bw/day were regarded as secondary to differences in terminal body weight rather than as primary effects of the test item (lower absolute and relative prostate gland weights, higher relative brain weight in females).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings consisted of:
- Liver: Enlarged at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (9/10 males, 2/10 females).
- Liver: Black-brown discoloration at 300 mg/kg bw/day (2/10 males) and 1000 mg/kg bw/day (10/10 males, 3/10 females).
- Thyroid gland: Enlarged at 1000 mg/kg bw/day (3/10 males).
- General: Emaciated appearance at 300 mg/kg bw/day (1/10 females) and 1000 mg/kg bw/day (5/10 females). However, as terminal body weights were within normal limits, this finding was not considered toxicologically significant.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were herefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thymus of males and the thyroid (see also 'any other information').
Kidneys:
- Tubular degeneration was present in males and females at 1000 mg/kg bw/day up to moderate degree.
- Tubular basophilia was present at increased incidence and/or severity in males starting at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree.
- Hyaline droplet accumulation was present at increased severity in males starting at 300 mg/kg bw/day up to moderate degree.
- Tubular mineralization was present at increased incidence and/or severity in females starting at 300 mg/kg bw/day up to moderate degree.
Liver:
- Hepatocellular hypertrophy was present in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Pigment deposition was present in males at 300 and 1000 mg/kg bw/day up to slight degree. This sometimes correlated with the macroscopic black-brown discoloration.
- Hepatocellular necrosis was present in a single male at 1000 mg/kg bw/day at slight degree. One female at 1000 mg/kg bw/day died prematurely, while in labour, most likely due to moderate centrilobular hepatocellular necrosis.
Thyroid gland:
- Follicular cell hypertrophy was present at increased incidence and/or severity in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day up to moderate degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Colloid alteration was present in males and females at 1000 mg/kg bw/day up to minimal degree.
Thymus:
- Lymphoid atrophy was present in males at 1000 mg/kg bw/day at minimal degree. This correlated with the decreased organ weight.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 96.2% and 98.7%). No test item was detected in the Group 1 formulation.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 2.8% and 2.1% in the low and high dose goups, respectively).

 

Text Table 1 Mean Percent Organ Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

              Absolute

5

-27

-41

-

-

-

              Relative to body weight

3

-19

-35

-

-

-

 

 

 

 

 

 

 

THYROID GLAND

 

 

 

 

 

 

              Absolute

13

38**

38**

6

13

19*

              Relative to body weight

0

40**

40**

0

17

17**

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

              Absolute

28*

26*

63**

-2

15

25**

              Relative to body weight

25**

38**

81**

0

11

39**

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

              Absolute

12

16

16

2

4

8

              Relative to body weight

9

27**

28**

3

0

19**

*: P<0.05, **: P<0.01. - no remarkable findings

Text Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Tubular degeneration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

-

-

-

2/5

        Slight

-

-

-

1/5

-

-

-

-

        Moderate

-

-

-

2/5

-

-

-

-

    Tubular basophilia

 

 

 

 

 

 

 

 

        Minimal

2/5

3/5

2/5

-

-

1/5

-

2/5

        Slight

-

-

2/5

3/5

-

-

-

1/5

a = Number of tissues examined from each group.

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Hyaline droplet accumulation

 

 

 

 

 

 

 

 

        Minimal

3/5

3/5

-

-

-

-

-

-

        Slight

2/5

1/5

1/5

3/5

-

-

-

-

        Moderate

-

1/5

4/5

2/5

-

-

-

-

    Tubular mineralization

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

2/5

3/5

2/5

-

        Slight

-

-

-

-

-

-

1/5

2/5

        Moderate

-

-

-

-

-

-

1/5

1/5

LIVERa

5

5

7

10

5

5

5

5

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

3/5

4/7

1/10

-

-

-

2/5

        Slight

-

1/5

1/7

8/10

-

-

-

3/5

    Pigment deposition

 

 

 

 

 

 

 

 

        Minimal

-

-

1/7

2/10

-

-

-

-

        Slight

-

-

1/7

-

-

-

-

-

    Hepatocellular necrosis

 

 

 

 

 

 

 

 

        Slight

-

-

-

1/10

-

-

-

-

THYROID GLANDa

5

5

5

6

5

5

5

5

    Follicular cell hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

1/5

2/5

1/6

3/5

3/5

1/5

-

        Slight

-

3/5

3/5

5/6

1/5

1/5

4/5

2/5

        Moderate

-

-

-

-

-

-

-

3/5

    Colloid alteration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

1/6

-

-

-

4/5

THYMUSa

5

5

6

5

5

1

-

5

    Lymphoid atrophy

 

 

 

 

 

 

 

 

        Minimal

-

-

-

3/5

-

-

-

-

a = Number of tissues examined from each group.

Conclusions:
Based on effects observed on the liver (i.e. hepatocellular necrosis and substantial liver enlargement) and kidneys (i.e. tubular degeneration), a NOAEL of 300 mg/kg bw/day for parental toxicity was derived for both sexes.
Executive summary:

In an OECD 422 study, Wistar Han rats were treated with 1,3-bis(tert-butylperoxyisopropyl)benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent

controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14 or16 days of lactation (for 50-56 days). Females without offspring were treated for 41 days (two non-pregnant females).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Parental results

Exposure to the test item resulted in adverse changes in the liver and kidneys at 1000 mg/kg bw/day and a variety of non-adverse changes, starting at 100 mg/kg bw/day, as described below.

One female at 1000 mg/kg bw/day died prematurely, while in labour, most likely due to test itemrelated hepatotoxicity (i.e. moderate centrilobular hepatocellular necrosis). Several males and females treated at 1000 mg/kg bw/day showed piloerection after about one or two weeks of treatment. As the piloerection was not observed continuously and occurred in absence of other clinical signs of toxicity, it was considered not to reflect general ill health. Slight salivation observed after dosing among treated animals (at all dose levels) was considered a physiological response rather than a sign of systemic toxicity.

Male rats treated at 1000 mg/kg bw/day showed reduced body weight gain throughout treatment, accompanied by reduced food consumption mainly in the first two weeks, with slight weight loss in several animals in the last treatment week. To a lesser extent, reduced weight gain also occurred in males at 300 mg/kg bw/day (without a decrease in food consumption). As the resulting decreases in mean body weights were modest (less than 10% at the end of the treatment period), the effect on body weight (gain) was regarded as non-adverse within the context of this study.

Functional observation tests showed reduced motor activity in all females at 1000 mg/kg bw/day. For 3/5 females the numbers of total movements and ambulations were below the historical control ranges. There were no corroborative changes in other functional measures in the neuromuscular domain (including grip strength, gait and air righting reflex), supportive morphological correlates in examined neuronal tissues, or related clinical signs. Therefore, the lower motor activity in 1000 mg/kg bw/day females was considered not to reflect impaired neuromuscular function and was regarded as non-adverse within the context of this study.

Microscopic examination revealed test item-related changes in several organs as described below.

At 1000 mg/kg bw/day, test item-related renal toxicity was indicated by the presence of tubular degeneration in the kidneys of several males (1/5 slight, 2/5 moderate) and females (2/5 minimal). The increased plasma level of creatinine in males was likely related to the renal toxicity. Additional microscopic renal findings consisted of increases in the incidence and/or severity of tubular basophilia at 300 mg/kg bw/day in males (2/5 minimal, 2/5 slight) and 1000 mg/kg bw/day in males (3/5 slight) and females (2/5 minimal, 1/5 slight), hyaline droplet accumulation in males at 300 mg/kg bw/day (1/5 slight, 4/5 moderate) and at 1000 mg/kg bw/day (3/5 slight, 2/5 moderate), and tubular mineralization in females at 300 mg/kg bw/day (2/5 minimal, 1/5 slight, 1/5 moderate) increases in relative renal weights at 300 mg/kg bw/day in males and at 1000 mg/kg bw/day in both sexes.

The renal findings at 300 mg/kg bw/day were regarded as non-adverse since they were not associated with degenerative changes.

Hepatocellular necrosis was observed in the liver of one 1000 mg/kg bw/day male (at slight degree) and one 1000 mg/kg bw/day female (at moderate degree; this female died prematurely). This necrosis was considered an adverse change. Additional microscopic changes in the liver consisted of centrilobular hepatocellular hypertrophy in males at 100 mg/kg bw/day (3/5 minimal, 1/5 slight), at 300 mg/kg bw/day (4/7 minimal, 1/7 slight), at 1000 mg/kg bw/day (1/10 minimal, 8/10 slight) and in females at 1000 mg/kg bw/day (2/5 minimal, 3/5 slight), and pigment deposition in the liver in males at 300 mg/kg bw/day (1/7 minimal, 1/7 slight) and at 1000 mg/kg bw/day (2/10 minimal). The hepatocellular hypertrophy correlated with enlargement of the liver and the pigment deposition sometimes correlated with macroscopic black-brown discoloration. Some changes in clinical biochemistry parameters noted in 1000 mg/kg bw/day males may be related to the effect on the liver (higher alanine aminotransferase (ALAT) activity and lower cholesterol). The hepatocellular hypertrophy at 1000 mg/kg bw/day was associated with a degenerative change (hepatocellular necrosis) and therefore considered part of a group of effects indicating adversity. Furthermore, the magnitude of the liver enlargement at 1000 mg/kg bw/day (males had about 80% higher relative liver weights) was considered to exceed thresholds for adversity. The hepatic changes observed at 100 and 300 mg/kg bw/day were considered to be non-adverse as they occurred in the absence of any degenerative or inflammatory changes.

Microscopic changes in the thyroid gland included an increased incidence and/or severity of follicular cell hypertrophy in males at 100 mg/kg bw/day (1/5 minimal, 3/5 slight), at 300 mg/kg (2/5 minimal, 3/5 slight), at 1000 mg/kg (1/6 minimal, 5/6 slight) and in females at 300 mg/kg (1/5 minimal, 4/5 slight) and at 1000 mg/kg (2/5 slight, 3/5 moderate), and the presence of colloid alteration at 1000 mg/kg w/day in males (1/6 minimal) and females (4/5 minimal). This was associated with higher thyroid weights at 300 in males and 1000 mg/kg bw/day in both sexes. As these findings occurred in the absence of any degenerative change in the thyroid they were regarded as non-adverse. While increased follicular cell hypertrophy occurred in both sexes, serum levels of thyroid hormones were affected only in males. Starting at 300 mg/kg bw/day, serum levels of T4 were decreased dose-dependently (about 50% at 1000 mg/kg bw/day) and TSH levels were increased (on average nearly 10-fold at 1000 mg/kg bw/day). A decrease in T4, with increases in TSH and thyroid follicular cell hypertrophy and growth as compensatory responses, may be due to increased T4 turnover resulting from metabolic enzyme induction in the liver (hepatocellular hypertrophy).

Lymphoid atrophy in the thymus was observed in males at 1000 mg/kg bw/day (3/5 minimal). Based on its minimal severity, the lymphoid atrophy was regarded as non-adverse. It correlated with a decrease (of about 40%) in the weight of the thymus. The smaller decrease in thymus weight noted in 300 mg/kg bw/day males was not associated with microscopic changes, and therefore not regarded adverse.

Clinical chemistry results showed changes in some liver- or kidney-related parameters in 1000 mg/kg bw/day males (see above). Additional changes, all in males, consisted of higher plasma levels of total protein and potassium starting at 300 mg/kg bw/day and higher albumin at 1000 mg/kg bw/day.

Although these changes could not be related to adverse anatomic pathology findings, they were considered to be toxicologically relevant based on their magnitude (mean values in treated males exceeded historical control ranges).

Haematology showed a few changes in red blood cell parameters at 1000 mg/kg bw/day: lower haemoglobin concentration in males, and lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females. As these changes were slight (less than 10% difference from control values) they would not impact tissue oxygenation and were, therefore, regarded as non-adverse.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAELs) of 1,3-bis(tert-butylperoxyisopropyl)benzene were established:

Parental NOAEL: 300 mg/kg bw/day, based on adverse effects on the liver (i.e. hepatocellular necrosis and substantial liver enlargement) and kidneys (i.e. tubular degeneration) at 1000 mg/kg bw/day in both sexes.

Note: In this study, significant treatment-related changes in serum levels of total T4 (decreased) and TSH (increased) were observed at 300 and 1000 mg/kg bw/day (in males only). However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perkadox 14S-FL
- Molecular formula (if other than submission substance): C20H34O4
- Molecular weight (if other than submission substance): 338.49
- Smiles notation (if other than submission substance): CC(c1ccc(cc1)C(OOC(C)(C)C)(C)C)(OOC(C)(C)C)C
- Substance type: Organic peroxide
- Physical state: Solid
- Analytical purity: 99,7 - 99,9%
- Lot/batch No.: 5998
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: At room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the vehicle: Stability for at least 5 hours at room temperature, 13 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 to 200 mg/mL

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old; females were 12-14 weeks old
- Weight at study initiation: males between 275 and 319 g; females between 187 and 230 g
- Fasting period before study: no
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. The cages contained appropriate bedding. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: 6 days prior to start of the pre-test period (females) or 6 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analysed by the supplier for nutritional components and environmental contaminants.It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.
Periodic analysis of the water is performed.It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 47 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Sept 2017 To: 15 Nov 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 9 days (Groups 1-3 as a clear solution) or every 1-2 days (Group 4 as an opaque suspension), formulated in daily portions. Formulations were heated to a
maximum temperature of 50±5°C for maximally 10 minutes to obtain visual homogeneity. Formulations were stored in the refrigerator. When formulations were used on the day of preparation, formulations were released for dosing when they had reached a temperature of 40°C or lower. When
dosing formulations were prepared and stored in the refrigerator, formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. The dosing formulations and vehicle were continuously stirred until and during dosing.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 post-coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in Week 1 for concentration anaysis in all groups and for homogeity in groups 2 and 4 (low and high dose groups).
All samples were stored on dry ice immediately after sampling.
Concentration analysis was performed for duplicate sets of samples (approximately 500 mg). Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions (Group 1-3) and ± 15% for suspensions (Group 4) of target concentration.
Homogeneity analysis was performed on duplicate sets of samples (approximately 500 mg).
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was # 10%.
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14 or 16 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver were treated for 41 days.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 17-day dose range finder with oral administration of 1,3-bis(tert-butylperoxyisopropyl)benzene in rats, and in an attempt to produce graded responses to the test item.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were at least conducted immediately after dosing and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: all
- Parameters checked in accordance with Guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13).
- Dose groups that were examined: all
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.

IMMUNOLOGY: No

other: organ weights F0 generation according to Guidelines
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for one female that had died spontaneously.
Sperm parameters (parental animals):
Parameters examined in all P male parental generations:
testis weight, epididymis weight, prostate gland weight, seminal vesicles weight.

For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males
and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, plasma T4 level

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Following completion of the mating period (a minimum
of 28 days of administration).
- Maternal animals: - Females which delivered: PND 14 or16; - Females which failed to deliver: With evidence of mating: Post-coitum Day 25-26.

GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guidelines were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

GROSS NECROPSY
- performed according to Guidelines
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Paremetric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidene: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
about one (males) or two (females) weeks of treatment. In females piloerection occurred mostly in the post-coitum period. Salivation seen among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its modest severity (slight or moderate) and the time of occurrence (i.e. after dosing).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
The premature death of one female of the 1000 mg/kg bw/day group was considered to be related to treatment. All other animals survived until scheduled sacrifice.
The high dose female was found dead, while in labour, on Day 24 of the post-coitum period. On the day prior to death, she showed piloerection. Her body weight development and food consumption were unremarkable. The main macroscopic finding was the presence of dead fetuses in the uterus (10 in total). Main microscopic findings were moderate centrilobular hepatocellular necrosis in the liver and slight bone marrow atrophy. The hepatocellular necrosis was considered to be the main cause of death. As test item-related hepatocellular necrosis was also noted in a surviving male of the 1000 mg/kg bw/day group, the death of the high dose female was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males treated at 1000 mg/kg bw/day was reduced throughout the treatment period (statistically significant), with slight weight loss in several animals in the last week of this period. The resulting reductions in mean body weights were statistically significant at Days 8 and 15 of the mating period (8% difference from controls at the end of treatment). To a lesser extent, reduced weight gain was noted in males treated at 300 mg/kg bw/day (not statistically significant). Mean terminal body weight of 300 mg/kg bw/day males was 5% lower than that of controls.
Body weights and body weight gain of females were considered not to be affected by treatment up to and including 1000 mg/kg bw/day. The statistically significantly higher mean body weight gain noted in 1000 mg/kg bw/day females at Day 7 of the lactation period, mainly due to higher weight gain of one female, was regarded as a chance finding.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males at 1000 mg/kg bw/day consumed less food than controls in treatment weeks 1-2 and, to a lesser extent, weeks 3-4. Their food consumption after correction for body weight was reduced only in weeks 1-2.
Food consumption of females (before and after correction for body weight) was considered not to be affected by treatment. Occasional, slightly higher values noted at 1000 mg/kg bw/day (between Days 11-17 of the post-coitum period and Days 1-4 of the lactation period) were regarded as unrelated to treatment as mean food consumption of 1000 mg/kg bw/day females remained close to that of controls at the other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell parameters showed the following statistically significant differences between treated rats and controls (percentage differences from the control group mean are indicated between parentheses):
- Lower haemoglobin concentration in males at 1000 mg/kg bw/day (9%).
- Lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 1000 mg/kg bw/day (5 and 6%, respectively).
There were no treatment-related changes in white blood cell parameters or number of platelets. There were no test item-related differences in coagulation parameters. The lower mean activated partial thromboplastin time (APTT) values noted at 1000 mg/kg bw/day (statistically significant in males only) were considered to have arisen as a result of slightly high concurrent control values and, therefore, regarded as unrelated to treatment. Mean APTT values at 1000 mg/kg bw/day remained in the normal ranges.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in clinical chemistry parameter, mostly in 1000 mg/kg bw/day males, distinguished treated animals from control animals (percentage differences from the control group mean are indicated between parentheses). Mean values for these parameters in the affected group(s) of males were (slightly) out of the normal ranges.
- Higher alanine aminotransferase (ALAT) in males at 1000 mg/kg bw/day (48%).
- Higher total protein in males at 300 and 1000 mg/kg bw/day (5 and 10%, respectively).
- Higher albumin in males at 1000 mg/kg bw/day (10%).
- Higher creatinine in males at 1000 mg/kg bw/day (23%).
- Higher potassium in males at 1000 mg/kg bw/day (14%). Potassium was also higher (10%) in males at 300 mg/kg bw/day but the difference from controls was not statistically significant.
- Lower cholesterol in males at 1000 mg/kg bw/day (22%).
The other statistically significant variations noted in clinical chemistry values were considered unrelated to treatment due to the lack of a dose-related response, slightly high control value and/or a high value in a single animal.

Thyroid hormone analyses:
Mean serum T4 levels were statistically significantly decreased in F0 males at 300 and 1000 mg/kg bw/day (relative differences from controls: 32 and 51%, respectively), whereas mean serum TSH levels in these males were increased (4.3 and 9.7-fold, respectively; statistically significant at 1000 mg/kg bw/day only). The mean values in these groups of males were out of the historical control ranges.
Serum levels of T4 and TSH in F0 females were considered not to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females of the 1000 mg/kg bw/day group had lower mean numbers of total movements and ambulations compared to those of the control group (nearly 60% difference, statistically significant for total movements only). Motor activity was particularly low in 3 females which had values below the historical control ranges (2). The other two 1000 mg/kg bw/day females tested had activity values at or just below the concurrent control group ranges.
Activity habituation profiles were similar between the treated and control groups with a decreasing trend in activity over the duration of the test period Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
(2) Historical control data for motor activity in female Wistar Han rats (period 2015-June 2017):
Total movements: mean = 3502, P5 - P95 = 1478 - 5683 (n=195).
Ambulations: mean = 855, P5 - P95 = 301 - 1467 (n=195).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the thymus of males and the thyroid (see also 'any other information').
Kidneys:
- Tubular degeneration was present in males and females at 1000 mg/kg bw/day up to moderate degree.
- Tubular basophilia was present at increased incidence and/or severity in males starting at 300 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree.
- Hyaline droplet accumulation was present at increased severity in males starting at 300 mg/kg bw/ day up to moderate degree.
- Tubular mineralization was present at increased incidence and/or severity in females starting at 300 mg/kg bw/day up to moderate degree.
Liver:
- Hepatocellular hypertrophy was present in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day up to slight degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Pigment deposition was present in males at 300 and 1000 mg/kg bw/day up to slight degree. This sometimes correlated with the macroscopic black-brown discoloration.
- Hepatocellular necrosis was present in a single male at 1000 mg/kg bw/day at slight degree.
Thyroid gland:
- Follicular cell hypertrophy was present at increased incidence and/or severity in males starting at 100 mg/kg bw/day and in females starting at 300 mg/kg bw/day up to moderate degree. This correlated with the macroscopic enlargement and the increased organ weight.
- Colloid alteration was present in males and females at 1000 mg/kg bw/day up to minimal degree.
Thymus:
- Lymphoid atrophy was present in males at 1000 mg/kg bw/day at minimal degree. This correlated with the decreased organ weight.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
During the treatment period, all females had regular cycles, mostly of four days. The irregular cycle noted in one female of the 1000 mg/kg bw/day group (which was not pregnant) was not test item-related as it occurred prior to initiation of treatment (first cycle in the pretest period).
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
One male of the 300 mg/kg bw/day showed tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the observed non-pregnancy. This male had no normal spermatogenic staging profile.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (except abovementioned 300 mg/kg bw/day male).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Two females were not pregnant despite evidence of mating (one female at 300 mg/kg bw/day and one female at 1000 mg/kg bw/day). Another female at 1000 mg/kg bw/day was found dead on Day 24 post-coitum while in labour.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis (except one male at 300 mg/kg bw/day).

Details on results (P0)

No effects were observed on mating index, precoital time, number of implantation sites, anf fertility index. The non-pregnancy of one female at 300 mg/kg bw/day was due to infertility of the male she was paired with.The non-pregnancy of one high dose female was not associated with related histopathology changes in reproductive organs. These incidental cases of male infertility/non-pregnancy were regarded as unrelated to treatment as the incidence showed no dose-related trend and was within normal limits.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 100% for the 300 mg/kg bw/day group and 99% for the other groups.
One pup of the control group died spontaneously at PND 1, one pup of the 100 mg/kg bw/day group was euthanized for humane reasons at PND 2 and one pup of the 1000 mg/kg bw/day group went missing (presumably cannibalized) at PND 2. This incidental pup mortality was considered unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, treatment related lower body weights of pups were noted on PND 13. This was statistically significant for male pups, female pups and sexes combined, and resulted in a 20% lower mean body weight when compared to concurrent controls.
The remaining statistical significant changes (on PND 7 at 100 and 1000 mg/kg bw/day and on PND 13 at 100 and 300 mg/kg bw/day) were not considered toxicologically relevant as no dose response was apparent, all values were within normal limits7 of which the control group values were at the higher end.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were judged to be unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio, anogenital distance (absolute and normalized for body weight) in male and female pups and areola/nipple retention was considered to to be affected by treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproduction observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (mean accuracies between 96.2% and 98.7%). No test item was detected in the Group

1 formulation.

Homogeneity

The formulations of Groups 2 and 4 were homogeneous (coefficient of variation of 2.8% and 2.1% in the low and high dose goups, respectively).

Text Table 1 Mean Percent Organ Weight Differences from Control Groups

 

Males

Females

Dose level (mg/kg):

100

300

1000

100

300

1000

 

 

 

 

 

 

 

THYMUS

 

 

 

 

 

 

              Absolute

5

-27

-41

-

-

-

              Relative to body weight

3

-19

-35

-

-

-

 

 

 

 

 

 

 

THYROID GLAND

 

 

 

 

 

 

              Absolute

13

38**

38**

6

13

19*

              Relative to body weight

0

40**

40**

0

17

17**

 

 

 

 

 

 

 

LIVER

 

 

 

 

 

 

              Absolute

28*

26*

63**

-2

15

25**

              Relative to body weight

25**

38**

81**

0

11

39**

 

 

 

 

 

 

 

KIDNEYS

 

 

 

 

 

 

              Absolute

12

16

16

2

4

8

              Relative to body weight

9

27**

28**

3

0

19**

*: P<0.05, **: P<0.01. - no remarkable findings

Text Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Tubular degeneration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

-

-

-

2/5

        Slight

-

-

-

1/5

-

-

-

-

        Moderate

-

-

-

2/5

-

-

-

-

    Tubular basophilia

 

 

 

 

 

 

 

 

        Minimal

2/5

3/5

2/5

-

-

1/5

-

2/5

        Slight

-

-

2/5

3/5

-

-

-

1/5

a = Number of tissues examined from each group.

 

Males

Females

Dose level (mg/kg):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

5

5

5

5

5

    Hyaline droplet accumulation

 

 

 

 

 

 

 

 

        Minimal

3/5

3/5

-

-

-

-

-

-

        Slight

2/5

1/5

1/5

3/5

-

-

-

-

        Moderate

-

1/5

4/5

2/5

-

-

-

-

    Tubular mineralization

 

 

 

 

 

 

 

 

        Minimal

-

-

-

-

2/5

3/5

2/5

-

        Slight

-

-

-

-

-

-

1/5

2/5

        Moderate

-

-

-

-

-

-

1/5

1/5

LIVERa

5

5

7

10

5

5

5

5

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

3/5

4/7

1/10

-

-

-

2/5

        Slight

-

1/5

1/7

8/10

-

-

-

3/5

    Pigment deposition

 

 

 

 

 

 

 

 

        Minimal

-

-

1/7

2/10

-

-

-

-

        Slight

-

-

1/7

-

-

-

-

-

    Hepatocellular necrosis

 

 

 

 

 

 

 

 

        Slight

-

-

-

1/10

-

-

-

-

THYROID GLANDa

5

5

5

6

5

5

5

5

    Follicular cell hypertrophy

 

 

 

 

 

 

 

 

        Minimal

-

1/5

2/5

1/6

3/5

3/5

1/5

-

        Slight

-

3/5

3/5

5/6

1/5

1/5

4/5

2/5

        Moderate

-

-

-

-

-

-

-

3/5

    Colloid alteration

 

 

 

 

 

 

 

 

        Minimal

-

-

-

1/6

-

-

-

4/5

THYMUSa

5

5

6

5

5

1

-

5

    Lymphoid atrophy

 

 

 

 

 

 

 

 

        Minimal

-

-

-

3/5

-

-

-

-

a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproduction toxicity is at least 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

In an OECD 422 study, Wistar Han rats were treated with 1,3-bis(tert-butylperoxyisopropyl)benzene by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day (10 rats/sex/dose level). Concurrent

controls (10 rats/sex) received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 14 or16 days of lactation (for 50-56 days). Females without offspring were treated for 41 days (two non-pregnant females).

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted the reproductive parameters examined in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Reproduction NOAEL: at least 1000 mg/kg bw/day.