Registration Dossier

Administrative data

Description of key information

For 2212 -81 -9 an OECD 422 is available. Form this study the parental NOAEL is 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. The key NOAEL for repeated dose toxicity study in the read-across substance 25155 -25 -3, was established at 200 mg/kg/day in males and females rats considering the adverse effects observed at 800 mg/kg/day on the body weight gain in males and the kidneys in males and females during a 90-day study.

For further details on the read across rationale see section 13.2.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
see read across rationale in Section 13.2
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 535961 NM Horst / Netherlands
- Age at delivery: 7 weeks
- Weight at acclimatization:
Males: 229 to 266 g (mean 251 g)
Females: 118 to 155 g (mean 131 g)
- Fasting period before study: no
- Housing: groups of two, three or four in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet
- Water (e.g. ad libitum): community tap-water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Stability and Storage of Dose Formulations: For at least 8 days at room temperature (20 ± 5 °C).
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
Vehicle was pre-warmed to a temperature of approximately 40 °C. Luperox F was ground with mortar and pestle, weighed into a glass beaker on a tared precision balance and approximately 80% of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume and stirred for 30 minutes. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 0, 10, 40 and 160 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples (approximately 1 g each) were analyzed by HPLC coupled to an UV detector. The test item was used as the analytical standard.
The Luperox F concentrations in the dose formulations ranged from 81.5% to 104.5% with reference to the nominal and were within the accepted range of ±20%.
The homogeneous distribution of Luperox F in the preparations was approved because single results found did not deviate more than 8.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.
In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50, 200 and 800 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 (main groups), 5 (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The potential health effects of Luperox F (named 2,2-bis(t-butylperoxy isopropyl)benzene in the study report) was evaluated in an OECD 422 study. 2,2-bis(t-butylperoxy isopropyl)benzene was administered once daily orally (by gavage) at dosages of 0, 100, 300, and 1000 mg/kg/day, to male rats for 44 days in total and to female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post partum (last dosing). Treatment at 1000 mg/kg was associated with continuing body weight decrease during the study. Food consumption was decreased only during the pre-pairing period. Treatment at 1000 mg/kg/day was associated in kidneys with a diffuse tubular degeneration / regeneration in all males. A multifocal tubular degeneration / regenera¬tion was observed in some males at 300 mg/kg and some females at 1000 mg/kg. A slightly increased incidence of focal tubular degeneration / regeneration was noted in the females at 1000 mg/kg. A slightly increased incidence and severity of hyaline droplets in proximal convoluted tubules was noted in males at 1000 and 300 mg/kg. Behavioral investigations were considered to be not influenced by the treatment with the test item. No effects were noted for the parameters on clinical laboratory investigations or for macro¬scopic findings during scheduled necropsy. Based on these data, the No Observed Adverse Effect Level (NOAEL) was 100 mg/kg body weight/day.

- Post-exposure recovery period in satellite groups: 4 weeks for groups 1 and 4
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Treatment Period: Twice daily on days 1 to 3, once daily thereafter; in addition, animals were checked for salivation immediately after administration from treatment day 10 onwards
Recovery Period: Once daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Weekly Detailed Observations:
- Acclimatization Period: Once weekly
- Treatment Period: Once weekly (weeks 1-12)
- Recovery Period: Once (last week)

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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 : No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Acclimatization Period: Once in all animals
- Treatment Period: Once during week 13 in animals of the control and high dose groups using a direct ophthalmoscope

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 13 and 17
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes, 18 hours
- How many animals: all
- Parameters checked in table [No 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 13 and 17
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes, 18 hours
- How many animals: all
- Parameters checked in table [No. 2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 13 and 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No. 3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: last week of treatment and at the end of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: modified Irwin screen test, grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
- Seminology and Spermatid Count
Sperm analysis was performed in all main and recovery animals on weeks 13 and 17.

- Immunohistochemistry
Slides of the kidney from all male animals of the control and high-dose groups were stained with an anti-alpha-2-microgobuline-antibody.
Statistics:
The following statistical methods were used to analyze body weight, food consumption, grip strength, locomotor activity, clinical laboratory data, organ weights, sperm analysis data, and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied, if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Male no. 21 (group 2) was sacrificed on treatment day 58 in moribund condition following a gavage accident.

- Daily Observations
In group 4, salivation was observed at least transiently in all animals from treatment week 2 onwards. During the recovery period no clinical signs were recorded.
In group 3, salivation was observed at least transiently in almost all animals from treatment week 3 onwards.
In group 2, salivation was observed at least transiently in most animals from treatment week 3 onwards.
All other clinical signs recorded were considered to be incidental findings which are regularly recorded in animals of this age and strain.

- Weekly Detailed Observations
In group 4, salivation was observed in most males and several females from treatment week 2 onwards. During the recovery period no clinical signs related to treatment with the test item were recorded.
In group 3, salivation was observed in most males and single females from treatment week 4 onwards.
All other clinical signs recorded were considered to be incidental findings which are regularly recorded in animals of this age and strain.
During the recovery period, no clinical signs of toxicological relevance were observed.

BODY WEIGHT AND WEIGHT GAIN
In group 4, decreased body weights and lower body weight gain were recorded in males from treatment week 2 onwards. At the end of the treatment period the mean body weight of group 4 males was 15% lower than in the control group. At the end of the recovery period, the mean body weight of group 4 males was still 13% lower than in the control group.
In group 3, decreased body weights were recorded in males from treatment week 7 onwards. At the end of the treatment period the mean body weight of group 3 males was 5% lower than in the control group.
No effects of the treatment with the test item on body weight development were recorded in males of group 2 and in females at any dose level.

FOOD CONSUMPTION
In group 4, slightly lower absolute food consumption (mean over means over treatment -8%) was recorded in males. The relative food consumption of males in this group was slightly increased due to the lower body weights from treatment week 7 onwards. Slightly increased absolute (mean over means over treatment +10%) and relative (mean over means over treatment +12%) food consumption was recorded in females of group 4.
During the recovery period the food consumption of group 4 males and females was similar to the controls.
No effects of the treatment with the test item on food consumption were recorded in animals of groups 2 and 3.

OPHTHALMOSCOPIC EXAMINATION
No effects of the treatment on the eyes were recorded.

HAEMATOLOGY
In group 4, the following statistically significant changes in hematology parameters were observed:
• Decreased erythrocyte count in females (-8%)
• Decreased hemoglobin concentration in males (-5%) and females (-8%)
• Decreased hematocrit in females (0.40 vs. 0.43 in the control group)
• Increased hemoglobin concentration distribution width in males (+7%) and females (+27%)
• Increased relative reticulocyte count in males (0.033 vs. 0.021 in the control group) and females (0.038 vs 0.024 in the control group)
• Increased absolute reticulocyte count in females (+46%)
• Decreased low reticulocyte maturity index in females (0.392 vs. 0.458 in the control group)
• Increased platelet count in males (+27%) and females (+15%)
• Shortened prothrombin time in females (1.08 vs. 0.80 in the control group) and shortened activated partial thromboplastin time (25.0 sec vs. 32.1 sec in the control group)
In group 3, the following statistically significant changes in hematology parameters were observed:
• Shortened prothrombin time in females (1.01 vs. 0.80 in the control group) and shortened activated partial thromboplastin time (28.0 sec vs. 32.1 sec in the control group)

All changes mentioned above were within or very closed to the limits of the historical control values. They were also reversible, because they were either not observed after the recovery period or returned back to a magnitude where the values were within the range of the historical control data. Therefore, even if these changes seem treatment-related, they can be considered as not adverse effects.

CLINICAL CHEMISTRY
In group 4, the following statistically significant changes in clinical biochemistry parameters were observed:
• Increased protein concentration in males (+5%) and females (+18%)
• Increased albumin concentration in males (+10%) and females (+13%)
• Increased globulin concentration in females (+29%)
• Increased albumin/globulin ratio in males (1.92 vs. 1.76 in the control group)
• Increased cholesterol concentration in females (+99%)
• Increased phospholipids concentration in females (+61%)
• Increased triglyceride concentration in females (+58%)
• Decreased glucose concentration in males (-28%)
• Increased urea concentration in males (+18%)
In group 3, the following statistically significant changes in clinical biochemistry parameters were observed:
• Increased protein concentration in females (+10%)
• Increased albumin concentration in females (+8%)
• Increased globulin concentration in females (+14%)
• Increased cholesterol concentration in females (+38%)
• Increased phospholipids concentration in females (+24%)
In group 2, the following statistically significant changes in clinical biochemistry parameters were observed:
• Increased protein concentration in females (+4%)
• Increased globulin concentration in females (+8%)

Excepted the increased cholesterol and phospholipids concentrations in females of group 4, all changes mentioned above were within or very closed to the limits of the historical control values. They were also reversible, because they were either not observed after the recovery period or returned back to a magnitude where the values were within the range of the historical control data. Therefore, even if these changes seem treatment-related, they can be considered as not adverse effects.

URINALYSIS
In group 4, the increased ketone concentration in males (15.0 mmol/L vs. 0.5 mmol/L in the control group) was considered to be test item-related.
These changes mentioned above were reversible, because they were either not observed after the recovery period or returned back to a magnitude where the values were within the range of the historical control data.
All other changes reaching statistical significance were considered to be incidental, because they were either not dose-related and/or clearly within the range of the historical control data.

NEUROBEHAVIOUR
- Functional Observational Battery (Screen)
No effects on behavior or reflexes were observed at any dose level.
- Grip Strength
No effects on grip strength were observed.
- Locomotor Activity
No effects of the treatment with the test item on locomotor activity were recorded.

ORGAN WEIGHTS
In group 4, the following statistically significant changes in organ weights were observed:
• Increased liver weights in males (+39% absolute, +59% relative to body weight, +45% relative to brain weight) and females (+76% absolute, +89% relative to body weight, +80% relative to brain weight)
• Increased kidney weights in males (+10% absolute, +26% relative to body weight, +14% relative to brain weight)
In group 3, the following statistically significant changes in organ weights were observed:
• Increased liver weights in males (+18% absolute, +24% relative to body weight, +19% relative to brain weight) and females (+37% absolute, +43% relative to body weight, +37% relative to brain weight)
• Increased kidney weights in males (+11% absolute, +17% relative to body weight, +11% relative to brain weight)
In group 2, the following statistically significant changes in organ weights were observed:
• Increased liver weights in females (+19% absolute, +21% relative to body weight, +17% relative to brain weight)

At the end of the recovery period liver and kidney weights of high dose males were similar as in the control group while liver weights in females were still increased (+25% absolute, +28% relative to body weight, +31% relative to brain weight).

The increased liver weights in males and females were correlated histologically with hepatocellular hypertrophy. These findings are suggestive of an adaptative response to mixed function oxidase induction (Cattley et al., 2002).

The increased kidney weights in males were correlated histologically with hyaline droplets in tubular epithelial cells associated with alpha 2µ-globulin accumulation as demonstrated by immunohistochemistry. Alpha 2µ-globulin nephropathy is a specific finding in male rats (Khan et al., 2002], Williams et al., 2001) and has no relevance for human risk assessment (Swenberg et al., 1999).
Therefore, even if these changes in organ weights seem treatment-related, they can be considered as not adverse effects.

All other effects on organ weights reaching statistical significance were either considered to be secondary to body weight changes or incidental, because the changes were not dose-related and/or no other findings correlated with these findings.

GROSS PATHOLOGY
No macroscopic findings related to the treatment with the test item were observed.

All macroscopic findings recorded were considered to be incidental findings which are regularly recorded in animals of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Kidneys
Hyaline droplets in tubular epithelial cells were observed in males at all dose levels with a dose related mean severity (table 5). A minimal effect was observed in 6/9, 4/10 and 4/10 males in groups 2, 3 and 4, respectively and the effect was slight in 3/10 and 3/10 and moderate 2/10 and 3/10 in groups 3 and 4, respectively. The hyaline droplets were observed within the cytoplasm of proximal tubule lining cells. They were usually lightly eosinophilic and multiple; some of them were observed protruding from the cell. Following immunostaining with an anti alpha 2µ-globulin antibody, increased staining level in the proximal tubular cells (specific staining of small rounded and enlarged variably-shaped droplets) was present in 9/10 males of group 4 (no staining in the remaining male) while normal staining level was present in the proximal tubular cells (specific staining of small rounded droplets only) in 10/10 males of group 1.
In males and females of group 4 there were karyomegaly, focal/multifocal tubular basophilia and cytoplasmic pigmented vacuoles in tubular epithelial cells. Overall, males were more affected than females.
Karyomegaly was diagnosed for the presence of enlarged nuclei (2 - 4 times larger than normal) within proximal tubule lining cells, mainly within the outer cortex. Affected cells were occasionally bi- or poly-nucleated.
Focal/multifocal tubular basophilia was characterized by the presence of segments of proximal tubules lined by slightly basophilic cells. Their nuclei were normally-sized or slightly enlarged.
The cytoplasmic pigmented vacuoles involved the proximal tubular cells. They were yellowish and usually small.
At the end of the recovery period (table 6), hyaline droplets in tubular epithelium (1/5 male with a minimal grade), karyomegaly, focal/multifocal tubular basophilia and cytoplasmic pigmented vacuoles in tubular epithelium were still observed. Overall, incidence and/or severity of the findings were lower when compared to the end of the treatment period. Following immunostaining with an anti alpha 2µ-globulin antibody, the staining level was increased in the proximal tubular cells of 2/5 males (normal staining level in 2/5 males and no staining in the remaining male of group 4) while normal staining level was present in the proximal tubular cells of 5/5 males of group 1.

- Liver
Slight to marked hepatocellular hypertrophy was recorded in males and females of group 4 (able 7). Minimal hepatocellular hypertrophy was also recorded in males and females of group 3 in 1 out of 10 rats each. The changes were characterized by enlargement of hepatocytes that display a “ground glass” appearance. The hepatocellular enlargement was observed mainly in centrilobular areas (slight severity), centrilobular to midzonal areas (moderate severity) or extended to periportal areas (marked severity).
In animals of the recovery group there were no test-item related findings in the liver.
Associated to the increased liver weights recorded at necropsy, these findings are suggestive of a physiological adaptative induction of mixed function oxidase (Cattley et al., 2002).

- Thyroid
Minimal to moderate follicular cell hypertrophy was observed in males at all dose levels and in females of groups 3 and 4 with a dose-related mean severity (table 8). The changes were characterized by the presence of irregularly shaped follicles lined by tall columnar cells and containing a pale-staining colloid.
Minimal to slight follicular cell hypertrophy was still observed in males and females of the recovery group (table 9).
Associated to hepatocellular hypertrophy, the follicular cell hypertrophy in the thyroid is commonly seen in rats following administration of liver enzyme inducers where the underlying mechanism is an increased hepatic clearance of thyroid hormones followed by a compensatory increase in the pituitary secretion of TSH (Capen et al., 2002).

- Spleen
When compared to controls, a slight increased incidence and mean severity of splenic extramedullary hematopoiesis were recorded in males of groups 3 and 4 and in females of group 4 (table 10). In addition, minimal increased hemosiderosis was present in 3 out of 10 females of group 4.
Minimal increased hemosiderosis was still present in 2 out of 5 recovery females.
The minimal increased splenic hemosiderosis in females was consistent with increased red blood cell turn over.

- Stomach
Slight erosion/ulcer of the non-glandular mucosa was observed in 3 out of 10 females of group 4 only.
In animals of the recovery group there were no test-item related findings in the stomach.

- Other Findings
All other findings were those commonly seen in the laboratory rat. Their incidence/severity did not suggest any test-item relationship.

OTHER FINDINGS
- Seminology and Spermatid Count
Sperm Motility: No effects of the treatment with the test item on sperm motility were observed.
Morphology: No effects of the treatment with the test item on sperm morphology were observed.
Sperm Head Count: No effects of the treatment with the test item on sperm head counts were observed.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects observed
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Effects on the body weight gain in males and the kidneys in males and females
Critical effects observed:
not specified

Table 5     Incidence and Severity of Noteworthy Microscopic Findings in the Kidneys at Terminal Kill

 

Males

 

Females

Group

1

2

3

4

 

1

2

3

4

Dose (mg/kg bw/day)

0

50

200

800

 

0

50

200

800

Number of animals

10

9

10

10

 

10

10

10

10

n

10

9

10

10

 

10

10

10

10

 

Hyaline droplets, tubular epithelium

minimal

-

6

4

4

 

-

-

-

-

slight

-

-

3

3

 

-

-

-

-

moderate

-

-

2

3

 

-

-

-

-

Mean severity

0.0

0.7

1.6

1.9

 

0.0

0.0

0.0

0.0

 

Karyomegaly

minimal

-

-

-

1

 

-

-

-

8

slight

-

-

-

7

 

-

-

-

-

moderate

-

-

-

2

 

-

-

-

-

Mean severity

0.0

0.0

0.0

2.1

 

0.0

0.0

0.0

0.8

 

Tubular basophilia, focal/multifocal

minimal

-

-

-

6

 

-

-

-

2

slight

-

-

-

3

 

-

-

-

-

Mean severity

0.0

0.0

0.0

1.2

 

0.0

0.0

0.0

0.2

 

Cytoplasmic pigmented vacuoles, tubular epithelium

minimal

-

-

-

6

 

-

-

-

7

n:    Number examined; - : no animal affected

*:    Mean severity is ∑ number of animals x severity / number of examined organs in the group

Table 6     Incidence and Severity of Noteworthy Microscopic Findings in the Kidneys at Recovery Kill

 

Males

 

Females

Group

1

4

 

1

4

Dose (mg/kg bw/day)

0

800

 

0

800

Number of animals

5

5

 

5

5

n

5

5

 

5

5

 

Hyaline droplets, tubular epithelium

 minimal

-

1

 

-

-

 

Karyomegaly

 minimal

-

2

 

-

4

 slight

-

2

 

-

-

 moderate

-

1

 

-

-

 Mean severity

-

1.8

 

-

0.8

 

Tubular basophilia, focal/multifocal

 minimal

-

3

 

-

1

 

Cytoplasmic pigmented vacuoles, tubular epithelium

 minimal

-

2

 

-

3

n:    Number examined; - : no animal affected

*:    Mean severity is ∑ number of animals x severity / number of examined organs in the group

Table 7     Incidence and Severity of Noteworthy Microscopic Findings in the Liver at Terminal Kill

 

Males

 

Females

Group

1

2

3

4

 

1

2

3

4

Dose (mg/kg bw/day)

0

50

200

800

 

0

50

200

800

Number of animals

10

9

10

10

 

10

10

10

10

 

Liver

n

10

9

10

10

 

10

10

10

10

Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

 

minimal

-

-

1

-

 

-

-

1

-

slight

-

-

-

1

 

-

-

-

2

moderate

-

-

-

5

 

-

-

-

3

marked

-

-

-

4

 

-

-

-

5

Mean severity

0.0

0.0

0.1

3.3

 

0.0

0.0

0.1

3.3

n:    Number examined; - : no animal affected

*:    Mean severity is ∑ number of animals x severity / number of examined organs in the group

Table 8     Incidence and Severity of Noteworthy Microscopic Findings in the Thyroid at Terminal Kill

 

Males

 

Females

Group

1

2

3

4

 

1

2

3

4

Dose (mg/kg bw/day)

0

50

200

800

 

0

50

200

800

Number of animals

10

9

10

10

 

10

10

10

10

 

Thyroid glands

n

10

9

10

10

 

10

10

10

10

Hypertrophy, follicular cell

 

 

 

 

 

 

 

 

 

minimal

-

4

3

1

 

-

-

3

6

slight

-

2

4

3

 

-

-

-

2

moderate

-

-

2

6

 

-

-

-

-

Mean severity

0.0

0.9

1.7

2.5

 

0.0

0.0

0.3

1.0

n:    Number examined; - : no animal affected

*:    Mean severity is ∑ number of animals x severity / number of examined organs in the group

Table 9     Incidence and Severity of Noteworthy Microscopic Findings in the Thyroid at Recovery Kill

 

Males

 

Females

Group

1

4

 

1

4

Dose (mg/kg bw/day)

0

800

 

0

800

Number of animals

5

5

 

5

5

n

5

5

 

5

5

 

Hypertrophy, follicular cell

 minimal

-

1

 

-

1

 slight

-

1

 

-

-

 Mean severity

-

0.6

 

-

0.2

n:    Number examined; - : no animal affected

*:    Mean severity is ∑ number of animals x severity / number of examined organs in the group

Conclusions:
Repeated daily administration of Luperox F by oral gavage for a period of 91/92 days to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 or 800 mg/kg did not lead to any test item-related mortalities. Salivation was observed at all dose levels after the administration with a dose-related increase of incidence and severity. The treatment with the test item did not induce effects on the eyes, behavior, reflexes, grip strength or locomotor activity.
The food consumption was slightly reduced in males of group 4 which led to decreased body weights and lower body weight gain. Slightly increased food consumption in females of group 4 had no effect on body weight development in this group. No effects on food consumption and body weights were observed during the recovery period.
The changes recorded in hematology, clinical biochemistry and urine parameters which included effects on thrombin and prothrombin time, changes in albumin, globulin, cholesterol, phospholipids, and triglyceride concentration in the blood as well as an increase of ketone concentration in the urine were considered non-adverse, because most changes were still within or only slightly outside the historical controls and they were all reversible during the recovery period.
At necropsy, no macroscopic findings related to the treatment with the test item were observed.
A dose-related increase of liver weights recorded in males of groups 3 and 4 and in females at all dose levels correlated histologically with hepatocellular hypertrophy in both sexes of group 4 and in one male and female each of group 3. These findings are suggestive of an adaptive response to mixed function oxidase induction (Cattley et al., 2002). The liver findings were associated with follicular cell hypertrophy in the thyroid. This is commonly seen in rats following administration of liver enzyme inducers where the underlying mechanism is an increased hepatic clearance of thyroid hormones followed by a compensatory increase in the pituitary secretion of TSH (Capen et al., 2002). All findings related to liver and thyroids were almost completely reversible during the recovery period.
In the spleen, the slight increased extramedullary hematopoiesis observed in males and females correlated with slightly increased reticulocytes count recorded in group 4. The minimal increased splenic hemosiderosis in females was consistent with increased red blood cell turn over. These findings were almost completely reversible during the recovery period.
In the stomach, the slightly erosion/ulcer of the non-glandular mucosa observed in males and females of group 4 was most likely related to irritating properties of the test item. No findings were recorded in the stomach at the end of the recovery period.
Kidney weights were increased in males of groups 3 and 4. In the kidney of males, the cells lining the proximal tubule were the target cells of the test item. The hyaline droplets in males were associated with alpha 2µ-globulin accumulation as demonstrated by immunohistochemistry. Alpha 2µ-globulin nephropathy of male rats is a common finding following the administration of hydrocarbons (Khan et al., 2002) and has already been associated with karyomegaly within the proximal tubules (Williams et al., 2001). It is considered to be a rat-specific effect with no relevance for human risk assessment (Swenberg et al., 1999). The cytoplasmic yellowish vacuoles were possibly modified hyaline droplets. The segmental tubular basophilia was considered to be indicative of on-going cell degeneration/regeneration in group 4. In males and females of group 4 there were also minimal karyomegaly, minimal/slight focal/multifocal tubular basophilia and minimal cytoplasmic pigmented vacuoles in tubular epithelial cells. At the end of the recovery period, hyaline droplets in tubular epithelium of males, and karyomegaly, focal/multifocal tubular basophilia and cytoplasmic pigmented vacuoles in tubular epithelium of males and females were still observed; however, the incidence and/or severity of the findings were lower when compared to the end of the treatment period.
No effects of the treatment with the test item on sperm motility, morphology and sperm counts were observed.
Based on the results of this study, the NOAEL in males and females rats was established at 200 mg/kg/day considering the adverse effects observed at 800 mg/kg/day on the body weight gain in males and the kidneys in males and females.
Executive summary:

In a subchronic toxicity study performed according to the OECD test guideline no. 408 and GLP, Luperox F (96.7% of 2,2-Bis(t-butylperoxy isopropyl)benzene) was administered daily by oral gavage to-bred Wistar rats of both sexes at dose levels of 50, 200 and 800 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, corn oil, only. The groups comprised 10 animals per sex which were sacrificed after 91/92 days of treatment. Additional 5 rats per sex and group were used at 0 and 800 mg/kg. These animals were treated for 91 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed. Clinical signs, detailed cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Sperm analysis was performed in all males. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Due to test item-related morphologic changes in the kidneys, stomach, thyroid, spleen, and liver of high-dose animals, these organs from the mid- and low-dose group animals were examined.

No test item-related mortalities were recorded. Salivation was observed at all dose levels after the administration. The incidence and frequency of salivation increased with the dose level. No effects on behavior or reflexes were observed at any dose level. No effects on grip strength were observed. No effects of the treatment with the test item on locomotor activity were recorded. In group 4, slightly lower absolute food consumption was recorded in males. Slightly increased food consumption was recorded in females of group 4. During the recovery period, the food consumption of group 4 males and females was similar to the controls. No effects on food consumption were recorded in animals of groups 2 and 3. In group 4, decreased body weights and lower body weight gain were recorded in males from treatment week 2 onwards. At the end of the recovery period, the mean body weights of group 4 males where still slightly lower than in the control group. In group 3, slightly decreased body weights were recorded in males from treatment week 7 onwards. No effects on body weight development were recorded in males of group 2 and in females at any dose level. No effects of the treatment were recorded at ophthalmoscopic examinations. In group 4, signs of slight regenerative anemia which included decreased erythrocyte count, decreased hemoglobin concentration, decreased hematocrit, and an increase of immature red blood cells were recorded. In addition, platelet counts were increased in both sexes of group 4. Slightly prolonged prothrombin time and shortened activated partial thromboplastin time was recorded in females of groups 3 and 4. All these changes were not considered as adverse, because they were of low magnitude and not observed at the end of the recovery period. Total protein, albumin and/or globulin concentration was slightly increased in females at all dose levels and in males of group 4. Cholesterol and phospholipide concentration was increased in females of groups 3 and 4. In addition, the triglyceride concentration was increased in females of group 4. A decrease of the glucose concentration together with an increase of the urea concentration was recorded in males of group 4. All these changes were not considered as adverse, because they were of low magnitude and not observed at the end of the recovery period. In group 4, increased ketone concentrations were recorded in urines of males. These changes were reversible, because they were not observed at the end of the recovery period. A dose-related increase of liver weights was recorded in males of groups 3 and 4 and in females at all dose levels. Kidney weights were increased in males of groups 3 and 4. No effects of the treatment with the test item on sperm motility, morphology and sperm counts were observed. No macroscopic findings related to the treatment with the test item were observed. In the kidneys, hyaline droplets immunostained by an anti alpha 2µ-globulin antibody were recorded in the proximal tubules of males at all dose levels with a dose-related increase of the mean severity. Karyomegaly, tubular basophilia and pigmented vacuoles in the proximal tubules of the kidneys of males and females were recorded in group 4, mean severity being higher in males. Hyaline droplets in the tubular epithelium, karyomegaly, focal/multifocal tubular basophilia and cytoplasmic pigmented vacuoles in tubular epithelium were still observed at the end of the recovery period; however, incidence and/or severity of the findings were lower when compared to the end of the treatment period. Liver adaptive hepatocellular hypertrophy was recorded in males and females of groups 3 and 4 with a dose-related increase of the mean severity. In animals of the recovery group there were no test-item related findings in the liver. Thyroid follicular cell hypertrophy secondary to the liver enzyme induction was recorded with a dose-related increase of the mean severity in males at all dose levels and in females of groups 3 and 4. Minimal to slight follicular cell hypertrophy was also observed in males and females of the recovery group. In the spleen, increased extramedullary hematopoiesis secondary to increased red blood cell turn over was recorded in males of groups 3 and 4 and in females of group 4, and increased hemosiderosis in females of group 4. Minimal increased hemosiderosis was still present in 2 out of 5 recovery females of group 4. Erosion/ulcer of the non-glandular stomach was recorded in females of group 4. In animals of the recovery group there were no test-item related findings in the stomach.

Based on the results of this study, the NOAEL in males and females rats was established at 200 mg/kg/day considering the adverse effects observed at 800 mg/kg/day on the body weight gain in males and the kidneys in males and females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP guideline study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to regulation (CE) n°1272/2008, [1,3 -phenylenebis(1 -methylethylidene)]bis[tert-butyl] peroxide is not classified as toxic in case of repeated exposure as evidenced by the test results of structurally analogous 2,2 -Bis(t-butylperoxy isopropyl) benzene.