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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
EC Number:
218-664-7
EC Name:
[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
Cas Number:
2212-81-9
Molecular formula:
C20H34O4
IUPAC Name:
1,3-bis[1-(tert-butylperoxy)-1-methylethyl]benzene
Constituent 2
Chemical structure
Reference substance name:
Di-tert-butyl α,α,α',α'-tetramethyl-(p-phenylenedimethylene) diperoxide
EC Number:
220-479-1
EC Name:
Di-tert-butyl α,α,α',α'-tetramethyl-(p-phenylenedimethylene) diperoxide
Cas Number:
2781-00-2
Molecular formula:
C20H34O4
IUPAC Name:
1,4-bis[1-(tert-butylperoxy)-1-methylethyl]benzene
Test material form:
solid: flakes

Test animals

Species:
mouse
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals or test system 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

Administration / exposure

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
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
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

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 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.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Male no. 21 (group 2) was sacrificed on treatment day 58 in moribund condition following a gavage accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of the treatment on the eyes were recorded.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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 biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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 findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- 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 weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 pathological findings:
no effects observed
Description (incidence and severity):
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.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 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.
Description (incidence and severity):
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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.