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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
Study period:
2012-08-07 to 2012-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Age at study initiation: Male animals: 53 – 58 days old, Female animals: 53 – 58 days old
- Weight at study initiation: Male animals: 226 – 265 g, Female animals: 138 – 167 g
- Fasting period before study: no
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance
- Water: tap water ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 8 – 12 air exchanges / hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 2012-08-08 to 2012-12-04

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil) . Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for three days.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 32 mg/mL, 8 mg/mL and 2 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 19/4 and 19T3
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility. Five samples (5 mL, each) were taken from different places from each concentration (groups 2, 3 and 4) for analysis of concentration and homogeneity on 2 occasions during the first and last treatment week. Similarly, five samples were taken from different places from the control substance (group 1) on first occasion and one sample (5mL) was taken from the control substance on the second occasion and measured:
Date of samplings: August 14, and October 30, 2012
Date of measurement: August 15, and October 31, 2012.
The samples were stored in a refrigerator until the analysis. Measured concentrations varied between 99 and 106 % of the nominal concentrations and all formulations were considered to be homogeneous as tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) is soluble in sunflower oil. The suitability of the chosen vehicle for the test item (stability and homogeneity) was analytically proven. Recovery was 100 % (of nominal concentration) at 10 mg/ml, and 99 % at 100 mg/ml concentration. tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) was stable at concentrations of 10 and 100 mg/mL in sunflower oil in the refrigerator for 72 hours (at 5 +/- 3°C; recovery was 98 and 99 % of starting concentrations at 10 and 100 mg/mL, respectively) and after 4 hours at room temperature (recovery was 100 % of starting concentrations for both concentrations of 10 and 100 mg/mL).

HPLC Conditions:
Detector: 210 nm
Column: HyperPrep HS C 18 250*4.6 mm,
No.: 12187302B/10013
Mobil Phase: Acetonitrile: Water = 9 : 1 (v/v)
Flow: 1.2 mL/min
Injection volume: 50 μL
Temperature: 25 °C
Retention time: for TBPIN 5.7 min ±5%
Duration of treatment / exposure:
90 or 91 days (depending on day of necropsy)
Frequency of treatment:
Once a day on a 7 days/week basis, every day at a similar time (+/- 2 hours)
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose in the main study. Additionally, 5 animals per sex in the control and high dose group (recovery group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting based on findings obtained in a previous 28-Day Oral Gavage Toxicity Study with tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) in the Rat (Title: Tert-butyl peroxy-3,5,5-trimethylhexanoate, techn. pure" (TBPIN): 28-Day Oral Toxicity Study in Rats; Report no.:
SL-LT-035/10). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.
- Rationale for animal assignment:
All animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups were as uniformly as practicable. Grouping was aided by SPSS/PC software, verifying the homogeneity and variability between the groups and cages according to the actual body weight.
- Rationale for selecting satellite groups: assessment of reversibility, persistence or delayed occurrence of potential toxicological effects
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed in the treatment period with an accuracy of 1 g on Day 0, then weekly. Individual body weight changes were calculated. Fasted body weight was measured on day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION: Yes
The food consumption was determined in the treatment phase with an accuracy of 1 g on Day 7, then weekly by reweighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period
- Dose groups that were examined:Repeated on all control and high dose test animals prior to test termination (Day 89)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Anaesthetic used for blood collection: Yes, under isofluran anesthesia
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment (Days 90 and Day 91) and on recovery animals at the end of recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last exposure week (Day 86)
- Dose groups that were examined: all animals
- Battery of functions tested: Different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: Yes
- Time schedule for examinations: During the last two weeks of the treatment period (from Day 76 up to and including Day 89) and for recovery animals from Day 104 up to and including Day 117
- Dose groups that were examined: all female animals
- Examinations: A vaginal smear was prepared from each female. After drying, smears were stained with 1 % aqueous methylene blue solution and were examined by a light microscope. The type of cycle (regular or irregular), cycle length, number of cycle during the two weeks, number of animals with prolonged diestrus, number of animals with prolonged estrus were determined.

SPERM EXAMINATION
- Time for examinations: at Necropsy
- Quantitative examinations: The total number of homogenization of one side testis was enumerated. Testes and epididymides were frozen at the necropsy and enumeration was performed later.
- Qualitative examinations: Sperm motility was determined from ductus deferens of the same animals as enumeration at the necropsy. For the determination of the sperm motility the mean percentage of motile sperms was determined. The total sperm count and number of immotile sperms were recorded. Two samples were prepared from each animal.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, including organ weights
HISTOPATHOLOGY: Yes, including determination of α2μ-globulin in the kidney of male animals of control and high dose group.
Other examinations:
not applicable
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- sperm parameters
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations and in the course of the functional observation battery. Salivation was observed in the male and female animals of the 160 or 40 mg/kg bw/day groups with variable frequency within a group but in a dose related manner regarding the incidence and onset.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female rat died on Day 39 due to individual disease. There were no preceding clinical signs or body weight changes. Histological examinations revealed pulmonary alterations (acute alveolar emphysema, multifocal hemorrhages in the lungs), circulatory disturbances (passive hyperemia in the organs) in connection with a probably suffocation as the cause of death and a focal subchronic perihepatitis accompanied with local fibrosis in one of the liver lobes as an individual disease.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean liver and kidney weights (absolute and relative to the body and brain weights) were higher with respect to their controls in the male and female animals of 160 mg/kg bw/day group. Similar findings were also noted for the higher mean liver weight relative to body weight in male animals of 40 and 10 mg/kg bw/day groups and for the higher mean kidney weights relative to body and brain weights of male animals at 40 mg/kg bw/day. Although these differences reached statistical significances with respect to controls, all values remained within the historical control ranges.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The kidneys were judged to be pale in some animals (male and female) of 160 mg/kg bw/day groups and in two female animals at 10 mg/kg bw/day at the terminal necropsy. The liver was enlarged, pale and nutmeg-like patterned in several animals of 160 mg/kg bw/day group and in single male animals of 40 and 10 mg/kg bw/day groups. Since kidney and liver weight values remained within the historical control ranges and there were no related histopathological lesions in the kidneys and the liver these changes were considered to be signs of adaptation of the organ to the altered demands.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology investigations revealed test item related hyaline –like droplets in the epithel cells of some proximal convoluted tubules in the kidney of male animals treated with 160, 40 and 10 mg/kg bw/day, which was accompanied by dilatation of tubuli in the distal area, at the border of cortical – medullary region at 160 and 40 mg/kg bw/day. The severity of the lesions decreased in the low dose group as compared with the middle or high dose groups. The renal lesion was not present at the end of the recovery period and no additional renal lesions or increased cell turnover were determined. In accordance with literature data (ECETOC, 2002) the isolated and reversible finding hyaline-like droplets in male rats has to be considered as a toxicological relevant, but not adverse effect.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm analysis
Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 160 mg/kg bw/day dose.

EXAMINATION OF ESTROUS CYCLE
A test item influence on the estrous cycle was not detected. The percent of animals with regular and irregular estrous cycle was similar in the control and test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of cycles, days in pro-estrous, estrous or diestrus.

Determination of α2μ-globulin in the kidneys
Immunohistochemistry examinations demonstrated lower level of α2μ-globulin in male animals of the 160 mg/kg bw/day compared with control animals, therefore the presence of α2μ-globulin in the kidneys of male animals was deemed to reflect the normal physiological situation. Therefore, hyaline-like droplets at 160, 40 and 10 mg/kg bw/day doses in male rats could not be caused by α2μ-globulin.
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no test item related mortality at any dose level (160, 40 or 10 mg/kg bw/day). One control female rat (no. 107) died on Day 39. There were no preceding clinical signs or body weight changes. Histological examination revealed focal subchronic perihepatitis accompanied with local fibrosis in one of the liver lobes as sign of an individual disease and acute alveolar emphysema, multifocal hemorrhages in the lung as well as passive hyperemia in the other organs in connection with a probably suffocation as the cause of death.
Treatment period
Test item related salivation was observed in male and female animals administered with 160 or 40 mg/kg bw/day with variable frequency within a group but in a dose related manner regarding the incidence and onset (male: 15/15, 7/10; female 15/15, 4/10; respectively to doses). The behavior and physical condition of animals were considered to be normal at each dose level (160, 40 and 10 mg/kg bw/day) during the treatment period. Individual dermal alterations were observed in one female animal of 40 mg/kg bw/day group (scar between Days 49 and 57) and in one control male animal (alopecia between Days 20 and 46 and scar between Days 16 and 61). Alopecia and scar are common clinical signs in this strain of exerimental rats of this age and were only present in single animals of the low dose and the control group.
Recovery period
Clinical signs were not detected in the male or female animals of 160, 40 or 10 mg/kg bw/day group or in the control group during the recovery period.
The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period (treatment and recovery periods). Alopecia or scars were also detected in one female animal of 40 mg/kg bw/day (1/10) and in one control male animal (1/15) on the days of detailed weekly clinical observations. As mentioned above, alopecia and scar are common clinical signs in this strain of experimental rats of this age.

FUNCTIONAL OBSERVATION BATTERY
Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 160, 40 or 10 mg/kg bw/day). The behavior and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

BODY WEIGHT
Treatment period
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (160, 40 and 10 mg/kg bw/day) during the entire observation period. Statistical significances noted for the less mean body weight gain of male animals in groups of 160 mg/kg bw/day (between Days 35 and 42, 63 and 70) and 40 mg/kg bw/day (between Days 35 and 42, 63 and 70, as well as between Days 70 and 77) were transient and mean values remained within the historical control ranges. There were no significant differences in the total mean body weight gain (between Days 0 and 89) between the control and test item treated groups. A slightly higher mean body weight was observed in female animals at 160 mg/kg bw/day on Day 56, however this slight but statistically significant change was considered to be without any toxicological relevance.
Recovery group
The body weight and body weight gain were similar in the male and female animals of 160 mg/kg bw/day and control groups during the recovery period.

FOOD CONSUMPTION
Test item related effects on the mean daily food consumption were not detected.
Treatment period
The daily mean food consumption was comparable in the control and all test item treated groups (160, 40 or 10 mg/kg bw/day).
Recovery group
There were no significant differences in the mean daily food consumption between the control and 160 mg/kg bw/day groups during the four weeks post-treatment period.

OPHTHALMOLOGICAL EXAMINATION
The eyes were without any detected abnormalities in all animals before treatment and in the high dose group at termination of the treatment.

HEMATOLOGY
Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (160, 40 or 10 mg/kg bw/day).
Main group
At the termination of the treatment, statistical significances were noted in male animals for a slightly less mean corpuscular (erythrocyte) hemoglobin content (MCH) and mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 160 mg/kg bw/day with respect to the control group. The mean activated partial thromboplastin time (APTT) was longer in males of the 40 mg/kg bw/day group with respect to the control group. The mean hematocrit value (HCT) in female animals at 10 mg/kg bw/day showed some statistically significant variations when compared to the concurrent control.
Recovery groups
In the recovery group of 160 mg/kg bw/day dose, the hematocrit value was less and the platelet counts (PLT) were higher than in the control males. In female animals of 160 mg/kg bw/day recovery group, all examined hematological parameters were similar to the appropriate value in the control group.
The statistical significant values for MCH, MCHC, APTT, HCT and PLT were within the historical control ranges and therefore were not considered to be of biological relevance.

CLINICAL CHEMISTRY
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters.

Main group
In male animals administered with 160 mg/kg bw/day, a statistically significant, higher mean activity of alkaline phosphatase (ALP) and slightly higher mean potassium (K+) concentration were observed with respect to the control at the termination of the treatment. Furthermore, in the male animals, the activity of alkaline phosphatase was slightly higher than in the control group at 10 mg/kg bw/day. Statistical significances in ALP and K+ concentrations were without dose response.
In female animals of 160 mg/kg bw/day group, statistical significances were noted for the less concentrations of calcium (Ca2+), chloride (Cl-), albumin (ALB) and total protein (TPROT), lower albumin/globulin ratio (A/G), as well as for a slightly higher mean potassium level. The chloride concentration was below the control value in female animals of 40 mg/kg bw/day group.
Recovery groups
In group of 160 mg/kg bw/day, the activity of aspartate aminotransferase (AST; in male animals) and concentration of albumin (female) were slightly but statistically significantly less with respect to their control. These sporadic statistical differences (ALP, AST, K+, Ca2+, Cl-, ALB, TPROT, and A/G) were considered to be of little or no biological relevance. Although the mentioned differences between the control and test item treated groups were statistically significant but were small and all values remained within or marginal to the historical control ranges for these parameters. Therefore these findings were not considered to be of toxicological relevance.

NECROPSY
Main groups
Pale kidneys were observed in animals of 160 mg/kg bw/day (4/10 male, 2/10 female) and 10 mg/kg bw/day (2/10 female) groups at the terminal necropsy. However, kidney weight values remained within the historical control ranges and there were no related histopathological lesions in the kidneys, therefore these changes were considered to be signs of adaptation of the organ to the altered demands.
The liver was enlarged (3/10 male, 2/10 female), pale and nutmeg-like patterned (5/10 male, 4/10 female) in several animals of 160 mg/kg bw/day group. Enlarged, pale and nutmeg-like patterned liver was observed in single male animals of 40 and 10 mg/kg bw/day groups (1/10, both). However, liver weight values remained well within the historical control ranges and hepatocellular damage were not detected at the histopathological examination, therefore these changes were considered to be signs of adaptation of the organ to the altered demands. Smaller than normal testes and epididymides was considered to be an individual change in one 40 mg/kg bw/day dose treated male animal (1/10). Pale and smaller than normal Harderian gland was observed in two male rats administered with 40 mg/kg bw/day (2/10). There were no similar findings and no dose response at 160 mg/kg bw/day. Therefore these were judged to be also individual alterations. Slight, moderate or marked hydrometra was observed in several female animals in each group: 3/10 at 160 mg/kg bw/day, 4/10 at 40 mg/kg bw/day, 4/10 at 10 mg/kg bw/day and 6/10 in the control group. Hydrometra related to the female sexual cycle is a frequent observation in experimental rats.

Recovery group
There were no macroscopic changes in the male animals observed for four weeks after the termination of the treatment (control and 160 mg/kg bw). In the female animals, slight or moderate hydrometra (2/4 control) and larger than normal spleen (1/5; 160 mg/kg bw/day) were detected at the end of the recovery period. These necropsy findings were not considered to be biologically significant due to the sporadic occurrence and the missing dose response.

ORGAN WEIGHT
Main group
Slight, but statistically significant differences were detected in the higher mean kidney weights (absolute and relative to the body and brain weights) in male and female animals in 160 mg/kg bw/day group. Similar findings were also noted for kidneys weights relative to body weight and brain weight of male animals at 40 mg/kg bw/day with respect to the controls. The mean liver weights (absolute and relative to the body and brain weights) were statistically significantly higher in male and female animals in 160 mg/kg bw/day group reflected a test item influence. The mean liver weight relative to body weight was slightly but statistically significantly higher than in the control group in male animals of 40 and 10 mg/kg bw/day groups. In the male animals, the mean weight of adrenal glands (absolute) was slightly less at 160 and 40 mg/kg bw/day doses, and if referred to the brain weight at 160 mg/kg bw/day with respect to the controls. The mean heart weight relative to the body weight slightly exceeded the control value in male 160 mg/kg bw/day group and it was less than in the control group in
female animals in 40 mg/kg bw/day group. The thymus weights (absolute and relative to the body and brain weights) were slightly below the control value in female animals administered with 40 mg/kg bw/day
All these statistically differences with respect to the appropriate control were considered to be of no toxicological importance since the values remained well within the historical control ranges and related findings were not detected at the histopathological examinations or the findings indicated no dose response.

Recovery group
In the male animals of 160 mg/kg bw/day group, the mean kidney weight relative to the body weight was slightly but statistically significantly higher than in the control group. However, the value was within the historical control range and thus, considered to be of no toxicological relevance. In the female animals, all examined organ weights were comparable to their control.

HISTOPATHOLOGY
Main group
Hyaline-like droplets were observed in the epithel cells of some proximal convoluted tubules in the kidney of male animals treated with 160, 40 and 10 mg/kg bw/day (8/10, 10/10 and 5/10 animals, respectively), which was accompanied by dilatation of tubuli in the distal area, at the border of cortical – medullary region at 160 and 40 mg/kg bw/day (5/10 and 3/10 animals, respectively). The severity of the lesions decreased in the low dose group as compared with the middle or high dose groups. No hyaline droplets or dilatation of tubuli were found in the kidney of female animals of any groups (160, 40 and 10 mg/kg bw/day). The histopathological examination indicated that male rats were particularly sensitive for the appearance of hyaline-like droplets. Hyalinelike droplets could be related to a high concentration of the rat specific protein α2μ-globulin (Hard et al. 1991; Yamate et al. 1998). The determination of α2μ-globulin within this study clearly showed that the presence of α2μ-globulin in the kidneys of the male rats reflected the normal physiological situation and therefore hyaline-like droplets are not caused by α2μ-globulin. Beside hyaline droplets no additional renal lesion e.g. increased kidney weight, urinalysis parameters or regeneration of proximal tubular epithelium and no increased cell turnover were determined in the 90-day oral toxicity study and thus there were no evidence for the development of a hyaline droplet nephropathy (Chou and Poul 2005; Dill et al. 2003; Read et al. 1999). Hyaline-like droplets in the renal tubular epithel cells of male animals seemed to be not adverse on the basis of supporting literature data. As indicated by the ECETOC guidance document (2002) an effect is less likely to be adverse if there is no alteration in the general function of the test organism, organs or tissues affected and if it is transient, isolated or independent. In addition, the recovery group showed no hyaline-like droplets indicating the reversibility of the finding and supporting that the effect is not adverse since a change which is readily and completely reversible on cessation of treatment is considered to be of lower level of concern (ECETOC, 2002). Based on these criteria hyaline-like droplets in the tubular epithel has to be considered as a toxicological relevant, but no adverse effect.
Mild vacuolization was observed in tubular epithel cells in the proximal convoluted tubuli in two female animals in the 160 mg/kg bw/day group (2/10). The recovery group showed no vacuolization and thus the finding was considered to be a reversible alteration. Due to the low incidence i.e. mild vacuolization in 2/10 female animals and the reversibility these finding was considered to be of no toxicological importance.
Perilobular vacuolization of hepatocytes (minimal to moderate degree) was observed in a control and test item treated animals as follows: male animals: 1/10 at 160 mg/kg bw/day; 1/10 at 10 mg/kg bw/day and 1/10 control; female animals: 5/10 at 160 mg/kg bw/day; 4/9 in the control group.
The cytoplasmic vacuolization due to lipid accumulation is a common finding in the experimental rats. Increased synthesis or decreased transport of the lipids leads to lipidosis. In this study, perilobular vacuolization of hepatocytes was present with the same incidence in the control and high dose treated animals therefore it was considered to be independent from the test item.
In the lungs, minimal or mild focal alveolar emphysema was present in the control (2/10 male and 3/10 female) and in the 160 mg/kg bw/day (1 /10 male and 2/10 female) groups. Focal alveolar emphysema may be a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination. Since the focal alveolar emphysema were also present in the control group animals with comparable incidence these changes were considered to be not test item related.
The hyperplasia of bronchus associated lymphoid tissue (BALT) in some control (2/10 male and 3/10 female) and 160 mg/kg bw/day treated animals (2/10 male and 2/10 female) as well as the involution of the thymus in control (4/10 male and 1/10 female) and high dose treated animals (7/10 male and 1/10 female) are physiological phenomenon and therefore not test item related.
Dilatation of uterus was observed in several females: 6/10 in the control and 2/10 in 160 mg/kg bw/day group. Hydrometra - without inflammation or other pathological lesion - is also a physiological phenomenon in connection with the normal sexual cycle of females and therefore not test item related.
No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

Recovery group
No hyaline droplets were observed in the kidneys of male animals of the 160 mg/kg bw/day recovery group and thus the finding hyaline-like droplets was considered to be a reversible alteration.
Focal pulmonary alveolar emphysema (control: 2/5 male and 1/4 female; 160 mg/kg bw/day: 1/5 male and 1/5 female) and hyperplasia of bronchus associated lymphoid tissue (control: 1/5 male and 1/4 female; 160 mg/kg bw/day: 2/5 male and 1/5 female) were also present in the recovery animals. Since the effects were also present in the control group animals with comparable incidence, the finding was considered not to be test item related. Furthermore, the involution of the thymus in recovery control and treated animals in this age was considered also as a physiological phenomenon (control: 1/5 male and 1/4 female; 160 mg/kg bw/day: 3/5 male and 3/5 female) and therefore not test item related.

SPERM EXAMINATIONS
Sperm examinations did not point out any test item related influence on the sperm cells at 160 mg/kg bw/day. The total sperm count and sperms with not normal morphology (separated head and tail) were similar in 160 mg/kg bw/day and in the control groups. Statistical significances were noted for higher percentage of motile sperm cells and for the less percentage of immotile sperms in animals of 160 mg/kg bw/day. However these differences had no toxicological importance.

EXAMINATION OF ESTROUS CYCLE
A test item influence on the estrous cycle was not detected. The percent of animals with regular and irregular estrous cycle was similar in the control and test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of cycles, days in pro-estrous, estrous or diestrus.

DETERMINATION OF α2μ-globulin
Under the conditions of this study, tert. butylperoxy-3,5,5- trimethylhexanoate (TBPIN) did not cause an increased α2μ-globulin levels in male rats. In contrast, the values obtained by image analysis using previously a monoclonal antibody were generally lower as compared with control animals. The presence of α2μ-globulin in the kidneys of male Wistar rats of this strain is deemed to reflect the normal physiological situation.

REFERENCES

Chou CHSJ and Pohl HR (2005). Health effects classification and its role in the derivation of minimal risk levels: Renal effects. Regulatory Toxicology
and Pharmacology 42: 202-208.

Dill JA, Lee KM, Renne RA, Miller RA, Fuciarello AF, Gideon KM, Chan PC, Burka LT and Roycroft JH (2003). a2μ-Globulin nephropathy and
carcinogenicity following exposure to Decalin (Decahydronaphthalene) in F344/N rats. Toxicological Sciences 72: 223–234.

ECETOC Guidance (2002) Recognition of and Differentiation between, Adverse and Non-adverse effects in Toxicoloigal studies. Technical Report No. 85.

Hard GC, Snowden RT (1991). Hyaline droplet accumulation in rodent kidney proximal tubules: An association with histiocytic sarcoma. Toxicol
Pathol 19:88-97.

Read NG (1991). The role of lysosomes in hyaline droplet nephropathy induced by a variety of pharmacological agents on the male rat.
Histochemical Journal 23: 436-443.

Yamate J, Iwaki M, Nakatsuji S, Kuwamura M, Kotani T, Sakuma S (1998). Lysozyme-containing renal renal tubular hyaline droplets in F344 rats bearing a rat fibrosarcoma-derived transplantable tumor. Toxicol Pathol 26:699-703.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Key result
Dose descriptor:
NOEL
Effect level:
< 10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Salivation at 160 and 40 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this read across study, the no observed effect level (NOEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat was < 10 mg/kg bw /day for male rats and 10 mg/kg bw/day for female rats. The no observed adverse effect level (NOAEL) was 160 mg/kg bw/day for male and female rats.
Executive summary:

The objective of this read across study was to obtain information on the possible health hazards likely to arise from repeated exposure with tert. butylperoxy-3,5,5- trimethylhexanoate (TBPIN) at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 160, 40 and 10 mg/kg bw/day doses corresponding to concentrations of 0, 32, 8 and 2 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. 5 animals/ sex in the control and high dose groups were observed without administration for four weeks (recovery observations).

A sufficient stability of tert. butylperoxy-3,5,5-trimethylhexanoate (TBPIN) in the chosen vehicle was analytically verified up front. tert. butylperoxy- 3,5,5-trimethylhexanoate (TBPIN) was stable in the applied concentrations in sunflower oil at room temperature for 4 hours and in a refrigerator for 3 days. Concentrations of the test item in the dosing formulations varied from 99 % to 106 % of nominal concentrations at both analytical occasions, thereby confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. The kidneys were also evaluated histologically in all animals of the low and mid dose groups due to the necropsy and histopathological findings in the high dose group. The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

 

Results:

There was no test item related mortality. Toxic signs related to the test item were not detected at any dose level at the daily and detailed weekly clinical observations and in the course of the functional observation battery. Salivation was observed in the male and female animals of the 160 or 40 mg/kg bw/day groups with variable frequency within a group but in a dose related manner regarding the incidence and onset. The body weight development of male and female animals was not affected by the test item. No test item-related body weight, or body weight gain changes were observed with respect to controls at any dose level during the course of the study. The daily mean food consumption was similar in animals of the control and test item treated groups (160, 40 and 10 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment. No test item-related changes were observed in investigated hematology or blood coagulation parameters. Clinical chemistry examinations did not reveal any pathologic changes in the examined parameters. At necropsy the kidneys were judged to be pale in some animals (male and female) of 160 mg/kg bw/day groups and in two female animals at 10 mg/kg bw/day at the terminal necropsy. The liver was enlarged, pale and nutmeg-like patterned in several animals of 160 mg/kg bw/day group and in single male animals of 40 and 10 mg/kg bw/day groups. Since kidney and liver weight values remained within the historical control ranges and there were no related histopathological lesions in the kidneys and the liver these changes were considered to be signs of adaptation of the organ to the altered demands. The mean liver and kidney weights (absolute and relative to the body and brain weights) were higher with respect to their controls in the male and female animals of 160 mg/kg bw/day group. Similar findings were also noted for the higher mean liver weight relative to body weight in male animals of 40 and 10 mg/kg bw/day groups and for the higher mean kidney weights relative to body and brain weights of male animals at 40 mg/kg bw/day. Although these differences reached statistical significances with respect to controls, all values remained within the historical control ranges. Histopathology investigations revealed test item related hyaline –like droplets in the epithel cells of some proximal convoluted tubules in the kidney of male animals treated with 160, 40 and 10 mg/kg bw/day, which was accompanied by dilatation of tubuli in the distal area, at the border of cortical – medullary region at 160 and 40 mg/kg bw/day. The severity of the lesions decreased in the low dose group as compared with the middle or high dose groups. The renal lesion was not present at the end of the recovery period and no additional renal lesions or increased cell turnover were determined. In accordance with literature data (ECETOC, 2002) the isolated and reversible finding hyaline-like droplets in male rats has to be considered as a toxicological relevant, but not adverse effect.Immunohistochemistry examinations demonstrated lower level ofα2μ-globulin in male animals of the 160 mg/kg bw/day compared withcontrol animals, therefore the presence ofα2μ-globulin in the kidneys of male animals was deemed to reflect the normal physiological situation. Therefore, hyaline-like droplets at 160, 40 and 10 mg/kg bw/day doses in male ratscould not be caused byα2μ-globulin. A test item influence on the estrous cycle or influence on the sperm cells (count, mortality and morphology) was not detected.

Conclusion:

Based on these observations the no observed effect level (NOEL) for tert-Butylperoxy-3,5,5-trimethylhexanoat was < 10 mg/kg bw /day for male rats and 10 mg/kg bw/day for female rats. The no observed adverse effect level (NOAEL) was 160 mg/kg bw/day for male and female rats.

Reference

ECETOC Guidance (2002) Recognition of and Differentiation between, Adverse and Non-adverse effects in Toxicoloigal studies. Technical Report No. 85.