Registration Dossier

Administrative data

Description of key information

Based on the observations of the 90-day toxicity study on rats, the No Observed Adverse Effect Level (NOAEL) was determined as follows: 450 mg/kg bw/day for male and female animals.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-06 to 2013-06-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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: 36 – 41 days, Female animals: 36 – 41 days
- Weight at study initiation: Male animals: 136 – 167 g, Female animals: 104 – 134 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: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 2013-02-06 to 2013-06-04
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 and not stable 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: 14, 30, 90 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 19T2
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) on 2 occasions. One sample of 5 mL was taken from the control substance (group 1) on both occasions and measured:
Date of samplings: February 07, May 02, 2013
Date of measurement: February 08, May 03, 2013.
The samples were stored in a refrigerator until the analysis.
Measured concentrations varied between 103 and 117 % of the nominal concentrations and all formulations were considered to be homogeneous as tert. Butylperoxy-2-ethylhexanoate is soluble in sunflower oil.
The suitability of the chosen vehicle for the test item (stability and homogeneity) was analytically proven. Recovery was 104 % (of nominal concentration) at ~2 mg/ml, and 98 % at ~500 mg/ml concentration.
tert. Butylperoxy-2-ethylhexanoate was stable at concentrations of 2 and 500 mg/mL in sunflower oil in the refrigerator for 72 hours (at 5 +/- 3°C; recovery was 94 and 96 % of starting concentrations at 2 and 500 mg/mL, respectively) and after 24 hours at room temperature (recovery was 96 and 100 % of starting concentrations of 2 and 500 mg/mL).

HPLC Method:
Detector: 210 nm
Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm, No.: 10013/0605127W
Mobil Phase: Acetonitrile : Water (9 : 1 (v/v))
Flow Rate: 1.2 mL/min
Injection volume: 50 μL
Temperature: 25 °C
Retention time : 5,3 min ± 10 %
Duration of treatment / exposure:
90 or 91 days (depending on day of necropsy)
Frequency of treatment:
Once a day, 7 days/week, every day at a similar time (+/- 2 hours).
Dose / conc.:
70 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
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 previous repeated dose toxicity studies with tert. Butylperoxy-2-ethylhexanoate in the Rat [Tert-butyl peroxy-2-ethylheanoate, techn. pure (TBPEH): 28-day oral toxicity study in rats, Report no. SL-LT-028/10].
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
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: prior to the first exposure and once weekly thereafter
- Detailed clinicla observations checked in table [No.1] were included.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed in the treatment period on Day 0, then weekly. 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
- Time schedule for examinations: The food consumption was determined in the treatment phase on Day 7, then weekly by reweighing the non-consumed diet.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimation period and prior to test termination (Day 85)
- Dose groups that were examined: all control and high dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment (Day 90 and 91) and on the end of the recovery period (Day 118)
- Anaesthetic used for blood collection: Yes, under Isofluran anesthesia
- Animals fasted: Yes
- How many animals: all animals of 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 (Day 90 and 91) and on the end of the recovery period (Day 118)
- Animals fasted: Yes
- How many animals: all animals of 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
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength, motor activity

ESTROUS CYCLE
- Time schedule for examinations: during the last 2 weeks of the treatment and recovery period, respectively
- Dose groups that were examined: female animals of all dose groups
- Examinations: 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 EXAMINATIONS:
- Dose groups that were examined: male animals of the control and high dose group
- 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, see table No. 4 (including organs weights)
HISTOPATHOLOGY: Yes, see table No. 5
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 intergroup 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 frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female animal of the low dose group died on Day 4. Salivation was observed in male and female animals administered with 450, 150 or 70 mg/kg bw/day with variable frequency within a group and with a dose related manner.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female animal of the low dose group died on Day 4. Salivation was observed in male and female animals administered with 450, 150 or 70 mg/kg bw/day with variable frequency within a group and with a dose related manner.
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no test item related mortality at any dose level (450, 150 or 70 mg/kg bw/day).
One female rat of 70 mg/kg bw/day died on Day 4. There were no preceding clinical signs or body weight changes. Histological examination revealed an individual disease i.e. malignant tumor (sarcoma polymorphocellulare) in the liver and adrenal glands (one side) as the cause of death.
Treatment period
The behavior and physical condition of animals were considered to be normal at each dose level (450, 150 and 70 mg/kg bw/day) during the treatment period.
Test item related salivation was observed in male and female animals administered with 450, 150 or 70 mg/kg bw/day with variable frequency within a group and with a dose related manner. Salivation was noted for some animals before the administration (1/10 male in the 70 mg/kg bw/day group; 6/15, 1/10 and 1/15 female in the 450, 150 and control groups, respectively). One male animal of 450 mg/kg bw/day (1/15), showed transiently slight decreased activity between Days 51 and 61, which was an individual sign. There were no more animals (male or female) in the high dose group showing activity decrease therefore it was not considered to be related to the treatment.
Some animals of the control group (4/15 male and 7/15 female) also salivated in a slight or moderate degree.
Individual dermal alteration i.e. alopecia on the right side cheek was observed in one female animal of 450 mg/kg bw/day group (between Days 28 and 35, between Days 56 and 78, between Days 84 and 89). Alopecia is a common clinical signs in this strain of experimental rats of this age and was only present in single animal of the high dose group therefore was considered toxicologically not relevant.
Recovery period
Clinical signs were not detected in the male or female animals of 450 mg/kg bw/day groups or in the control group during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
Treatment period
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (450, 150 and 70 mg/kg bw/day) during the entire observation period.
Statistical significances noted for the slightly less mean body weight gain of male animals in groups of 150 mg/kg bw/day between Days 70 and 77 was transient and with low degree.
A slightly higher mean body weight gain was observed in the male animals of 450 and 70 mg/kg bw/day between Days 77 and 84, both, as well as in the female animals of 450 mg/kg bw/day between Days 35 and 42 with respect to the appropriate control.
There were no significant differences in the mean body weight or in the total mean body weight gain and values between the control and test item treated groups (450, 150 and 70 mg/kg bw/day), therefore these slight but statistically significant changes were considered to be without any toxicological relevance.
Recovery period
The body weight and body weight gain were similar in the male and female animals of 450 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.
The daily mean food consumption was comparable in the control and all test item treated groups during the entire treatment period (450, 150 and 70 mg/kg bw/day) and during the recovery period (450 mg/kg bw/day).

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

HAEMATOLOGY
Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (450, 150 or 70 mg/kg bw/day).
Main groups
At the termination of the treatment, statistical significances were detected in the white blood cell parameters of female animals i.e. in the mean white blood cell count (WBC) in 450 and 150 mg/kg bw/day groups and in the mean percentage of neutrophil granulocytes (NEU) and lymphocytes (LYM) in 450, 150 or 70 mg/kg bw/day groups. These statistical significances in WBC, NEU and LYM indicated only slight differences between the control and test item treated groups, the values were well within the historical control ranges and were not related to doses, therefore were not considered to be of biological relevance.
Recovery groups
In the recovery group of 450 mg/kg bw/day dose, all examined hematological parameters were similar to the appropriate value in the control group (male and female animals).

CLINICAL CHEMISTRY
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters (450, 150 and 70 mg/kg bw/day).
Main groups
In the male animals administered with 450 mg/kg bw/day, a slightly but statistically significant (p<0.01) higher mean concentration of phosphorous (Pi) and chloride (Cl-), less mean concentration of total protein and slightly higher A/G value were observed with respect to the control at the termination of the treatment. The chloride concentration statistically significantly (p<0.05) exceeded the control value in male animals of 150 mg/kg bw/day but remains well within the historical control range.
In the female animals of 450 mg/kg bw/day group, statistical significances were noted for the less concentration of glucose (GLUC), calcium (Ca2+) and sodium (Na+). The glucose concentration was below the control value in female animals of 150 and 70 mg/kg bw/day groups. In groups of 150 and 70 mg/kg bw/day, a slightly less concentration of creatinine and sodium, respectively, were detected with respect to the control.
Recovery groups
In the male animals of 450 mg/kg bw/day group, the concentration of creatinine and sodium were slightly higher and concentration of albumin (ALB) was slightly but statistically significantly less with respect to their control.
These sporadic statistical differences (Pi, Cl-, CREA, GLUC, Ca2+, Na+, TPROT, ALB and A/G ration) 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 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.

NEUROBEHAVIOUR
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, 450, 150 or 70 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.

ORGAN WEIGHTS
There were no test item related organ weight alterations in the examined organs (450, 150 or 70 mg/kg bw/day) groups.
Main groups
Slight, but statistically significant differences were detected in the less mean brain weights in male animals in 450, 150 and 70 mg/kg bw/day groups. Similar findings were also noted for heart weights (450 and 150 mg/kg bw/day) and heart weight relative to body weight of male animals at 450 mg/kg bw/day with respect to the controls.
The mean thymus weights relative to the brain weight were statistically significantly higher in female animals in 450 mg/kg bw/day group comparing to the control group.
All these statistically significant differences with respect to the appropriate control were with small degree, values remained well within the historical control ranges and related findings were not detected at the histopathological examination therefore these changes were not considered to be toxicologically relevant.
Recovery groups
In the male animals of 450 mg/kg bw/day group, the mean testes weight relative to the body weight was slightly but statistically significantly higher than in the control group. In the female animals, all examined organ weights were comparable to their control at the end of the recovery period.

GROSS PATHOLOGY
Macroscopic changes related to the test item were not detected at the necropsy in 450, 150 or 70 mg/kg bw/day groups.
Necropsy findings of dead animal of 70 mg/kg bw/day (1/10 female; sanguineous liquid in the abdominal cavity, pale liver and eyes) referred to an individual disease, which was also supported by the results of the histopathological examination.
Main groups
Point-like hemorrhages in the thymus were noted for two male animals (2/10) of 70 mg/kg bw/day group which were considered to be acute changes due to the exsanguination procedure and not related to the treatment.
In the female animals, ovarian cyst (1/10 in 150 mg/kg bw/day; 1/10 in the control group) and hydrometra (3/10 in 450 mg/kg bw/day; 3/10 in 150 mg/kg bw/day; 1/10 in control group) were observed at the terminal necropsy. Ovarian cyst and hydrometra are common findings in female animals of this species and strain with similar age and these were judged to be also individual alterations.
Recovery groups
There were no macroscopic findings in male animals of the recovery groups at 450 mg/kg bw/day and in the control group.
In the female animals, compact formation (1/5, control group) and hydrometra (1/5 at 450 mg/kg bw/day) were observed at the end of the recovery period.
Both findings (renal alteration and hydrometra) were individual alterations occurring also in not treated animals of this species and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item related microscopic alterations were not detected in the examined organs or tissues of animals in 450 mg/kg bw/day group (male or female).
Main groups
In the lungs, minimal or mild focal alveolar emphysema was present in the control (2/10 male and 1/10 female) and in the 450 mg/kg bw/day (2 /10 male) groups. Acute pulmonary hemorrhages were detected in two male animals (2 /10) of the control group. Focal alveolar emphysema and hemorrhages in the lungs may be a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The hyperplasia of bronchus associated lymphoid tissue (BALT) in some control (2/10 male and 1/10 female) and 450 mg/kg bw/day treated animals (3/10 male and 1/10 female) is a physiological phenomenon.
Dilatation of uterus was observed in some females: 1/10 in the control and 3/10 in 450 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.
Ovarian cyst occurred in one control female animal (1/10) and was considered to be an individual disorder, which is a common finding in this strain of experimental rats.
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, urinary system, pancreas 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 groups
One side nephroblastoma was detected in one control female animal (1/5) as an individual disorder.
Focal pulmonary alveolar emphysema (1/5 male and 1/5 female, controls), acute hemorrhage (1/5 male, control; 1/5 female at 450 mg/kg bw/day) and hyperplasia of bronchus associated lymphoid tissue (2/5 female control; 1/5 male and 1/5 female at 450 mg/kg bw/day group) were also present in the recovery animals. Furthermore, dilatation of the uterine horn was present in one female rat of 450 mg/kg bw/day group.

ESTROUS CYCLE
A test item influence on the estrous cycle was not detected.
There were no significant differences between the control and test item treated groups in the examined parameters (percentage of animals with regular and irregular estrous cycle, in the mean number of cycles, days in pro-estrous, estrous or diestrus, animals in prolonged estrous or in diestrus).

SPERM EXAMINATIONS
Sperm examinations did not point out any test item related influence on the sperm cells at 450 mg/kg bw/day.
The total sperm count, motile sperms and sperms with not normal morphology (separated head and tail) were similar in animals examined in the 450 mg/kg bw/day and in the control groups (10/10, 10/10, respectively).
Key result
Dose descriptor:
NOAEL
Effect level:
450 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
Critical effects observed:
no
Conclusions:
Based on the observations, the No Observed Adverse Effect Level (NOAEL) was determined as follows: 450 mg/kg bw/day for male and female animals.
Executive summary:

The objective of the study was to obtain information on the possible health hazards likely to arise from repeated exposure with tert. Butylperoxy-2-ethylhexanoate 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), 450, 150 and 70 mg/kg bw/day doses corresponding to concentrations of 0, 90, 30 and 14 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. 5 animals per sex in the control and high dose groups were observed without administration for four weeks (recovery observations).

A sufficient stability of tert. Butylperoxy-2-ethylhexanoate in the chosen vehicle was analytically verified up front. Tert. Butylperoxy-2-ethylhexanoate was stable in the applied concentrations in sunflower oil at room temperature for 24 hours and in a refrigerator for 3 days. Concentrations of the test item in the dosing formulations varied from 103 % to 117 % 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 thymus was also evaluated histologically in two animals of the low dose group due to the necropsy findings.

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 450, 150 and 70 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 (450, 150 and 70 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment.

A test item influence on the estrous cycle was not detected.

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. Specific macroscopic alterations related to the test item were not detected at the necropsy observations. The mean weights (absolute and relative to the body and brain weights) of examined organs were not affected by the test item at any dose level (450, 150 or 70 mg/kg bw/day).

Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 450 mg/kg bw/day dose.

Histopathology investigations did not reveal any test item related toxic or other lesions in the investigated organs.

 

Conclusion:

Under the conditions of the present study tert. Butylperoxy-2-ethylhexanoate did not cause adverse effects in male or female Hsd.Brl.Han: Wistar rats after the consecutive 90-day oral (gavage) administration. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows: 450 mg/kg bw/day for male and female animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There are two repeated dose toxicity studies available (28-day and 90-day study) which were conducted according to GLP and the respective OECD/EU test guideline.

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

Key study: 90-day repeated dose toxicity

The objective of the study was to obtain information on the possible health hazards likely to arise from repeated exposure with tert. Butylperoxy-2-ethylhexanoate 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), 450, 150 and 70 mg/kg bw/day doses corresponding to concentrations of 0, 90, 30 and 14 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. 5 animals per sex in the control and high dose groups were observed without administration for four weeks (recovery observations).

A sufficient stability of tert. Butylperoxy-2-ethylhexanoate in the chosen vehicle was analytically verified up front. Tert. Butylperoxy-2-ethylhexanoate was stable in the applied concentrations in sunflower oil at room temperature for 24 hours and in a refrigerator for 3 days. Concentrations of the test item in the dosing formulations varied from 103 % to 117 % 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 thymus was also evaluated histologically in two animals of the low dose group due to the necropsy findings.

The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only.

 

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 450, 150 and 70 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 (450, 150 and 70 mg/kg bw/day).

There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment.

A test item influence on the estrous cycle was not detected.

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. Specific macroscopic alterations related to the test item were not detected at the necropsy observations. The mean weights (absolute and relative to the body and brain weights) of examined organs were not affected by the test item at any dose level (450, 150 or 70 mg/kg bw/day).

Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 450 mg/kg bw/day dose.

Histopathology investigations did not reveal any test item related toxic or other lesions in the investigated organs.

 

Under the conditions of the present study tert. Butylperoxy-2-ethylhexanoate did not cause adverse effects in male or female Hsd.Brl.Han: Wistar rats after the consecutive 90-day oral (gavage) administration. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows: 450 mg/kg bw/day for male and female animals.

 

Supporting study: 28-day repeated toxicity

Tert.-Butylperoxy- 2-ethylhexanoat was tested in a 28 day repeated dose toxicity study by oral application in rats according to EU method B.7 and OECD guideline 407. The test item was administered at 100, 316 and 1000 mg/kg bw/day. The substance caused a decrease in the number of platelets in the blood of the mid and the high dose females and some minor liver changes (higher organ weights in the high dose groups of both sexes, elevated plasma alkaline phosphatase level in the high dose females). There was also a borderline indication for renal tubular alterations in the high dose females. There was a sex difference in the response to the test substance with different effects in both sexes and a somewhat higher susceptibility of the females. The NOAEL was 316 mg/kg bw/day in males and 100 mg/kg bw/day in females.

 


Conclusion:
The repeated dose toxicity study covering a longer exposure period, i. e. the 90 -day toxicity study, is preferred for the assessment of substance-related effects after long-term administration. Therefore, the NOAEL obtained in this study (450 mg/kg bw/d) would be taken into account for DNEL derviation. However, there are data available from an EOGRTS where exposure duration lasted for up to 17 weeks and which revealed an NOAEL of 100 mg/kg bw/d based on affected fertility parameters. Thus, this more sensitive endpoint is applied as PoD for DNEL derivation.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.