tert-pentyl 2-ethylperoxyhexanoate

Endpoint summary

Description of key information

Risk assessment was assessed using the results of the 90-day study conducted on the read-across substance CAS# 3006-82-4.

The DNELs calcuated using this study were the most conservative of studies available when considering the repeat dose study on the read-across CAS# 3006-82-4 and OECD 421 available on CAS# 686-31-7.
CAS# 3006-82-4 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
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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2013-02-06 to 2013-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP regulations and the respective OECD/EU guideline.

Justification for type of information:

see cross-referenced read-across:supporting information in Section 13.2 attachment

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

Deviations:

no

Qualifier:

according to guideline

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

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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).

Remarks:

Doses / Concentrations:
70, 150, 450 mg/kg bw/day
Basis:
actual ingested

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

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

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:

not specified

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March-24 April 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; coherence between data, results and conclusion

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. Animals were bred by Harlan Laboratories Netherland and supplied by Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
- Age at study initiation: 6 weeks
- Weight at study initiation: approximately 141 g for males and 137 g for females
- Housing: clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor
- Diet (e.g. ad libitum): 4 RF 21
- Water (e.g. ad libitum): via bottle
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C+/- 2°C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours each day

IN-LIFE DATES: From: 21 March To: 24 April 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF FORMULATIONS:

DOSE PREPARATION
- Rate of preparation of dose (frequency): daily or a few days before administration on the basis of the stability data

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was chosen due to the chemical/phisical properties of the compound
- Concentration in vehicle: 20, 60 and 200 mg/mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC-MS/MS validated method was applied to verify concentration and homogeneity of the formulations. Prior to commencement of treatment,
analysis was performed to confirm that the proposed formulation procedure was acceptable for content check and homogeneity.
Stability data of Tert-pentyl 2-ethylperoxyhexanoate in corn oil at 1 and 200 mg/mL over a 24 hour period and 8 days at +4°C were available.
Samples of the formulations, prepared on Weeks 1 and 4 of treatment, were also analysed to check the concentration and homogeneity.
The results of analyses were within the acceptance range (90-110%).
The method was validated and the stability of tert-pentyl 2-ethylperoxyhexanoate formulations at +4°C was also evaluated.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once a day, 7 days a week

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female rats/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The aim of this study was to investigate the toxicity of Tert-pentyl 2-ethylperoxyhexanoate when given orally by gavage to rats for 4 consecutive
weeks. Three groups of 5 rats per sex each received the test item at 100, 300 and 1000 mg/kg/day, while a similar constituted group received the
vehicle alone (corn oil) and acted as control.
The following parameters were monitored during the in vivo phase: mortality, body weight, clinical signs, including neurotoxicity assessment,
food consumption and clinical pathology investigation.
The animals were subjected to detailed macroscopic observations at necropsy, including organ weights and histopathological examination.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least 3 times a week

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just
prior to necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The interval between allocation and
treatment initiation was less than a full week. The group mean daily intake per rat was calculated.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment, just prior to necropsy
- Anaesthetic used for blood collection: Yes (isofluorane anaesthesia )
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Parameters checked in table [No. 6] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks of treatment, just prior to necropsy
- Animals fasted: Yes
- How many animals: 5 males and 5 females from each group
- Parameters checked in table [No.7] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks of treatment, just prior to necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (animals received approximately 10 mL/kg of drinking water by gavage)
- Parameters checked in table [No.8] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once per week
- Dose groups that were examined: all goups
- Battery of functions tested: sensory activity / grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Terminal body weight
Absolute organ weight
Relative organ weight to body weight

Statistics:

Standard deviations were calculated as considered appropriate.
For continuous variables (body weight, clinical pathology investigations, absolute and relative organ weights) the significance of the differences
amongst group means was assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test
before Dunnett’s test. If the data were found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
Statistical analysis of histopathological findings was carried out by means of the non parametric Kolmogorov Smirnov test.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was detected especially in the mid- and high dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation was detected especially in the mid- and high dose groups.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

kidney and/or liver weights increased in the mid and high dose groups when compared to the controls.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: no mortality occurred. Salivation was found in animals receiving 300 and 1000 mg/kg/day.

BODY WEIGHT: no effects. No significant differences were recorded in body weight between the groups. A slight
reduced mean body weight was only observed at the end of treatment period in high dose
females when compared to the control group

FOOD CONSUMPTION: no effects

HAEMATOLOGY: no effetcs

CLINICAL CHEMISTRY: Some males treated with 1000 mg/kg/day showed increments of cholesterol, triglycerides, glucose, urea, globulin and protein and decrement of phosphorus, creatinine and bile acids.
A number of females from the same group showed high levels of alkaline phosphatase, cholesterol, triglycerides, bile acids and chloride and
decrease of aspartate aminotransferase.
The above findings were sporadic, of low severity and/or inconsistent between sexes, therefore they were considered of no toxicological significance.

URINALYSIS: no effects

NEUROBEHAVIOUR: no effects

ORGAN WEIGHTS: At the end of dosing period, a statistically significant increase was recorded in kidney and/or liver weights of the mid- and high
dose groups (animals dosed at 300 and 1000 mg/kg/day, respectively) when compared to the controls.
Males: absolute and relative liver weight increased up to 35% (high dosage)
absolute and relative kidneys weight increased up to 18.8% (high dosage)
Females: absolute and relative liver weight increased up to 18.8 % and 28.7%, respectively (high dosage)

GROSS PATHOLOGY: no effects

HISTOPATHOLOGY: NON-NEOPLASTIC: no effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Increase of liver and kidney weight with no histhological findings.

Key result

Critical effects observed:

not specified

The doses were chosen based on the results of the DRF (see the RSS for more information).

Conclusions:

No evidence of toxic effects was seen in animals receiving the test item up to 1000 mg/kg/day.
Minor effects were restricted to changes in clinical chemistry investigations (increased circulating bile acids, triglycerides, cholesterol) and increasedliver weight with no histopathological findings. These effects may be considered an adaptive response to a metabolic stress.
On the basis of the results obtained in this study, the NOAEL of tert-pentyl 2-ethylperoxyhexanoate was 1000 mg/kg/day for both males and females.

Executive summary:

The aim of this study was to investigate the toxicity of Tert-pentyl 2-ethylperoxyhexanoate when given orally by gavage to rats for 4 consecutive weeks. The study was performed according to the OECD 407 guideline abnd was in compliance with the GLP.

 

Three groups of 5 rats per sex each received the test item at 100, 300 and 1000 mg/kg/day, while a similar constituted group received the vehicle alone (corn oil) and acted as control.

The following parameters were monitored during the in vivo phase: mortality, body weight, clinical signs, including neurotoxicity assessment, food consumption and clinical pathology investigation. The animals were subjected to detailed macroscopic observations at necropsy, including organ weights and histopathological examination.

 

At clinical examination, salivation was the relevant sign recorded in treated animals mainly in the mid- and high dose groups. Neurotoxicity assessement, such as weekly detailed clinical observations, sensory reactivity to different stimuli and motor activity did not show differences between the groups. No significant changes of body weight and food consumption were observed between groups. No changes of toxicological relevance were observed in haematological parameters (including coagulation tests), in biochemical parameters and in urinanalysis after 4 weeks of treatment.

 

No significant differences were recorded in body weight between the groups. A slight

reduced mean body weight was only observed at the end of treatment period in high dose

females when compared to the control group. Liver and/or kidneys weights were significantly increased in treated animals mainly at the highest dosage.

At necropsy, reduced size of prostate glands in several high dose males and increased incidence of red colour of the pituitary in treated females, were recorded, but no treatment-related microscopic changes were seen in treated animals.

 

Minor effects were restricted to changes in clinical chemistry investigations (increased circulating bile acids, triglycerides, cholesterol) and increased liver weight with no histopathological findings. These effects may be considered an adaptive response to a metabolic stress.

 

In conclusion no evidence of toxic effects was seen in animals receiving the test item up to 1000 mg/kg/day. On the basis of the results obtained in this study, the NOAEL of Tert-pentyl 2-ethylperoxyhexanoate was 1000 mg/kg/day for both males and females.

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:

Klimisch 1 study, GLP compliant

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

Repeated dose toxicity was assessed using the results of the 90-day study conducted on the read-across substance CAS# 3006-82-4. 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 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.

CAS# 3006 -82 -4 Tert.-Butylperoxy- 2-ethylhexanoate was tested in 28 -day oral (gavage) toxicity study in male and female Fischer, F344/DuCrl. rats according to Council Regulation (EC) No. 440/2008, Annex B.7. and OECD Guideline 407, including recovery groups for counted and high dose animals. Dose levels administered in this study were selected based on data obtained during 7 -day-dose-range-finding toxicity study. Two control (with and without recovery) and four dose groups (n=5 animals per group and sex; low mid, high dose and high dose recovery) were investigated in this study. The test item was administered at 100 mg, 316 mg and 1000 mg per body weight dose levels. The vehicle is Corn oil.

The test substance caused a decrease in the number of platelets in the blood of the mid and the high dose males and some minor liver changes in both sexes (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. The effect on the circulating platelets was not accompanied by any notable alteration of their bone marrow precursors, the megakaryocytes, or any other parameter of the bone marrow. The liver changes may be based on enzyme induction, as no histological alterations were found in this organ. The effects noted were at most mild in severity, never became life threatening, but persisted partly until the end of the recovery period. In the study report, the No-observed-adverse-effect-level (NOAEL) of tert.-Butylperoxy- 2-ethylhexanoat was determinated at 316 mg per kg body weight in the males and at 100 mg per kg body weight and day in the females. However, when administered up to 450 mg/kg/day for 90 -days, no adverse effects were noted (see above summary).

CAS# 686 -31 -7 was given orally by gavage to Fischer rats for 4 consecutive weeks to three groups of 5 rats per sex each at 100, 300 and 1000 mg/kg/day, while a similar constituted group received the vehicle alone (corn oil) and acted as control (Monetini, 2012). At clinical examination, salivation was the relevant sign recorded in treated animals mainly in the mid and high dose groups. Neurotoxicity assessement, such as weekly detailed clinical observations, sensory reactivity to different stimuli and motor activity did not show differences between the groups. No significant changes of body weight and food consumption were observed between groups. No changes of toxicological relevance were observed in haematological parameters (including coagulation tests), in biochemical parameters and in urinanalysis after 4 weeks of treatment. No significant differences were recorded in body weight between the groups. A slight reduced mean body weight was only observed at the end of treatment period in high dose females when compared to the control group. Liver and/or kidneys weights were significantly increased in treated animals mainly at the highest dosage. At necropsy, reduced size of prostate glands in several high dose males and increased incidence of red colour of the pituitary in treated females, were recorded, but no treatment-related microscopic changes were seen in treated animals. Minor effects were restricted to changes in clinical chemistry investigations (increased circulating bile acids, triglycerides, cholesterol) and increased liver weight with no histopathological findings. These effects may be considered an adaptive response to a metabolic stress. In conclusion no evidence of toxic effects was seen in animals receiving the test item up to 1000 mg/kg/day. On the basis of the results obtained in this study, the NOAEL of tert-pentyl 2-ethylperoxyhexanoate was 1000 mg/kg/day for both males and females.

The possible effects of CAS# 686 -31 -7 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower (<10%) than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxicity was considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Justification for classification or non-classification

According to EU directive 67/548/EEC and according to EU Regulation (EC) N0. 1272/2008 (CLP), tert-amyl peroxy-2-ethylhexanoate is not classified for repetated dose toxicity.