Registration Dossier

Administrative data

Description of key information

A repeat dose study in rats was conducted in rats orally dosed with the test item for 4 weeks (OECD 407). In this GLP subacute toxicity study, effects were seen in liver, forestomach, and in kidney. Slightly elevated liver weights noted in the females treated with 300 or 1000 mg/kg/day were considered to be adaptive, non-adverse changes of metabolic origin. Forestomach mucosal necrosis was recorded in males treated with 100 mg/kg/day and both sexes treated with 300 mg/kg/day and 1000 mg/kg/day. The microscopic effects on the forestomach were considered to be local injuries that resulted in subsequent adverse reactions. Increased severity of hyaline droplets in the renal proximal tubules in males treated with 1000 mg/kg/day. This was considered to be a protein specific to male rats similar to alpha-2 microglobulin, and generally derived from hyperfunction of the liver. Although this is considered to be an adverse finding in rats, this protein has no counterpart in humans and is therefore of no toxicological relevance. The no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach. However, the no-observed-adverse-effect-level (NOAEL) for systemic toxicityof O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was considered to be 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-Apr-2012 to 18-Dec-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted accrding to GLP in compliance with internationally accepted test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
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

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: Groups 1 to 4, 5 males and 5 females in each group. Group 10, 1 male and 1 female reserve animals
- Age (at Delivery): Ca. 7 weeks
- Body Weight Range (at Acclimatization): 210.3 to 233.0 g (mean 220.8 g) in males and 131.3 to 153.9 g (mean 145.1 g) in females
- Identification: during acclimatization cage card and tail mark (later ear tattoo), during treatment cage card and individual ear tattoo
- Randomization: Randomly allocated to groups by body weight.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
DOSE FORMULATIONS

The test item was used as supplied by the Sponsor. The purity was not corrected.

The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study D45848), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was weighed into a glass beaker on a tared Mettler balance.

The vehicle was added and the mixtures were stirred using a magnetic stirrer. The dose formulations were used at room temperature (20 ± 5 °C).

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

These analyses were repeated under GLP for this study.

STORAGE OF DOSE FORMULATIONS

The dose formulations were stored in glass beakers in the refrigerator (5 ± 3°C)

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range finding toxicity study in Wistar rats, Harlan Laboratories study D45848
- Dose Volume: 5 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1), 20 mg/mL/day (Group 1), 60 mg/mL/day (Group 3) and 200 mg/mL/day (Group 4),
- Duration of Pre-Randomization Phase: 1 day
- Duration of Acclimatization Phase: 7 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

The analysis was performed by Harlan Laboratories Ltd. using a HPLC method provided by the Sponsor.

After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration of the first formulation were taken to confirm stability (4 hour and 8 days).

The samples were delivered to the analytical department (Harlan Laboratories Ltd., Analytics, Zelgliweg 1, 4452 Itingen / Switzerland) at ambient temperature and stored there at -20 ± 5 °C until analysis.

The test item was used as analytical standard.

RESULTS

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R2) calculated exceeded 0.99.

The O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate and, therefore, the absence of the test item in the vehicle control samples (PEG300) was confirmed.

The application formulations investigated during the study contained the test item in the range of 88.2% to 109.5%, thereby meeting the required content limit of ±20% with reference to the nominal content. Single results did not deviate more than 4.7% (acceptance criterion: <15%) from the corresponding mean, demonstrating that the dose formulations were homogenous. Recoveries met the variation limit of 10% from the time-zero (homogeneity) mean, thereby showing that the test item was stable in application formulations when kept four hours at room temperature or for eight days refrigerated.

In conclusion, the results indicate the accurate use of the test item O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate and PEG300 as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficiently stable.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
other: Purity not corrected
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
In this subacute toxicity study, O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Because of possible test-item-related-findings that were noted in animals of the high dose group, the stomach and liver from both sexes and kidneys from males of the low and intermediate groups were also examined to establish a no-effect level.
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY

Observations for viability / mortality were recorded twice daily.

DAILY OBSERVATIONS

The animals were observed for clinical signs once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.

WEEKLY BEHAVIORAL OBSERVATIONS

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 3) thereafter. More details on the investigations can be found in the section “Any other information on materials and methods incl. tables” below.

FUNCTIONAL OBSERVATIONAL BATTERY (SCREEN)

During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results are present in the summary and individual tables of the weekly detailed clinical observations under week 4.

- Grip Strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

- Locomotor Activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

FOOD CONSUMPTION

The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

BODY WEIGHTS

Body weights were recorded weekly during acclimatization and treatment periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

CLINICAL LABORATORY INVESTIGATIONS

Blood Sampling at 4 Weeks: 25-May-2012

Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn using a micro-hematocrit glass capillary tube.

Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

HEMATOLOGY

The following hematology parameters were determined:

Complete Blood Cell Count
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Reticulocyte count
- Reticulocyte maturity index (low, medium, high fluorescence)
- Leukocyte count, total
- Differential leukocyte count:
- Neutrophils
- Eosinophils
- Basophils
- Lymphocytes
- Monocytes
- Large unstained cells
- Platelet count

Hemoglobin Derivatives
- Methemoglobin
- Heinz bodies (slides were prepared but not evaluated)

Coagulation
- Prothrombin time (= thromboplastin time)
- Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY

The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Phospholipids
- Aspartate aminotransferase
- Alanine aminotransferase
- Lactate dehydrogenase
- Alkaline phosphatase
- Bile acids
- Gamma-glutamyl-transferase
- Creatine kinase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
Sacrifice and pathology:
NECROPSY

Sacrifice after 4 Weeks: 25-May-2012

All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

A blood sample was taken from each animal by heart puncture (ca. 2 mL) into appropriate tubes (Vacutainer glass tubes containing SST-Gel and Clot Activator or the equivalent) for serum preparation and then placed on ice. Following centrifugation, the serum was divided in 2 aliquots of at least 350 µL each and transferred to plastic (polypropylene) tubes. These samples were stored at approximately -80 °C and protected from light prior to transfer for possible analysis of hormone activity. In the absence of thyroid related changes in this study, these optional evaluations were not performed.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

- Adrenal glands
- Aorta
- Bone (sternum, femur including joint)
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Esophagus
- Eyes w/optic nerve (fixed in Davidson's solution)
- Harderian gland (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Larynx
- Lacrimal gland, exorbital
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Mammary gland area
- Nasal cavity
- Ovaries
- Pancreas
- Pharynx
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Salivary glands - mandibular, sublingual
- Sciatic nerve
- Skeletal muscle
- Skin
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Tongue
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with cervix, vagina and oviducts
- All gross lesions

ORGAN WEIGHTS

The organs from animals listed below were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

- Adrenal glands
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Epididymides (fixed in Bouin's solution)
- Heart including auricles
- Kidneys
- Liver
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Spleen
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Uterus, with cervix, vagina and oviducts

The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

HISTOTECHNIQUE

All organ and tissue samples, as defined under Histopathology below, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY

Slides of all organs and tissues listed below collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist.

- Adrenal glands
- Bone marrow (femur)
- Brain - including section of medulla/pons, cerebral and cerebellar cortex
- Cecum
- Colon
- Duodenum
- Epididymides (fixed in Bouin's solution)
- Eyes w/optic nerve (fixed in Davidson's solution)
- Heart including auricles
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Kidneys
- Liver
- Lungs, filled w/formalin at necropsy
- Lymph nodes – mesenteric and mandibular
- Ovaries
- Pituitary gland
- Seminal vesicles/Prostate gland incl. coagulating glands
- Rectum
- Sciatic nerve
- Skeletal muscle
- Spinal cord - cervical, midthoracic, lumbar
- Spleen
- Stomach
- Testes (fixed in Bouin's solution)
- Thymus
- Thyroid (incl. parathyroid gland, if possible)
- Trachea
- Urinary bladder, filled w/formalin at necropsy
- Uterus, with cervix, vagina and oviducts
- All gross lesions

Because of possible test-item-related-findings that were noted in animals of the high dose group, the stomach and liver from both sexes and kidneys from males of the low and intermediate groups were also examined to establish a no-effect level. The stage of estrus at necropsy was recorded.

Attempts were made to correlate gross observations with microscopic findings. A peer review was performed. The findings of the study pathologist and the peer-reviewing pathologist compared favorably. The peer review report is included in the raw data.
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, clinical laboratory data, organ weights and ratios as well as macroscopic findings:

- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

- Fisher's exact-test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, male mean food consumption reduced during W1 and 2, in females throughout treatment slightly lower mean food consumption considered test item related. Males at 300 mg/kg bw/day slight mean food consumption reduced in W1 and 2.
Food efficiency:
not examined
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:
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:
effects observed, treatment-related
Description (incidence and severity):
Forestomach mucosal necrosis recorded in males at 100 mg/kg/day and both sexes at 300 and 1000 mg/kg/day considered to be local injuries that resulted in subsequent adverse reactions
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA

All animals survived the scheduled treatment or recovery periods.

DAILY OBSERVATIONS

There were no findings evident in any male or female at any dose level.

WEEKLY BEHAVIORAL OBSERVATIONS

During week 1, slightly decreased activity, hunched posture and ruffled fur were noted in one male treated with 1000 mg/kg/day. These findings were not seen during subsequent weekly observations at weeks 2 and 3, nor were they evident in any other male at this dose level. The females of all dose groups were without findings during weeks 1-3 of treatment.

FUNCTIONAL OBSERVATIONAL BATTERY

There were no findings evident in any male or female at any dose level during the functional observational battery performed at week 4.

- Grip Strength: The mean hind-limb grip strength of males at all dose groups were significantly lower (all p<0.05) than those of the control males. In the absence of similar changes in the mean fore-limb grip strength or of a clear dose response relationship, these differences were considered to be incidental. The mean fore- and hind-limb grip strength values of the test item-treated females were unaffected.

- Locomotor Activity: There were no differences of mean locomotor activity at any interval nor was the total locomotor activity affected.

FOOD CONSUMPTION

There was a slight reduction of mean food consumption in males treated with 300 mg/kg/day and 1000 mg/kg/day during the first and second measurement intervals (days 1 - 8 and 8 - 15). The differences to the control values were -7.9% and -9.9% on days 1 - 8, respectively and -6.6% and -3.9% on days 8 - 15, respectively. These differences were considered to be related to the treatment with the test item.

In females treated with 1000 mg/kg/day, slightly lower mean food consumption was noted at all measurement intervals, varying between -8.6% to -11.4% lower when compared with the control females. These differences were considered to be test item related.
The remaining treated females were unaffected.

BODY WEIGHTS

The mean body weights of the test item-treated males and females compared favorably with those of the respective controls.

In males treated with 1000 mg/kg/day, marginally lower mean body weight gain values were noted, but the differences were not statistically significant and a clear dose-response relationship was not present.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

There were no test item-related changes in the mean hematology parameters at any dose level.

Although the mean absolute and/or relative numbers of ‘large unstained cells’ frequently attained statistical significance at all dose levels, all values remained within their respective historical control ranges and were therefore considered to be of no toxicological relevance.

There were a few cases of statistically significant differences (ie elevated mean absolute reticulocyte count in males at 300 mg/kg/day, reduced mean absolute leukocyte count in males at 100 mg/kg/day, mean absolute neutrophil count in females at 1000 mg/kg/day (all p<0.05)), these also remained within the ranges of the historical control data.

The mean hemoglobin level noted in the females at 1000 mg/kg/day was significantly lower (p<0.05) than that of the controls, but this was not accompanied by concomitant changes in related parameters and therefore considered to be unrelated to the treatment with the test item.

CLINICAL BIOCHEMISTRY

There were no test item-related changes in the mean clinical biochemistry parameters at any dose level.

All differences in the mean clinical biochemistry parameters were either unrelated to dose (differences in bile acids in males at 300 mg/kg/day or elevated creatinine values noted in females at 100 mg/kg/day and 1000 mg/kg/day), within the range of the historical control values or considered irrelevant (reduced total bilirubin in females at 300 mg/kg/day and reduced aspartate aminotransferase activity in females at 1000 mg/kg/day).

3. PATHOLOGY

ORGAN WEIGHTS

Slightly elevated liver weights noted in the females treated with 300 or 1000 mg/kg/day were considered to be adaptive changes of metabolic origin.

The mean absolute and relative organ weights of the test item treated males compared favorably with those of the control males.

In females treated with 1000 mg/kg/day, significantly elevated mean absolute liver weights and mean liver-to-body weight ratios (both p<0.05) were noted when compared with controls. The mean liver-to-brain weight ratio was also significantly elevated (p<0.01) in these females. The mean kidney-to-brain weight ratio was significantly elevated (p<0.05) but this was considered to be related to an incidental difference in brain weights.

At 300 mg/kg/day, significantly elevated mean absolute liver weights and mean liver-to-brain weight ratios (both p<0.01) were seen and the mean liver-to-body weight ratio was significantly elevated (p<0.05) when compared with the controls.

At 100 mg/kg/day, the mean absolute and relative uterus/oviduct weights were significantly elevated (all p<0.05). In the absence of any dose response relationship, these changes were considered to be incidental.

MACROSCOPIC FINDINGS

There were no gross lesions that could be attributed to treatment with the test item.
All gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

MICROSCOPIC FINDINGS

Microscopically, the treatment-related findings were recorded in stomach and liver of both sexes of animals, and kidneys of male animals.

- Stomach: Multifocal or diffuse mucosal necrosis of forestomach, together with hyperkeratosis/para-keratosis, was recorded in males treated with 100 mg/kg/day and both sexes treated with 300 mg/kg/day and 1000 mg/kg/day. Such lesions were observed frequently along with reactive squamous hypertrophy/hyperplasia and were associated with dyskeratosis and mucosal/submucosal edema and inflammatory cell infiltration as well. See also table 1 in section “Any other information on results incl. tables” below.

- Liver: Centrilobular hepatocellular hypertrophy was recorded in one male and one female treated with 1000 mg/kg/day. There were no further indicators of liver injury. See also table 2 in section “Any other information on results incl. tables” below.

- Kidneys: Increased severity of hyaline droplets in the proximal tubules was recorded along with increased incidence and severity of tubular basophilia in male animals treated with 1000 mg/kg/day. No toxicologically relevant findings were noted in females; the kidneys of females treated with 100 or 300 mg/kg/day were not examined. See also table 3 in section “Any other information on results incl. tables” below.

- Vaginal Estrus Stages: No specific direction of estrus cycle was observed in animals treated with 1000 mg/kg/day, and therefore it was judged that there were no treatment-related effects on the estrus cycles. See also table 4 in section “Any other information on results incl. tables” below.

- Other Findings: The remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
> 0 - < 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The no-observed-effect-level (NOEL) was considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
> 0 - < 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach.
Critical effects observed:
not specified

TABLES OF MICROSCOPIC FINDINGS

 

Table 1: Incidence and Mean Severity Gradeof Main Findings in Stomach

 

Finding
Incidence /
Mean Severity
Grade

Group 1
Control

Group 2
100 mg/kg/day

Group 3
300 mg/kg/day

Group 4
1000 mg/kg/day

 

5M

5F

5M

5F

5M

5F

5M

5F

Forestomach necrosis, multifocal/diffuse
Incidence/Mean Severity

0

0

2/1.5

0

5/1.4

4/1.8

5/1.4

5/2.2

 

 

Table 2: Incidence and Mean Severity Grade of Main Findings in Liver

 

Finding
Incidence /
Mean Severity
Grade

Group 1
Control

Group 2
100 mg/kg/day

Group 3
300 mg/kg/day

Group 4
1000 mg/kg/day

 

5M

5F

5M

5F

5M

5F

5M

5F

Hepatocellular hypertrophy, centrilobular 
Incidence/Mean Severity

0

0

0

0

0

0

1/1.0

1/1.0

 

 

Table 3: Incidence and Mean Severity of Main Findings in Kidneys

 

Finding
Incidence /
Mean Severity
Grade

Group 1
Control

Group 2
100 mg/kg/day

Group 3
300 mg/kg/day

Group 4
1000 mg/kg/day

 

5M

5F

5M

 

5M

 

5M

5F

Hyaline droplets, proximal tubules
Incidence/Mean Severity

4/1.0

0

4/1.0

 

5/1.0

 

5/1.8

0

Tubular basophilia
Incidence/Mean Severity

1/1.0

2/1.0

2/1.0

 

2/1.0

 

4/1.5

3/1.0

 

 

Table 4: Incidence of Estrus Stages in Vagina

 

Finding
Incidence

Group 1
Control

Group 2
100 mg/kg/day

Group 3
300 mg/kg/day

Group 4
1000 mg/kg/day

 

5F

 

 

5F

Proestrus

0

1

Estrus

1

3

Metestrus

4

1

Diestrus

0

0

 

Conclusions:
Oral administration of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate to Wistar rats at doses of 100, 300 and 1000 mg/kg/day, for 28 days resulted in no deaths, no clinical signs during daily or weekly observations, no effects upon the functional observational battery (including the fore- and hindlimb grip strength and locomotor activity), no effects upon body weights, no effects upon the hematology or clinical biochemistry parameters, and no macroscopical changes.

Test item-related findings were generally restricted to reductions in mean daily food consumption in males and females at 1000 mg/kg/day and males at 300 mg/kg/day, and slightly elevated (and adaptive) liver weights in females at 300 mg/kg/day and 1000 mg/kg/day. Microscopically, O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate produced forestomach mucosal necrosis and centrilobular hepatocellular hypertrophy of both sexes and enhanced hyaline droplets in renal proximal tubules as well as increased incidence and severity of tubular basophilia in the kidney in male animals. Of these findings, centrilobular hepatocellular hypertrophy was considered to be an adaptive change, enhanced hyaline droplets in renal proximal tubules and increased incidence and severity of tubular basophilia in the kidney of males was considered to be metabolic change specific to the male rat and of no toxicological relevance. However, the forestomach mucosal necrosis was considered to be adverse.

The no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was considered to be 1000 mg/kg/day.
Executive summary:

GENERAL

 

In this subacute toxicity study, O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment.

 

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

 

Because of possible test-item-related-findings that were noted in animals of the high dose group, the stomach and liver from both sexes and kidneys from males of the low and intermediate groups were also examined to establish a no-effect level.

 

MORTALITY/VIABILITY: All animals survived the scheduled treatment or recovery periods.

 

CLINICAL SIGNS (DAILY AND WEEKLY) : There were no findings evident during daily observations in any male or female at any dose level.

 

Slightly decreased activity, hunched posture and ruffled fur were noted in one male treated with 1000 mg/kg/day during week 1 of the detailed behavioral observations. These findings were not seen during subsequent weekly observations, nor were they evident in any other male at this dose level. The females of all dose groups were without findings during weeks 1 - 3 of treatment.

 

FUNCTIONAL OBSERVATIONAL BATTERY: There were no findings evident in any male or female at any dose level during the functional observational battery performed at week 4.

 

GRIP STRENGTH: There were no test item-related differences in the mean fore- and hindlimb grip strength values.

 

LOCOMOTOR ACTIVITY: There were no differences of mean locomotor activity at any interval nor was the total locomotor activity affected.

 

FOOD CONSUMPTION: At 1000 mg/kg/day, the mean daily food consumption of the males was reduced during days

1 - 8 and 8 - 15 of treatment (-9.9% and -3.9%, respectively). In females, slightly lower mean food consumption was noted at all measurement intervals. The differences ranged from -8.6% to -11.4% when compared with the control females. These differences were considered to be test item related. There was a slight reduction of mean food consumption in males treated with 300 mg/kg/day during the first and second measurement intervals (days 1 - 8 and 8 - 15), amounting to -7.9% and -6.6%, respectively. Females at 300 mg/kg/day and all animals at 100 mg/kg/day were unaffected.

 

BODY WEIGHTS: The mean body weights of the test item-treated males and females compared favorably with those of the respective controls.

 

CLINICAL LABORATORY INVESTIGATIONS

 

- Hematology: There were no test item-related changes in the mean hematology parameters at any dose level.

- Clinical Biochemistry: There were no test item-related changes in the mean clinical biochemistry parameters at any dose level.

 

ORGAN WEIGHTS: Slightly elevated liver weights noted in the females treated with 300 or 1000 mg/kg/day were considered to be adaptive changes of metabolic origin. All other organ weights were considered to be within the range of typical biological variation.

 

MACROSCOPIC/MICROSCOPIC FINDINGS: There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

 

Forestomach mucosal necrosis was recorded in males treated with 100 mg/kg/day and both sexes treated with 300 mg/kg/day and 1000 mg/kg/day. In the forestomach of the affected rats, hyperkeratosis/parakeratosis, reactive squamous hypertrophy/hyperplasia, dyskeratosis and/or mucosal/submucosal edema and inflammatory cell infiltration were observed. These were considered to be local injuries that resulted in subsequent adverse reactions.

 

In the liver, centrilobular hepatocellular hypertrophy was recorded at a minimal severity in one male and one female treated with 1000 mg/kg/day. There were no further indicators of liver injury, hence, this lesion was considered to be one of metabolic adaptation (as would enzyme induction), and was deemed to be not adverse.

 

Increased severity of hyaline droplets in the renal proximal tubules was recorded in male animals treated with 1000 mg/kg/day. This was considered to be induced by metabolic overload of protein specific to male rats and similar to alpha-2 microglobulin, derived from hyperfunction of the liver. Increased incidence and severity of renal tubular basophilia were considered to be a secondary tubular injury caused by the enhanced hyaline droplet deposition.

 

CONCLUSION

 

Transient reductions in mean daily food consumption were noted in the high dose males during the first two weeks of treatment, whereas females showed slightly lower mean food consumption at all measurement intervals. These differences were considered to be test item related.

 

Slightly elevated liver weights noted in the females treated with 300 or 1000 mg/kg/day were considered to be adaptive, non-adverse changes of metabolic origin. Although minimal centrilobular hepatocellular hypertrophy was recorded two rats treated with 1000 mg/kg/day, and was considered to be metabolic adaptation, and not adverse.

 

Forestomach mucosal necrosis was recorded in males treated with 100 mg/kg/day and both sexes treated with 300 mg/kg/day and 1000 mg/kg/day. In the forestomach of the affected rats, hyperkeratosis/parakeratosis, reactive squamous hypertrophy/hyperplasia, dyskeratosis and/or mucosal/submucosal edema as well as inflammatory cell infiltration were observed. These were considered to be local injuries that resulted in subsequent adverse reactions.

 

Increased severity of hyaline droplets in the renal proximal tubules was recorded in male animals treated with 1000 mg/kg/day. This was considered to be a protein specific to male rats similar to alpha-2 microglobulin, and generally derived from hyperfunction of the liver. Increased incidence and severity of renal tubular basophilia were considered to be secondary injuries caused by enhanced hyaline droplet deposition. Although this is considered to be an adverse finding in rats, this protein has no counterpart in humans and is therefore of no toxicological relevance.

 

The no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level (NOAEL) were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was considered to be 1000 mg/kg/day.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This is the only oral gavage 28-day GLP study available to evaluate potential effects of the test substance following repeated dosing.
System:
urinary
Organ:
kidney

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

In the subacute toxicity study, effects were noted in liver, forestomach, and in kidney. Slightly elevated liver weights noted in the females treated with 300 or 1000 mg/kg/day were considered to be adaptive, non-adverse changes of metabolic origin. Minimal centrilobular hepatocellular hypertrophy recorded in two rats treated with 1000 mg/kg/day were considered to be metabolic adaptation, and not adverse. Forestomach mucosal necrosis was recorded in males treated with 100 mg/kg/day and both sexes treated with 300 mg/kg/day and 1000 mg/kg/day. The microscopic effects on the forestomach (hyperkeratosis / parakeratosis, reactive squamous hypertrophy / hyperplasia, dyskeratosis and/or mucosal / submucosal edema and inflammatory cell infiltration) were observed. These were considered to be local injuries that resulted in subsequent adverse reactions. Increased severity of hyaline droplets in the renal proximal tubules was recorded in male animals treated with 1000 mg/kg/day. This was considered to be a protein specific to male rats similar to alpha-2 microglobulin, and generally derived from hyperfunction of the liver. Increased incidence and severity of renal tubular basophilia were considered to be secondary injuries caused by enhanced hyaline droplet deposition. Although this is considered to be an adverse finding in rats, this protein has no counterpart in humans and is therefore of no toxicological relevance.

The no-observed-effect-level (NOEL) and the local no-observed-adverse-effect-level were considered to be below 100 mg/kg/day, based on the adverse irritative findings in the forestomach. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity of O-(2-ethylhexyl) O,O-tert-pentyl peroxycarbonate was considered to be 1000 mg/kg/day.

Thus, the effects, an NOEL and an NOAEL were reported, and the effects seen were not sufficient for classification.

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

Apparently well-conducted OECD guideline GLP study.

Justification for classification or non-classification

Data are conclusive, and the systemic effects seen are not sufficient for classification.