Di-tert-pentyl peroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2012 - 09 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-pentyl peroxide

EC Number:

234-042-8

EC Name:

Di-tert-pentyl peroxide

Cas Number:

10508-09-5

Molecular formula:

C10H22O2

IUPAC Name:

2-methyl-2-[(2-methylbutan-2-yl)peroxy]butane

Test material form:

other: Liquid

Details on test material:

- Name of test material: Luperox DTA
- Synonym: Di-tert-amylperoxide
- Physical state: slightly yellowish liquid
- Lot/batch No.: 11650 920B10-011
- Purity: 98.2%
- Expiry date: December 2012
- Storage condition: at room temperature.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France.
- Age at study initiation: approximately 6 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 215 g (range: 198 g to 232 g) and the females had a mean body weight of 169 g (range: 155 g to 179 g).
- Fasting period before study: no
- Housing: the animals were housed by five, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 23 March 2012 to 20 April 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was administered as a solution in the vehicle.
It was mixed with the required quantity of vehicle. No correction factor was applied.
The frequency of dose formulation preparation was based on available stability data. The dose formulations were stored at room temperature, protected from light, and delivered to the study room in brown flasks.


VEHICLE
- Justification for use and choice of vehicle: test item soluble in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID.
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogeneous
Stability: stable after 4 days at ambient temperature and protected from light

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a 7 day toxicity study (CIT/Study No. 38492 TSR). In this preliminary study, the test item was given at 100, 300 or 1000 mg/kg/day to Sprague-Dawley rats. No mortalities or clinical signs were observed in the study. There were no obvious effects of the test item on body weight and there was a minimal trend to a dose-related reduction in mean food consumption in males. There were no test item-related macroscopic findings and the only relevant effect seen at necropsy was higher liver weights in test item-treated males, with statistical significance at 1000 mg/kg/day.

The same dose-levels as in the preliminary study have thus been chosen for the present study.


- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule mortality: once a day during the acclimation period and at least twice a day during the treatment period.
- Clinical signs: once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before group allocation, then on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: each animal was evaluated once in week 4.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.

Sacrifice and pathology:

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- liver (males and females) and kidneys (males only) from the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
- immunostained kidneys from the control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period,
- on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(for clinical signs but no mortality)

Mortality:

mortality observed, treatment-related

Description (incidence):

(for clinical signs but no mortality)

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:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL SIGNS:
All animals treated at 1000 mg/kg/day had ptyalism from day 11. This was considered to be related to treatment with the test item but non adverse. Incidental findings included reflux at dosing, scabs, soft feces, thinning of hair and chromodacryorrhea and were generally observed in isolated animals.

BODY WEIGHT (GAIN):
There were no toxicologically relevant effects on mean body weight and mean body weight change.

FOOD CONSUMPTION:
There were no toxicologically relevant effects on mean food consumption.

NEUROBEHAVIOURAL EXAMINATION:
There were no test item-related effects at Functional Observation Battery, including motor activity.

HAEMATOLOGY:
At 1000 mg/kg/day, there were in males statistically significantly higher mean white blood cell count, due to higher basophil but mainly lymphocyte counts, when compared to controls. There were also statistically significantly lower mean hemoglobin concentration and packed cell volume. Females of that group had a statistically significantly shortened prothrombin time than in the other groups.
These findings were considered to be test item-related but non adverse in the absence of any pathological correlates or in view of the amplitude of difference from controls.

At 100 and 300 mg/kg/day, there were no test item-related effects.

CLINICAL CHEMISTRY:
At 1000 mg/kg/day, there were statistically significant low mean glucose and high mean cholesterol levels in both sexes, associated in females with a statistically significantly higher mean triglyceride concentration, when compared with controls. The trend to a high mean cholesterol level appeared in a dose-related manner from 100 mg/kg/day, especially in males. These variations were considered to be test item-related but non adverse in the absence of any correlating adverse effect at pathology or in view of the amplitude of difference from controls.

At 1000 mg/kg/day, there were also in females statistically significant variations in mean chloride and calcium levels when compared with controls. In the absence of any other correlating findings in this sex, these variations were considered not to be toxicologically relevant.

The statistically significantly low alkaline phosphatase activity recorded at 1000 mg/kg/day in both sexes (350 and 198 IU/L in males and females, respectively, vs. 491 and 314 IU/L in controls, p<0.01) was considered not to be toxicologically relevant.

URINALYSIS:
At 1000 mg/kg/day, all males had a few cells in all fields of urine microscopically observed (one in controls) and a higher mean urine volume (also seen in two females) when compared with controls. These finding were considered to be test item-related but non adverse. The cells observed in urine could be related to the effects seen in male kidneys at microscopy.

At 100 and 300 mg/kg/day, there were no test item-related effects.
The higher urine specific gravity noted in males at 100 mg/kg/day (1044, vs. 1030 in controls, p<0.05) was considered to be unrelated to the treatment with the test item as not observed in the highest dose-levels.

ORGAN WEIGHTS:
When compared with controls, there were minimal to slight increases in mean absolute and relative liver weights in males at all dose-levels and in females at 300 and 1000 mg/kg/day. These increases were statistically significant in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day.

The mean absolute and relative kidney weights were increased in males given the test item at all dose-levels, reaching statistical significance for the relative weight at 300 and 1000 mg/kg/day.

These variations in liver and kidney weights were considered to be related to the test item administration in view of the correlated microscopic changes.

Other statistically significant changes for heart, prostate with seminal vesicles, spleen and uterus were considered not to be toxicologically significant as there were no microscopic correlates.

GROSS PATHOLOGY:
The few macroscopic findings noted at the end of the treatment period were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration.

HISTOPATHOLOGY: NON-NEOPLASTIC:
The test item administration induced centrilobular hypertrophy in the liver from 300 mg/kg/day in males and females, and tubular hyaline droplets in the kidney of males at all dose-levels.

. Liver
Minimal to slight centrilobular hypertrophy was seen in 3/5 males and 5/5 females at 1000 mg/kg/day. This finding was also seen with minimal severity in 1/5 males and 2/5 females at 300 mg/kg/day. There were no associated degenerative changes at any of the dose-levels.

. Kidney
Moderate hyaline droplets were noted in the proximal tubular epithelium of the renal cortex from 5/5 males at 1000 mg/kg/day, and were associated with evidence of regeneration characterized by multifocal minimal to slight tubular basophilia. Hyaline droplets were observed with lesser severity at 100 and 300 mg/kg/day. Hyaline droplets were variably sized, round, dense eosinophilic and intracytoplasmic. Moderate to marked staining with an anti-a2µ globulin antibody was seen in droplets located in the cytoplasm of tubular cells and in the lumen of tubules from all high-dose males. Minimal to slight staining with this antibody was also seen in all control males, but it was located in small droplets in the cytoplasm of tubular cells only and was not seen within the lumen.
Minimal tubular basophilia was seen in 4/5 control males but was less extensive than in rats given the test item at 1000 mg/kg/day. At 100 and 300 mg/kg/day, incidence and severity of tubular basophilia were similar to that seen in controls.
Hyaline droplets were not observed in any of the females.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings in the Sprague-Dawley rat.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the NOAEL is 300 mg/kg bw/d in males based on the association of renal alpha2µ-globulin hyaline droplets with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover.
The NOAEL in females is 1000 mg/kg.
There were no other effects that could be considered as adverse at any doses. Renal alpha2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human.

Executive summary:

The toxicity of di-tert-amyl peroxide following daily oral administration (gavage) to rats for 4 weeks was evaluated in an OECD 407 study.

The substance was dissolved in corn oil and administrated to groups of five male and five female Sprague-Dawley rats at 0, 100, 300 or 1000 mg/kg/day.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis were performed on all animals. Blood was also taken for a possible further thyroid hormone investigation, but no analysis was performed.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopicpost-mortemexamination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues (including liver and kidneys) from control- and high-dose animals sacrificed at the end of the treatment period, on liver (males and females) and kidneys (males only) from the low- and mid-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

There were no unscheduled deaths. The only relevant clinical sign was ptyalism noted from day 11 in all animals treated at 1000 mg/kg/day and was considered to be non adverse. There were no toxicologically relevant effects on mean body weight, mean body weight change or mean food consumption. There were no test item-related effects at Functional Observation Battery, including motor activity, or at macroscopicpost-mortemobservation.

 

At 1000 mg/kg/day in males, some hematological findings were considered to be test item-related but non adverse (higher mean white blood cell count, mainly due to higher mean lymphocyte count, lower mean hemoglobin concentration and mean packed cell volume).

At blood biochemistry, some variations were noted and considered to be test item-related but non adverse (lower mean glucose level in males and females respectively, higher mean cholesterol level in females, with a higher mean triglyceride concentration).

At urinalysis, all males had a few cells in all fields of urine microscopically observed (only one in controls) and a higher mean urine volume. These urinary findings were considered to be non adverse. The cells observed in urine could be related to the effects seen in male kidneys at microscopy.

 

At pathology, there were minimally to slightly statistically higher mean liver and mean kidney weights in males, and mean liver weight in females when compared with controls (up to +45% for the liver and up to +20% for the kidney). Microscopic examination revealed centrilobular hypertrophy in the liver of males and females, and tubular hyaline droplets (which were shown to bea2µ globulin at immunohistochemistry) and basophilia in the kidney of males. These kidney findings were considered to be adverse in the current study for rats at 1000 mg/kg. Nevertheless, renal hyaline droplets are considered not to be relevant for human.

 

At 100 and 300 mg/kg/day, there were no test item-related effects on hematology, blood biochemistry or urinalysis parameters, except a trend to a high mean cholesterol level from 100 mg/kg/day, especially in males. At pathology, there were minimally to slightly higher mean liver and kidney weights in males at both dose-levels and mean liver weight in females at 300 mg/kg/day when compared with controls, which were statistically significant only in males at 300 mg/kg/day (up to +25% for the liver and up to +16% for the kidney). At microscopic examination, there was centrilobular hypertrophy in the liver at 300 mg/kg/day in 1/5 males and 2/5 females. In the kidney of males, tubular hyaline droplets were seen with dose-related severity from 100 mg/kg/day. In the absence of associated tubular basophilia, these renal findings were considered not to be adverse at 100 and 300 mg/kg/day.

 

Under the experimental conditions of this study, the dose-level of 1000 mg/kg/day was considered to be an adverse Effect Level in rats as at 1000 mg/kg/day renal a2µ globulin hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. Renal a2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human.