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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP; OECD guideline was followed

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
June 1989
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl peroxypivalate
EC Number:
213-147-2
EC Name:
tert-butyl peroxypivalate
Cas Number:
927-07-1
Molecular formula:
C9H18O3
IUPAC Name:
tert-butyl 2,2-dimethylpropaneperoxoate

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Spargue Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Pilot study: 28.2 - 30.6 g at randomization (males), 26.0 - 28.3 g at randomization (females); Toxicity study: 28.4 - 34.6 g at randomization (males), 25.5 - 29.4 g at randomization (females); Supplemental toxicity study: 29.0 - 35.3 g at randomization (males), 25.8 - 30.7 g at randomization (females); Micronucleus assay: 28.0 - 34.4 g at randomization (males), 26.0 - 29.3 g at randomization (females)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material.
- Diet: free access to rodent chow
- Water: free access to tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C
- Humidity: 50 +/- 20 %
- Photoperiod: 12 hour light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
190, 380, 760 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
- Vehicle control and the low test dose: 15 animals
- High test dose: 20 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Doses / concentrations: 60 mg/kg

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study and a supplemental toxicity study.

TREATMENT AND SAMPLING TIMES: Test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes.

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to acapped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Scoring for micronuclei with microscope.
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<=0.05, Kastenbaum-Bowman Tables).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution. All analyses were performed seperately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the relative to the vehicle control (p<=0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or uncofirmed positive and a repeat assay recommended. The test item was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000, 5000 mg test item/kg (total volume of 20 mL test ite/vehicle miture/kg bw
- Solubility: test item is insoluble in water; test item was soluble in corn oil at a maximum concentration of 500 mg/mL
- Clinical signs of toxicity in test animals: Mortality occurred within three days of dose administration as follows: 5/5 males and 5/5 females at 5000 mg/kg and 1/2 males at 1000 mg/kg. Clinical signs, which were noted within four hours or later after dose administration, included lethargy in male mice at 100 and 1000 mg/kg. All other animals appeared normal throughout the observation period.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The number of micronucleated polchromatic erythrocytes per 1000 polychromatic erythrocytes in test item-treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of the dose level or bone marrow collection time.
- Ratio of PCE/NCE: Reductions of up to 28 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in test item-treated mice relative to their respective vehicle controls.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

Tert-butyl peroxypivalate was tested in mouse micronucleus assay according OECD guideline no. 474 and EU method B.12. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study and a supplemental toxicity study. The second phase, the micronucleus study, evaluated the potential of the test item to increase the incidene of micronucleated polychromatic erythocytes in bone marrow of male and female mice. In both phases of the study, test and control items were administered in a constant volume of 20 mL/kg body weight by single intraperitoneal injection.

In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test item/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality was observed in 1/2 male mice at 1000 mg/kg and 5/5 male mice and 5/5 female mice at 5000 mg/kg. Clinical signs following dose administration included lethary in male mice at 100, 1000 mg/kg.

In the toxicity assay, male and female mice were dosed with 400, 800, 1200, or 2000 mg test item/kg body weight. Mortality was observed in 5/5 male mice and 5/5 female mice at 1200 and 2000 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 400 and 800 mg/kg and prostration in male and female mice at 1200 and 2000 mg/kg. Due to the lack of a test item dose level with an intermediate level of toxicity, a supplemental toxicity assay was performed.

In the supplemental toxicity assay, male and female mice were dosed with 900, or 1100 mg test item/kg body weight. mortality was observed in 2/5 male mice and 1/5 female mice at 900 mg/kg and 5/5 male mice and 5/5 female mice at 1100 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 900 and 1100 mg/kg and prostration in male mice at 900 mg/kg and in mice at 1100 mg/kg. The high dose for the micronucleus test was set at 760 mg/kg which was estimated to be approximately 80% of the LD50/3.

In the micronucleus assay, male and female mice were dosed with 190, 380 or 760 mg/kg body weight of tert-butyl peroxypivalate. Mortality was observed in 3/20 male and 1/20 female mice receiving 760 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at all test item dose levels. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examinedmicroscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 28 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some ofte test item-treated groups relative to the respective vehicle controls.

No significant increase in micronucleated polychromatic erythrocytes in test item-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration.

Tert-butyl peroxypivalate did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

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