tert-pentyl 2-ethylperoxyhexanoate

Genetic toxicity: in vivo

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:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP, OECD study 474 (July 1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-pentyl 2-ethylperoxyhexanoate

EC Number:

211-687-3

EC Name:

tert-pentyl 2-ethylperoxyhexanoate

Cas Number:

686-31-7

Molecular formula:

C13H26O3

IUPAC Name:

2-methylbutan-2-yl 2-ethylhexaneperoxoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material (as cited in study report): t-amyl 2-Ethylhexaneperoxoate
- Physical state: liquid
- Analytical purity: 96.5 %
- Lot/batch No.: 11615 616101-128
- Expiration date of the lot/batch: February 2002
- Storage condition of test material: at +4°C and protected from light

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Crédo, I'Arbresle, France
- Age at study initiation: approximately 6 weeks old
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: individually in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum A04 C pelleted maintenance diet (SSNIFF Spezialdiät GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum drinking water filtered by a 0.22µ membrane
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 30 to 70
- Air changes (per hr): at least 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

Volume: 10 ml/kg

Duration of treatment / exposure:

2 administrations separated by 24 hours.

Frequency of treatment:

once daily for 2 days

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): commomly used in this test and recommanded by the OECD guideline
- Route of administration: oral
- Frequency: one administration only
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

Femurs of the animals were removed and all the bone marrow was flushed out using fetal calf serum.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square
test value (Lovell and coll., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the x2 value (Lovell and coll., 1989).
When there was significant within-group heterogeneity, then that group was compared with the
control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969). The student 11t11 test was used for the PEINE ratio comparison.
Probability values of p0.05 was considered as significant.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

systemic toxicity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY (6 animals)
In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Hypoactivity and/or dyspnea and sometimes staggering gait were noted in all animals during at least the 4 hours following treatment. Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by C02 inhalation in excess. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no mortality was noted, the top dose-level selected for the main test was 2000 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, t-amyl 2-Ethylhexaneperoxoate did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.

Executive summary:

T-amyl 2-Ethylhexaneperoxoate was tested in a Mammalian Erythrocyte Micronucleus Test, according to the OECD n° 474 Guideline and EC 92/69/EEC B.12 guidelines in compliance with the Principles of Good Laboratory Practice.

In a bone marrow micronucleus assay, groups of 5 males and 5 females Swiss Ico:OF1 (IOPS Caw) mouse were treated by gavage with t-amyl 2-Ethylhexaneperoxoate (purity 96.5%) at doses of 0, 500, 1000, and 2000 mg/kg. 

One group of 5 males and 5 females received the vehicle under the same experimental conditions, and acted as control group. One group of 5 males and 5 females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

Bone marrow cells were harvested at 24 and/or 48 hours post-treatment. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

At 2000 mg/kg/day, dyspnea, hypoactivity, sedation, staggering gait and/or pi1oerection were noted in both males and females. At 24 hours following the second treatment only piloerection persisted in males.

The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item were considered as equivalent to those of the vehicle control group.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.

Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under these experimental conditions, t-amyl 2-Ethylhexaneperoxoate did not induce any noteworthy increase in the number of micronucleated with structural chromosome aberration, both with and without S9 mix, in any experiment. 

In conclusion, t-amyl 2 -ethylhexaneperoxoated did not induce an increase in micronucleus mouse bone marrow when tested to 2000 mg/kg.