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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:
10 April 1996 - 31 may 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: 2,5-dimethyl-2,5-di(t-butylperoxy)hexane
Purity: 92.5%
Lot No.: 7827530475
Receipt Date: March 22, 1996
Physical Description: Light yellow liquid
Storage Conditions: Room temperature

Test animals

Species:
mouse
Strain:
other: IRC
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Spr- Age at study initiation: 6-8 weeks
- Weight at study initiation:
Pilot study:
Males, 30.6 - 33.9 grams at randomization
Females, 25.0 - 28.4 grams at randomization
Micronucleus assay:
Males, 27.0 - 35.0 grams at randomization
Females, 24.0 - 29.9 grams at randomization
- Assigned to test groups randomly: yes, under following basis: equalization of group mean body weight.
- Fasting period before study: no data
- 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. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): Mice had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants (Harlan TEKLAD Certified Rodent Chow 7012C)
- Water (e.g. ad libitum): Mice had free access tap water (Washington Suburban Sanitary Commission, Potomac Plant).
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 ± 3.3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 10 April 1996 - 31 may 1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: max 500 mg/ml
- Amount of vehicle (if gavage or dermal): 20ml/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article-vehicle mixture, the vehicle alone and the positive control (CP) will be given as a single administration. The rate of administration will be 20 ml/kg to deliver the targeted dose. All mice in the experimental groups will be weighed and the dose volume will be based on individual body weight.
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
Twenty-four, 48, and 72 hours after dose administration, five animals per sex per test article-treated and vehicle control groups will be sacrificed by carbon dioxide asphyxiation. The positive control group will be sacrificed 24 hours after dose administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Vehicle Control: 15
Low test dose (1250 mglkg): 15
Mid test dose (2500 mg/kg): 15
High test dose (5000 mg/kg): 20
Positive Control, 60 mg/kg: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): known positive
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For the pilot study, mice were randomly assigned to one group of five males and five females and to 4 groups of two males each. Each mouse was given a sequential number and identified by ear tag. All mice were weighed immediately prior to dose administration and the dose volume based on individual body weights. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 ml/kg body weight. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were observed after dose administration for clinical signs of chemical effect.
Twenty-four, 48, and 72 hours after dose administration, five animals per sex per test article-treated and vehicle control groups will be sacrificed by carbon dioxide asphyxiation. The positive control group will be sacrificed 24 hours after dose administration.

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 a capped 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:
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 115 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
The test is valid when: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1 000 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 (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979). All analyses were performed separately for each sex and sampling time. In order to quantify the test article effect on erythropoiesis, 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 substance 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 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 unconfirmed positive and a repeat assay recommended. The test article 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
Remarks:
Reductions of up to 21% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance-treated groups relative to their respective vehicle controls.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
For the pilot study the substance was administered by oral gavage to male mice at 1, 10, 100, or 1000 mg test article/kg and to male and female mice at 5000 mg/kg which was administered in a total volume of 20 ml test substance-vehicle mixture/kg body weight. Mortality was observed in 1/5 male mice at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in one male mouse at 1000 mg/kg and lethargy and diarrhea in male mice and female mice at 5000 mg/kg, and crusty eyes and prostration in one male mouse at 5000 mg/kg. All other animals appeared normal throughout the observation period. Due to less than 50% mortality in the pilot study, a toxicity study was not required. The highest dose level for the micronucleus study was set at 5000 mg/kg.

RESULTS OF DEFINITIVE STUDY
See tables

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results of the assay indicate that under the conditions described in this report the substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. It was concluded to be negative in the mouse micronucleus assay.
Executive summary:

The test substance was tested in the mouse micronucleus assay.

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 ml/kg body weight by a single oral gavage.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test substance was soluble in com oil at a concentration of 500 mg/ml, the maximum concentration tested. In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test article/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality occurred in 1/5 males at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in one male mouse at 1000 mg/kg and lethargy and diarrhea in male and female mice at 5000 mg/kg, and crusty eyes and prostration in one male mouse at 5000 mg/kg.

Due to less than 50% mortality at 5000 mg/kg in the pilot assay, the high dose for the micronucleus test was set at 5000 mg/kg.

In the micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight. No mortality occurred in male or female mice in the micronucleus study. Clinical signs observed after dose administration included diarrhea in male and female mice at all test substance dose levels, lethargy in male and female mice at 2500 and 5000 mg/kg and crusty eyes in one male mouse at 5000 mg/kg.

Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 21 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to the vehicle control groups. No significant increase in micronucleated polychromatic erythrocytes in test subsatnce 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 (p>0.05, KastenbaumBowman).

The results of the assay indicate that under the conditions described in this report the substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. It was concluded to be negative in the mouse micronucleus assay.