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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:
2001-12-21 and 2002-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl peracetate
EC Number:
203-514-5
EC Name:
tert-butyl peracetate
Cas Number:
107-71-1
Molecular formula:
C6H12O3
IUPAC Name:
tert-butyl peracetate
Details on test material:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the refrigeratore in the dark between +10 and +30°C

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Drawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 27.6 - 31.5 g (male), 23.5 - 28.1 g (female)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: certified laboratory rodent chow (Harlan TEKLAD certified Rodent 7012C); ad libitum
- Water: tap water (Washington Suburban Sanitary Commission, Potomac Plant); ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle: corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test article and compatibility of the vehicle with the test system
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered at a dose volume of 20 mL/kg bw.
Frequency of treatment:
single intraperitoneal injection
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg)

Examinations

Tissues and cell types examined:
- Bone marrow cells were examined microscopically for micronucleated polychromatic erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection based on a pilot toxicity study followed by a toxicity study.

TREATMENT AND SAMPLING TIMES: Bone marrow cells were collected after 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: 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 slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenmald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Bone marrow cells, polychromatic and normochromatic erythrocytes were analyzed for the presence of micronuclei. For analysis slides were coded using a random number table. Using medium magnification (10x40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10x100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total reythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
The test article was considered to induce positive results if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control 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 is recommended. The test article is considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a valid test - mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the positive control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group.
Statistics:
According to Kasten-Bowman Tables

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no toxicity was observed up to 1200 mg/kg which is the highest dose used in the micronucleus study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot Toxicity Study: Mortality and clinical signs included prostation were observed in males and females at the highest dose of 2000 mg/kg. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg and in males and females at 2000 mg/kg. Ataxia, paralysis and irregular brathing were noted in males and females at 2000 mg/kg.In addition, pilorection was observed in males at 10 mg/kg.

Toxicity Study: Mortality was observed at concentrations of 1400 mg/kg and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg.

Definitive study - no mortality was observed.
Clinical signs including lethargy and piloerection were noted in 300, 600, and 1200 mg/kg dose groups. Slight reductions (up to 4%) in the ratio of polychromatic to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. No significant increase in micronucleated polychromatic erythrocytes (PCEs) were observed in any test substance treated group (0.7 or less per 1000 PCEs). A significant increase (p<0.05) in micronucleated polychromatic erythrocytes was observed in the positive control group (~ 20 per 1000 PCEs). The validity criteria were met.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test substance (50% active ingredient) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Hence, the test substance was concluded to be negative in the micronucleus in vivo test.
Executive summary:

The test article (50% active ingredient) was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess toxicity of the test article and set dose levels for the definitive study consisted of a pilot study followed by a toxicity study. The second phase, the definitive micronucleus study, was designed to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. In both phases of the study, test and control articles were administered at a dose volume of 20 mL/kg body weight by a single intraperitoneal injection.

The test article was soluble in corn oil at 200 mg/mL, the maximum concnetration tested in the solubility test. All test article dosing formulations were delivered to the test system as light yellow solutions.

Cyclophosphamide monhydrate (CP) was used as the positive control article at a dose of 50 mg/kg bw.

In the pilot toxicity study, male mice (2/group) were dosed with 1, 10, 100 or 1000 mg test article/kg body weight and male and female mice (5/sex/group) were dosed with 2000 mg test article/kg body weight. Mortality was observed in 4/5 males and 5/5 females at 2000 mg/kg bw. Clinical signs immediately following dose administration included: prostration in males and females at 2000 mg/kg bw. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg bw and in males and females at 2000 mg/kg bw. Ataxia, paralysis and irregular breathing were noted in males and females at 2000 mg/kg bw. In addition, pilorection was observed in males at 10 mg/kg bw.

In the toxicity assay, male and female mice (5/sex/group) were dosed with 1200, 1400, 1600 or 1800 mg test article/kg body weight. Mortality was observed at concentrations of 1400 mg/kg bw and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg bw.

In the definitive micronucleus study, male and female mice (5/sex/group) were dosed with 300, 600 or 1200 mg test article/kg body weight, vehicle or positive control article. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy and pilorection in males and females at 300, 600 and 1200 mg/kg bw. Bone marrow cells were collected 24 and 48 hours after treatment. Slight reductions (up to 4%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. These reductions suggest that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman).

The validity critria for the positive control (Cyclophosphamide monohydrate) and the negative control (corn oil) were met.

It can be concluded that the test substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. The test substance was negative in the mouse micronucleus assay.

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