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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to EPA guideline equivalent to OECD guideline 474.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
other: Environmental Protection Agency - Health Effect Test Guidelines, EPA Report 56016-83-001.
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The specific test system used for this study employed peripheral blood erythrocytes from mice following improved procedures for this test suggested by Schlegel and MacGregor (Mutation Research, 104, 367-369, 1982).
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Valeric acid
EC Number:
EC Name:
Valeric acid
Cas Number:
Molecular formula:
pentanoic acid
Details on test material:
- Name of test material (as cited in study report): n-Valeric acid
- Lot/batch No.: 47-167

Test animals

Swiss Webster
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories, Inc. (Portage, MI).
- Age at study initiation: Four-week old
- Weight at study initiation: The body weights of the male mice dosed with n-Valeric Acid ranged from 20.2 g to 24.6 g (mean = 22.5 ± 1.3 S.D.) and for female mice, weights ranged from 15.9 g to 21.7 g (mean = 18.6 ±1.7 S.D.)
- Assigned to test groups randomly: [yes, under following basis: ] randomized by weight and animals outside a range of 2 standard deviations from the mean weight for each sex were not used.
- Fasting period before study:
- Housing: Five mice/sex/cage were housed in shoe-box type plastic cages, measuring 30 x 20 x 12.5 cm. Two or three additional animals were added to dosage groups in which toxicity was expected to decrease survival but only 5 animals were evaluated for micronuclei per group. Each cage was labeled with animal I.D. numbers, sex and treatment doses. Ab-Sorb-Dri® bedding (Garfield, NJ) was placed in the cages and changed at 4- to 5-day intervals.
- Diet:ad libitum(basic diet of Purina Certified Rodent Chow Checkers (Pelleted) #5002,Ralston-Purina Company, St. Louis, MO.
- Water: ad libitum. Municipal Authority of Westmoreland County (Greensburg, PA)
- Acclimation period: 5 to 6 days

- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.

IN-LIFE DATES: Starting Dates:
Definitive Toxicity Study - May 21, 1985
Micronucleus Test - June 4, 1985

Completion Dates:
Definitive Toxicity Study - May 24, 1985
Micronucleus Study - June 7, 1985

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used: corn oil
- Positive control: Triethylenemelamine (CAS #51-18-3) at a dose of 0.3 mg/kg

Details on exposure:
Intraperitoneal toxicity study: Animals were dosed with the test and control materials by intraperitoneal (I.P.) injection. I.P. injection is a typical route of dosing for this test system, and it was used because this route achieves rapid and significant availability of test chemicals to the bone marrow target cells.

Micronucleus Study:
Micronucleus determinations were conducted using a minimum of 5 animals/sex/group and dosages of approximately 80%, 50% and 25% of the averagemale-female LD50 value. Additional animals were added to the highest dose group, because deaths were possible from inspection of the LD50 study, but only 5 of the surviving animals were examined for micronuclei.
Duration of treatment / exposure:
single intraperitonal injection
Frequency of treatment:
sampling after 24, 48 and 72 hours
Post exposure period:
3 days
Doses / concentrations
Doses / Concentrations:
83, 166, 266 mg/kg
analytical conc.
No. of animals per sex per dose:
Approximately five
Control animals:
yes, concurrent vehicle
Positive control(s):
TEM triethylenemelamine
- Justification for choice of positive control(s): Triethylenemelamine (CAS #51-18-3) was used as the positive control agent to demonstrate the sensitivity and responsiveness of the animals in the definitive test.
- Route of administration: intraperitoneal (I.P.) injection
- Doses / concentrations: 0.3 mg/kg


Tissues and cell types examined:
Polychromatic Erythrocyte to Normochromatic Erythrocyte Ratios
Details of tissue and slide preparation:
Not applicable
Evaluation criteria:
Data were compared for significant increases above the vehicle control frequencies using the Fisher's Exact Test (Sokal and Rohlf, 1981). Data for
males and females were combined for analyses when statistical tests showed that there was no significant difference in micronuclei frequencies between sexes. There was a statistically significant difference between male and female micronuclei values at 30 and 72 hour and data for these periods were analyzed separately. A positive result in the micronucleus test would be interpreted by at least one statistically significant (p < 0.05) increase above the vehicle control with an indication of a dose-related effect of treatment. A test would be considered inconclusive if only one dose produced effects statistically different from the control and no dose-effect relationship was apparent. A test result would be considered negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with n-Valeric Acid.
Fisher's Exact Test

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:
RESULTS of Toxicity Study for Dosage Selection
The calculated LD50 values were 308 mg/kg for males, 356 mg/kg for females and 332 mg/kg for the average male and female data. For the definitive micronucleus test, the average LD50 value was used to determine the test doses.

The PCE/NCE ratio of the vehicle control and the highest test dose with adequate numbers of survivors (> 3) was quantified and compared to determine possible bone marrow cytotoxicity. The PCE/NCE ratio of males dosed with 200 mg/kg, the highest dose with 3 or more surviving animals, was not remarkably lower than the vehicle control values. Females administered the same dose had a slight decrease in the PCE/NCE ratio which was not considered to be an excessive cytotoxic effect. The lack of weight gain for survivors at the 400 mg/kg dosage level indicated that the test material was likely to produce some deaths at high dosages in the definitive study, so three additional animals were added to the highest dosage group.

- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): No remarkable dose-related decreases in the PCE/NCE ratios relative to the control values were observed in this study at any of the three periods. PCE/NCE ratios for TEM were lower than the negative control at all three sampling times. The cytotoxicity of TEM to bone marrow is an expected and typical finding.
- Appropriateness of dose levels and route: I.P. injection is a typical route of dosing for this test system, and it was used because this route achieves rapid and significant availability of test chemicals to the bone marrow target cells.
- Statistical evaluation: Fisher's Exact Test

No remarkable or treatment-related increases were observed with any of the treatment groups sampled at the three respective sampling intervals following injection of the test chemical. Slight increases (1.7- to 2.0-fold) above vehicle control values were observed at the 30 hour sampling interval at the lowest treatment dose for males and females. However, these increases did not show a concentration-related trend, and the increases were not statistically different from the concurrent control values. TEM produced at least 10-fold increases in the incidence of micronuclei at both the 30 and 48 hour sampling times indicative of the sensitivity of the animals used for these tests.

Statistical comparisons were performed with pooled male and female data for the 48 hour sample and with individual data for males and females at 30 and 72 hours. The 48-hour data showed no significant difference between sexes and could be combined, while the other two sampling intervals had significant differences between sexes which precluded a combined analysis. Following these analyses, no statistically significant or treatment-related increases in the numbers of micronuclei were observed at any of the harvest intervals. Past experiences with this test suggest that the greatest increases in micronuclei would be expected at the 30-hr or 48-hr harvest, but the data did not reveal any treatment-related increases for these sampling times. In addition, the incidence of micronuclei for the groups administered n-Valeric Acid or the vehicle was within the expected range of variability for this test system noted in previous tests in our laboratory.

According to the evaluation criteria in the protocol and accepted procedures to classify effects in this test system, n-Valeric Acid was considered to be inactive in the production of chromosome damage in vivo under the conditions of this assay. The test evaluated reasonably high doses in males and females and no treatment-related increases were observed. n-Valeric Acid was considered non-clastogenic in the in vivo micronucleus test.

TEM used as a positive control agent for this study produced highly significant increases in numbers of micronuclei for all three sampling times. The quantitative level of micronuclei produced by TEM ranged from 3.7 to 22 times greater than the vehicle control value at the three sampling intervals. Numbers of micronuclei in the vehicle control animals were in a low and acceptable range for this test system at all sampling times.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
n-Valeric Acid was not an active agent in producing treatment- related increases in micronuclei in male or female Swiss-Webster mice. Dosage levels up to 80% of the LD50 dose for males and females did not produce treatment-related effects in this test. n-Valeric Acid was interpreted to be inactive as a clastogenic agent -in -viv o under the conditions of the micronucleus test system.
Executive summary:

n-Valeric Acid was evaluated for potential clastogenic (chromosome-damaging) activity with the -in vivo micronucleus test system employing male and female Swiss-Webster mice. Test doses for the micronucleus test were chosen from data obtained in a study to determine the toxicity of n-Valeric Acid administered by intraperitoneal injection using corn oil as a vehicle. n-Valeric Acid produced treatment-related toxicity following injection of doses between 50 mg/kg to 400 mg/kg. The LD50s were 308 mg/kg for males and 356 mg/kg for females. For determining dosage levels for the micronucleus test, an average LD50 value of 332 mg/kg was

calculated from the data for males and females.

For the definitive micronucleus test, a dosage range of 83 mg/kg to 266 mg/kg representing 80%, 50% and 25% of the average LD50 value was used for both males and females. Concurrent positive (triethylene- melamine) and negative (corn oil) control agents, administered by intraperitoneal injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that n- Valeric Acid did not produce either treatment-related or statistically-significant increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals sampled at 30, 48 and 72 hours postdosing. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test.

The lack of positive increases in the incidence of micronuclei in animals exposed to n-Valeric Acid indicates that n-Valeric Acid does not possess clastogenic activity in -vivo under the conditions of the micronucleus test system. Previous tests on n-Valeric Acid conducted at BRRC revealed positive effects in both the -in -vitro sister chromatid exchange test (BRRC Report 47-160) and chromosome damage assays (BRRC Report 47-221) performed with a rat liver S9 metabolic activation system. Negative studies on n-Valeric Acid included a CHO/HGPRT gene mutation test (BRRC Report 47-160) and an Ames (Salmonella) mutation test (BRRC Report 47-131).