3-nitrobenzoic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program

Author:

Kristine L. Witt

Year:

2000

Bibliographic source:

Environmental and Molecular Mutagenesis

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Micronucleus study was performed in vivo to determine the mutagenic nature of m-nitrobenzoic acid

GLP compliance:

not specified

Type of assay:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

3-nitrobenzoic acid

EC Number:

204-508-5

EC Name:

3-nitrobenzoic acid

Cas Number:

121-92-6

Molecular formula:

C7H5NO4

IUPAC Name:

3-nitrobenzoic acid

Details on test material:

- Name of test material: m-Nitrobenzoic acid
- Molecular formula: C7H5NO4
- Molecular weight: 167.1195 g/mol
- Substance type: Organic
- Physical state: Powder (Solid)
- Purity: No data
- Impurities (identity and concentrations): No data

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose level of 2% (2857.1 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

90 days

Frequency of treatment:

No data

Post exposure period:

No data

Doses / concentrations

Remarks:

2% (2857.1 mg/Kg bw/day)

No. of animals per sex per dose:

No data

Control animals:

not specified

Positive control(s):

Urethane
- Justification for choice of positive control(s): No data
- Route of administration: Drinking water
- Doses / concentrations: 0.2%

Examinations

Tissues and cell types examined:

Polychromatic and normochromatic erythrocytes were screened for the presence of micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: VA. Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. Drops of blood were spread on precleaned standard glass microscope slides, air dried, and were stained immediately before scoring with either Hoechst 33258/pyronin Y or acridine orange. All slides were coded prior to scoring by a person not involved in reading the slides. Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy.

METHOD OF ANALYSIS: Criteria for identification of MN were that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain). Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method, in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 erythrocytes.

OTHER: No data

Evaluation criteria:

The erythrocytes were observed for micronuclei. The MN results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group.

Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an
unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study.

Although statistical analyses were used as an important aid in evaluating the test results, statistical significance was not the only determining factor in arriving at an overall call for a chemical. A decision to classify a test as negative, equivocal, or positive for induction of micronuclei in this in vivo assay was based on a broader evaluation of a number of factors that determined the biological relevance of the results, including the appropriateness of the concurrent control data, the magnitude of the observed response and the presence of a dose-dependent increase in the frequency of micronucleated cells.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Results and discussion

Test results

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

No data

Applicant's summary and conclusion

Conclusions:

Test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.

Executive summary:

Micronucleus study was performed in vivo to determine the mutagenic nature of test substance. The study was performed using B6C3F1 mice at dose level of 2% (2857.1 mg/Kg bw/day) for 90 days. Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. Drops of blood were spread on precleaned standard glass microscope slides, air dried, and were stained immediately before scoring with either Hoechst 33258/pyronin Y or acridine orange. All slides were coded prior to scoring by a person not involved in reading the slides. Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy. Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative. Based on the observations made, test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.