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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
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 publiication

Data source

Reference
Reference Type:
publication
Title:
Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU)
Author:
Taijiro Matsushima, Makoto Hayashi, Atsuko Matsuoka, Motoi Ishidate, Kunihiko F.Miura, Hidesuke Shimizu, Yuji Suzuki, Kanehisa Morimoto, Hiroko Ogura, Kanae Mure, Kimiko Koshi and Toshio Sofuni
Year:
1999
Bibliographic source:
Mutagenesis vol.14 no.6 pp.569–580, 1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro micronucleus test was performed to determine the toxic nature of 3,5-Diaminobenzoic acid
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Name of test material: 3,5-diaminobenzoic acid
- EC name: 3,5-diaminobenzoic acid
- Molecular formula: C7H8N2O2
- Molecular weight: 152.1522 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:
No data
Species / strain
Species / strain:
other: Chinese hamster lung cell line (CHL)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Eagle’s minimum essential medium supplemented with 10% heat inactivated (56°C for 30 min) calf serum
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction prepared from phenobarbital- and 5,6-benzoflavone-pretreated male Sprague–Dawley rats was used
Test concentrations with justification for top dose:
1500 – 3500 µg/mL (0, 1000, 1500, 2000, 2500, 3000 or 3500 µg/mL)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Controls
Negative controls:
yes
Solvent controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and conditions:
METHOD OF APPLICATION: in medium
Cells seeded: 1X104–1X105 cells

DURATION
- Preincubation period: No data
- Exposure duration:
Without S9: 24, 48, 78 hrs, 6 hrs + 42 hrs (recovery) and 6 hrs + 66 hrs (recovery)

With S9: 6 hrs + 42 hrs (recovery) and 6 hrs + 66 hrs (recovery)
- Expression time (cells in growth medium): 66 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 66 hrs

SELECTION AGENT (mutation assays): Acridine orange or Giemsa

SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: The number of micronucleated cells per 1000 intact interphase cells was recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The candidate MNs were categorized into three groups: very small (pin point) inclusions stained homogeneously (type 1) (those were not included for result evaluation), typical, i.e., smaller in diameter than ¼ of the normal main nucleus (type 2), and large, i.e., between ¼ and ½ the diameter of the normal main nucleus (type 3). The number of mitoses and abnormal cells (e.g., multinucleated cells, cells with abnormal nucleus) that appeared in the same microscopic field was also recorded.

As the negative control values ranged from 0.1 to 2.0% (Table II), chemicals yielding statistically significant MN frequencies with .4.0% were judged to be clear positives. When a compound induced a statistically significant MN frequency of < 4.0%, it was judged a weak positive. When no statistical significance was obtained, it was judged negative.
Statistics:
The frequencies of cells with type 2 and/or type 3 MN in the treatment groups were compared with those of the concurrent negative control by Fisher’s exact test. The concentration–response relationship was evaluated by the Cochran– Armitage trend test. A result was considered statistically significant when the P-value of the Fisher’s exact test was smaller than 0.05 divided by the number of treatment groups and the P-value of the trend test was also smaller than 0.05.

Results and discussion

Test results
Species / strain:
other: Chinese hamster Lung Cell line (CHL)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
not specified
Additional information on results:
No data

Applicant's summary and conclusion

Conclusions:
3, 5 -Diaminobenzoic acid failed to induce mutation in Chinese hamster lung cell line (CHL) both with and without S9 metabolic activations system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro micronucleus test was performed to determine the toxic nature of 3, 5 -Diaminobenzoic acid. The study was performed using Chinese hamster lung cell line (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was used at concentration of 1500 – 3500µg/mL (0, 1000, 1500, 2000, 2500, 3000 or 3500µg/mL). The suration of exposure was 24, 48, 78 hrs, 6 hrs + 42 hrs (recovery) and 6 hrs + 66 hrs (recovery) without S9 and 6 hrs + 42 hrs (recovery) and 6 hrs + 66 hrs (recovery) with S9.The number of micronucleated cells per 1000 intact interphase cells was recorded. The candidate MNs were categorized into threegroups: very small (pin point) inclusions stained homogeneously (type 1) (those were not included for result evaluation), typical, i.e., smaller in diameter than ¼ of the normal main nucleus (type 2), and large, i.e., between ¼ and ½ the diameter of the normal main nucleus (type 3). The number of mitoses and abnormal cells (e.g., multinucleated cells, cells with abnormal nucleus) that appeared in the same microscopic field was also recorded. The statistically significant increase in MN frequency was obtained by short treatment (6166 h) with S9 mix, but it was not dose-dependent and the maximum frequency was only 1.7%, which was within the variations of the negative control value of the laboratory tested. The MN test showed clear negative results with 24, 48 and 72 h continuous treatments at up to 3500 mg/mL. 3, 5 -Diaminobenzoic acid failed to induce mutation in Chinese hamster lung cell line (CHL) both with and without S9 metabolic activations system and hence is not likely to classify as a gene mutant in vitro.