3,5-diaminobenzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro micronucleus test was performed by Matsushima et al (1999) to determine the toxic nature of 3, 5 -Diaminobenzoic acid (CAS no 535 -87 -5). 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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

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

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

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cell line (CHL)

Details on mammalian cell type (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 / cell type 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 / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental 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.

Species / strain:

mammalian cell line, other: Chinese hamster Lung Cell line (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

No data

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Various peer reviewed publications were reviewed to determine the mutagenic nature of 3, 5 -Diaminobenzoic acid. The studies are as mentioned below:

In vitro micronucleus test was performed by Matsushima et al (Mutagenesis, 1999) to determine the toxic nature of 3, 5 -Diaminobenzoic acid (CAS no 535 -87 -5). 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 was24, 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.

In a supporting data, Gene mutation toxicity study was performed by Dybing et al ( Biochemical pharmacology, 1977) to determine the mutagenic nature of 3, 5-Diaminobenzoic acid. The test chemical was studied at dose levels of 10µg/plate using Salmonella typhimurium strain TA1538 with S9 metabolic activation system. The colonies on each plate (histidine revertants) were counted after 2-day incubation at 37°C. Values from plates without mutagen (representing spontaneous revertants, averaging 25 colonies per plate) were subtracted. For bacterial toxicity tests, mutagen and bacteria (after a 1: 200,000 dilution) were plated on full agar plates containing 8 g/liter of Difco nutrient broth. 3, 5-Diaminobenzoic acid failed to induce mutation in Salmonella typhimurium strain TA1538 in the presence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Shibai- Ogata et al ( Mutagenesis, 2011) performed in vitro Micronucleus test to determine the mutagenic nature of 3, 5 Diaminobenzoic acid. Three thousand cells were each seeded with 100µL of medium in each well of 96-well microplates to avoid the cultures reaching confluence before completion of the treatment procedures and then pre-cultured for 24 h before testing of the chemicals.Cells were continuously treated with test chemicals by employing four treatment procedures: brief treatment with or without S9 mix and two prolonged (24 or 48 h) treatments. All incubations were carried out in a 5% CO2 humidified atmosphere at 37C. After the prescribed incubation time, the cells were washed with PBS once, fixed with 100% ethanol for at least 30 min, followed by replacement of PBS and stored at 4C until the staining procedures. Cytotoxicity was assessed as the reduction of cell viability. The microplates were stained with Hoechst 33342 and CellMask Red for automated analysis, and MN were identified and counted automatically in fluorescence images. 3, 5 diaminobenzoic acid did not induce mutation during brief treatment with or without S9 mix and during prolonged (24 hrs) treatments. No mutation was noted during prolonged (48 hrs) treatment upto 5000µM dose level. Based on the observations made, 3, 5 diaminobenzoic acid failed to induce mutation in Chinese hamster lung cells (CHL) in the presence and absence of S9 mix and hence is not likely to classify as a gene mutant in vitro.

In the study performed by Yoshimi et al ( Mutagenesis, 1988), the hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) is known to be sensitive to various classes of DNA-reactive carcinogens and is regarded as a reliable short-term test for the detection of chemical carcinogens.The genotoxicity of 3,5-diaminobenzoic acid, was examined by a DNA repair test with rat hepatocytes. The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO and the positive control used was N-2-fluorenylacetamide. The hepatocytes were exposed to the test chemical for 20 hrs. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The test chemical was evaluated to be positive only when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls. The results of the hepatocyte/DNA repair test suggests that 3,5-diaminobenzoic acid induced DNA repair in rat hepatocytes and hence is positive for genotoxicity in vitro.

Based on the key study and its supporting data summarized, 3, 5 -Diaminobenzoic acid is not likely to exhibit genetic toxicity. Thus, the chemical is not classified as a genetic toxicant.

Justification for classification or non-classification

Based on the key study and its supporting data summarized, 3, 5 -Diaminobenzoic acid (CAS no 535 -87 -5) is not likely to exhibit genetic toxicity. Thus, the chemical is not classified as a genetic toxicant.