m-toluidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Short description of key information:
There is an Ames test performed according to OECD TG 471 and yielded a negative result (MHLW 2002). An HPRT test was performed with m-toluidine according to the respective guideline and yielded a negative result (WOLLNY 2014).
There is an in-vitro Chromosome Aberration test with CHL/IU cells performed according to OECD TG 473 and yielded a negative result (MHLW 1995)

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Procedure: plate incorporation method.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

+/- S9-mix: 0, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 2-aminoanthracene

Details on test system and experimental conditions:

preincubation methodology

Evaluation criteria:

positive when assay plates with the test substance show a significant increase in revertantcolony count as compared with that on negative control plates and when this effect is reasonably reproducible or dosedependent

Statistics:

yes, but method not mentioned

Species / strain:

other: Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

m-Toluidine was not mutagenic in Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA with and without an exogenous metabolic activation system.

Remarks on result:

other: other: Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA

Remarks:

Migrated from field 'Test system'.

m-Toluidine was not mutagenic in Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA with and without an exogenous metabolic activation system.

Conclusions:

Negative in TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA with and without an exogenous metabolic activation system.

Executive summary:

Genotoxicity of m-toluidine was studied by a Reverse Mutation test in bacteria (Ames test) according to OECD TG 471 and GLP. m-Toluidine was not mutagenic in Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA with and without an exogenous metabolic activation system (MHLW 2002).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

other: Chinese hamster lung fibroblasts (V79): the cells have a stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Pretest on toxicity
4 h in the presence and 4 and 24 h in the absence of a metabolic activation system:
8.4 µg - 1072 µg/ml (approximately equal to 10mM)
GENE MUTATION ASSAY
--EXPERIMENT I
4 h without S9 mix:
67.0, 134.0, 268.0, 536.0, 1072 µg/ml
4 h with S9 mix:
67.0, 134.0, 268.0, 536.0, 1072 µg/ml
--EXPERIMENT II
24 h without S9 mix:
134.0, 268.0, 536.0, 804.0, 1072 µg/ml
4 h with S9 mix:
67.0, 134.0, 268.0, 536.0, 1072 µg/ml

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The assay was performed in 2 independent experiments, using 2 parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points that is three times above the spontaneous mutation frequency.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relevant cytotoxic effects indicated by a relative cloning efficientcy 1 or cell density below 50% occurred only in the absence of metabolic activation at 1072 µg/ml in experiment 1 (4 hr treatment) and experiment 2 (24 hr treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

the positive controls were functional

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Executive summary:

The study was performed to investigate the potential of m-toluidine to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditiions. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours (33.5 µg/ml - 1072 µg/ml). The second experiment was performed with a treatment time of 4 hours with (33.5 µg/ml - 1072µg/ml) and 24 hours without (67.0 µg/ml -1072 µg/ml) metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficientcy 1 or cell density below 50% occurred only in the absence of metabolic activation at 1072 µg/ml in experiment 1 (4 hr treatment) and experiment 2 (24 hr treatment).

No relevant and reproducible dose dependent increase in mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, m-toluidine is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster CLHL/IU cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9-mix: 0, 0.13, 0.26, 0.52 mg/ml (continuous treatment)
-S9-mix: 0, 0.3, 0.6, 1.1 mg/ml (short-term treatment)
+S9-mix: 0, 0.3, 0.6, 1.1 mg/ml (short-term treatment)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9-mix: Mitomycin C; +S9-mix: Cyclophosphamide

Details on test system and experimental conditions:

as requested by the guideline

Evaluation criteria:

the test substance is considered positive when assay cultures with the test substance show a significantly higher incidence of cells with chromosome aberrations as compared with the negative controls and when this effect is reasonably reproducible or dose-dependent

Statistics:

yes, according to Ishidate, 1987

Species / strain:

other: Chinese hamster Lung /IUL cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9-mix: >0.52 mg/ml (continuous treatment); -S9-mix: >1.1 mg/ml (short-term treatment); + S9-mix: >1.1 mg/ml (short-term treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Executive summary:

m-Toluidine was tested for clastogenic activity in a chromosome aberration test in Chinese hamster CHL/IUL cells according to OECD TG 473 under GLP conditions in the presence and in the absence of a metabolic activation system. No mutagenic activity of m-toluidine was detected. The concurrent substances used as positive controls were functional. Thus, m-toluidine has no clastogenic property.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No valid in vivo genotoxicity test available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genotoxicity of m-toluidine was studied by a Reverse Mutation test in bascteria (Ames test) according to OECD TG 471 and GLP. m-Toluidine showed no mutagenic activity in Salmonella typhimurium TA 100, TA1535, TA98, TA1537, Eschericha coli WP2uvrA with and without an exogenous metabolic activation system. Appropriate positive control substances were functional (MHLW 2002)

A study was performed to investigate the potential of m-toluidine to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditiions. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours (33.5 µg/ml - 1072 µg/ml). The second experiment was performed with a treatment time of 4 hours with (33.5 µg/ml - 1072µg/ml) and 24 hours without (67.0 µg/ml -1072 µg/ml) metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficientcy 1 or cell density below 50% occurred only in the absence of metabolic activation at 1072 µg/ml in experiment 1 (4 hr treatment) and experiment 2 (24 hr treatment).

No relevant and reproducible dose dependent increase in mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, m-toluidine is considered to be non-mutagenic in this HPRT assay.

m-Toluidine was tested for clastogenic activity in a chromosome aberration test in Chinese hamster CHL/IUL cells according to OECD TG 473 under GLP conditions in the presence and in the absence of a metabolic activation system. No mutagenic activity of m-toluidine was detected. The concurrent substances used as positive controls were functional. Thus, m-toluidine has no clastogenic property.

OVERALL m-Toluidine was tested for mutagenicity using point mutation tests in bacteria and in mammalian cells as well as a test for chromosme aberrations. All tests were performed according to the respective guidelines under GLP conditions. No mutagenic activity was detected. Thus, it can be concluded that m-toluidine is a non-mutagenic substance

Short description of key information:
There is an Ames test performed according to OECD TG 471 and yielded a negative result (MHLW 2002). An HPRT test was performed with m-toluidine according to the respective guideline and yielded a negative result (WOLLNY 2014).
There is an in-vitro Chromosome Aberration test with CHL/IU cells performed according to OECD TG 473 and yielded a negative result (MHLW 1995)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All relevant in-vitro tests (key studies) are negative.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.