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Genetic toxicity in vitro

Description of key information

Data for the various test chemicals was reviewed to determine the mutagenic nature of Methyl 2-(methylamino)benzoate ( 85-91-6). The studies are as mentioned below:

AMES Assay

Test substance was considered to be non mutagenic in Salmonella typhimurium strains with and without metabolic activation system in the plate incorporation and preincubation method performed.

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source.
Qualifier:
according to guideline
Guideline:
other: Data is from WHO Food additives series 56 study report available on JECFA - Monographs & Evaluations
Principles of method if other than guideline:
Gene toxicity in vitro study was performed for the test compound methyl N-methylanthranilate using S typhimurium strain TA98 and TA100
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
3-5000 µg/plate
Vehicle / solvent:
No data
Untreated negative controls:
not specified
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:
Test was carried out both in direct plate assay and pre-incubation assay.
Evaluation criteria:
An increase in the number of revertants was noted
Statistics:
No data
Species / strain:
S. typhimurium, other: TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the direct plate assay, cytotoxicity observed at 3330 and 5000 µg/plate (+/- S9) in both the strains.
In pre-incubation assay, cytotoxicity observed 333-5000 µg/plate (+/- S9) in both the strains except in TA98 at 333µg/plate +S9.
Remarks on result:
other: No mutagenic effec were observed
Conclusions:
Test substance was considered to be non mutagenic in Salmonella typhimurium strains TA100 and TA98 with and without metabolic activation system in the plate incorporation and preincubation method performed.
Executive summary:

Gene toxicity in vitro study was performed for the test compound methyl N-methylanthranilate using S typhimurium strain TA98 and TA100. Plate incorporation and preincubation assay was performed at dose levels of 3-5000 µg/plate. with and without S9 metabolic activation system. The given test substance Methyl-N-Methyl-anthranilate failed to induce mutation in Salmonella typhimurium strains TA100 and TA98 with and without metabolic activation system S9 in the plate incorporation and preincubation method performed. The test material Methyl-N-Methyl-anthranilate is therefore not likely to be classified as a gene toxicant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data from handbook or collection of data
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: As mention below
Principles of method if other than guideline:
Weight of evidence prepared from various studies mention below
To evaluate the mutagenic potential of test chemical in Chinese hamster lung(CHL)cells by in vitro mammalian chromosome aberration test.


GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not specified
Species / strain / cell type:
other: Chinese hamster lung(CHL)cells
Details on mammalian cell type (if applicable):
CHL / IU cells derived from Chinese hamster lungs obtained from Dainippon Pharmaceutical Co., Ltd. (September 2003, passage: 14th generation,
frozen: 17th) were used for the test within 4 weeks after thawing succession
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
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:
1,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL
2,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Isotonic sodium chloride solution
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Isotonic sodium chloride solution
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix, Mitomycin C +S9 mix, Benzo [a] pyrene
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: short-time treatment method :6h
continuous treatment method:24h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

NUMBER OF CELLS EVALUATED: 200 metaphases were scored per experimental group.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:
Structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells.
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: No mutagenic effect were observed
Conclusions:
In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material.SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence ofmetabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0,50,75,100,125,150 µg/mL concentration in In the continuous treatment method were used . The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation inChinese hamster lungs cells inthe presence and absence of S9and 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

Data for the various test chemicals was reviewed to determine the mutagenic nature of Methyl 2-(methylamino)benzoate ( 85-91-6). The studies are as mentioned below:

AMES Assay and DNA repair Assay

Gene toxicity in vitro study was performed for the test chemical using S typhimurium strain TA98 and TA100. Plate incorporation and preincubation assay was performed at dose levels of 3-5000 µg/plate with and without S9 metabolic activation system. The given test substance failed to induce mutation in Salmonella typhimurium strains TA100 and TA98 with and without metabolic activation system S9 in the plate incorporation and preincubation method performed. The test material is therefore not likely to be classified as a gene toxicant in vitro.

 

In the same study, Salmonella mutagenicity assay was performed to evaluate the mutagenic nature of the test chemical. Direct plate incorporation assay was performed at dose levels of 10 -3330 µg/plate. The given test substance failed to induce mutation in Salmonella typhimurium strains TA102, TA1535, TA1537 with and without metabolic activation system S9 in the plate incorporation at concentration of 10-3330 µg/plate. Therefore, the test chemical is not likely to classify as a gene mutant in vitro.

 

In the same study, another gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical . Preincubation assay was performed at dose levels of 3 -1000 µg/plate. The given test substance Methyl-N-Methyl-anthranilate was found to be non-mutagenic in Salmonella typhimurium strains TA102, TA1535, TA1537 with and without S9 metabolic activation system in the preincubation assay performed at concentration of 3-1000 µg/plate. Thus the test chemical is not likely to classify as a gene mutant in vitro.

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 test chemical, 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 test chemical is negative for genotoxicity in vitro.

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material.SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence of metabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

 

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0, 50, 75,100,125,150 µg/mL concentration in In the continuous treatment method were used. The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation inChinese hamster lungs cells inthe presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

Based on the studies summarized, the test chemical Methyl 2-(methylamino)benzoate ( 85-91-6) is not likely to classify as gene mutant in vitro.  

Justification for classification or non-classification

Based on the data summarized and CLP criteria , Methyl 2-(methylamino)benzoate ( 85-91-6)did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.