Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl-and acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Chromosome aberration study:

The test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate does not induce gene mutation in mammalian cell lines in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE report is based on three gene mutation in vitro toxicity studies as- 1., 2. The Ames Salmonella typhimurium mutagenicity test was conducted for the test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA102, and TA1535

Remarks:

RA 1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

1/2. 5–5000 μg/plate

Vehicle / solvent:

1 and 2. No data3. - Vehicle(s)/solvent(s) used: Yes, no detailed data available - Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

mitomycin C

Remarks:

RA 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

mitomycin C

other: 2-aminofluorene for all the strains with S9 mix

Remarks:

RA 2

Details on test system and experimental conditions:

1 and 2. METHOD OF APPLICATION: in agar (plate incorporation) with preincubation modificationDURATION- Preincubation period: No data available- Exposure duration: 48 h - Expression time (cells in growth medium): 48 h- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, the cytotoxicity study were carried out by accounting for the microcolonies(histidine auxotroph) in the background lawn. The sparse or absent growth indicated the toxic nature of dyes toward the tester strains.OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1 and 2. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, and TA1535

Remarks:

RA 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 500 μg

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, and TA1535

Remarks:

RA 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 500 μg

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate. The studies are as mentioned below:

Ames mutagenicity test was conducted for two test chemicals to evaluate its genotoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA102, and TA1535 with dose concentration of 5–5000 µg/plate in plate incorporation assay. The plates were incubated for 48 h. The doses of test chemical, together with the appropriate concurrent positive controls, were tested in triplicate on each tester strain with and without S9 metabolic activation. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response. Both the test chemicals did not induce gene mutation in the Salmonella typhimuriumTA98, TA100, TA102, and TA1535 both in the presence and absence of S9 activation system and hence the chemical is not likely to be a gene mutant.

Based on the data summarized, Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Reference 2

Endpoint:

in vitro gene mutation 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 is from peer reviewed publication

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

1. Thymidine kinase (TK) locus2. hprt locus in CHO-K1-BH4 cells and the gpt locus in AS52 cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 1

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.- Properly maintained: No data available - Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 and CHO-AS52 / 2

Details on mammalian cell type (if applicable):

- Type and identity of media: No data- 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:

Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

1. 0.001-6 µg/mL2. 250-3000 µg/mL

Vehicle / solvent:

1.- Vehicle(s)/solvent(s) used: Water- Justification for choice of solvent/vehicle: The test chemical was soluble in water2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene and dimethylbenz[a]- anthracene (+S9)

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

2

Details on test system and experimental conditions:

1, METHOD OF APPLICATION: in mediumCells at start: 6000000 cellsDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selectionSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200cells/plate for viable count determinations DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated culturesOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available2. METHOD OF APPLICATION: in mediumCells at the strat of experiment: 1 X 106 cells/100-mm dishDURATION- Preincubation period: No data- Exposure duration: 5 hrs- Expression time (cells in growth medium): 7 days- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): 7 daysSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Parallel culturesof 400 cells/60-mm dish were established for cytotoxicity determinationOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data2,Details on test system and conditionsMETHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium):48 h

Rationale for test conditions:

No data

Evaluation criteria:

1 . Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.2. Results were expressed as 6-thioguanine resistant mutants/106 clonable cells.

Statistics:

Not specified

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C / 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 and CHO-AS52

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

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate does not induce gene mutation in mammalian cell lines in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino] phenyl]ethenyl]-1,3,3-trimethyl- & acetate. The studies are as mentioned below:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of structurally and functionally similar test chemical.The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 0.001 -6 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence andabsence of S9 activation system and hence is not likely to be gene mutant in vitro.

Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of the test chemical. The study was performed using Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml without S9 and 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml with S9. 1X 10⁶cells/100 mm dish were exposed to the test chemical for 5 hrs in serum-free nutrient mixture F12. After treatment, cells were washed with Ca+ +/Mgt +-free phosphate-buffered saline (PBS) and allowed to recover overnight in fresh F12 medium with 5% fetal bovine serum (FBS). The cells were then plated in F12 medium with 5% FBS and incubated for 7 days with three passages to permit expression of induced mutants. Parallel cultures of 400 cells/60-mm dish were also established for cytotoxicity determination. At the end of the phenotypic expression period, 2 X 10⁶cells were plated at a concentration of 2 X 10⁵celIs/100-mm dish in hypoxanthine-free F12 containing 5% dialyzed FBS and 10 µM 6-thioguanine. Cloning efficiency cultures, grown in medium without 6-thioguanine, had 200 cells/60-mm plate. All cultures were incubated for 7 days before colonies were fixed, stained and counted. Results were expressed as 6-thioguanine resistant mutants/10⁶clonable cells. At the concentrations tested, the results were negative in CHO-K1-BH4 cells, while 66 mutants per 10⁶clonable cells were obtained in AS52 cells at a test chemical concentration producing a high level of toxicity. This positive response, however, was not consistently found in subsequent experiments. Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9. GV-treated cells, especially AS52 cells, became tenaciously attached to the plates such that it required longer trypsinization times to dissociate them during subculture. When observed with an inverted microscope, the exposed cells appeared larger and more spindle-shaped than control cells. The test chemical did not induce gene mutation in Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Based on the data summarized, Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate does not induce gene mutation in mammalian cell lines in the presence and absence of S9 metabolic activation system and hence it 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:

Ames test:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate. The studies are as mentioned below:

Ames mutagenicity test was conducted for two test chemicals to evaluate its genotoxic effects when exposed to Salmonella typhimurium strains TA98, TA100, TA102, and TA1535 with dose concentration of 5–5000 µg/plate in plate incorporation assay. The plates were incubated for 48 h. The doses of test chemical, together with the appropriate concurrent positive controls, were tested in triplicate on each tester strain with and without S9 metabolic activation. A dose-related increase (at least 2-fold) in revertant colonies was used to define a statistically significant mutagenic response. Both the test chemicals did not induce gene mutation in the Salmonella typhimuriumTA98, TA100, TA102, and TA1535 both in the presence and absence of S9 activation system and hence the chemical is not likely to be a gene mutant.

Chromosome aberration study:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of structurally and functionally similar test chemical.The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 0.001 -6 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence andabsence of S9 activation system and hence is not likely to be gene mutant in vitro.

Mammalian cell gene mutation assay was conducted to evaluate the genotoxic potential of the test chemical. The study was performed using Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of0, 0.02, 0.04, 0.05, 0.06, 0.08, 0.10, 0.125, 0.25, 0.50, 1.00 µg/ml without S9 and 0, 0.10, 0.20, 0.25, 0.30, 0.40, 0.50, 1.00, 1.50 µg/ml with S9. 1X 10⁶cells/100 mm dish were exposed to the test chemical for 5 hrs in serum-free nutrient mixture F12. After treatment, cells were washed with Ca+ +/Mgt +-free phosphate-buffered saline (PBS) and allowed to recover overnight in fresh F12 medium with 5% fetal bovine serum (FBS). The cells were then plated in F12 medium with 5% FBS and incubated for 7 days with three passages to permit expression of induced mutants. Parallel cultures of 400 cells/60-mm dish were also established for cytotoxicity determination. At the end of the phenotypic expression period, 2 X 10⁶cells were plated at a concentration of 2 X 10⁵celIs/100-mm dish in hypoxanthine-free F12 containing 5% dialyzed FBS and 10 µM 6-thioguanine. Cloning efficiency cultures, grown in medium without 6-thioguanine, had 200 cells/60-mm plate. All cultures were incubated for 7 days before colonies were fixed, stained and counted. Results were expressed as 6-thioguanine resistant mutants/10⁶clonable cells. At the concentrations tested, the results were negative in CHO-K1-BH4 cells, while 66 mutants per 10⁶clonable cells were obtained in AS52 cells at a test chemical concentration producing a high level of toxicity. This positive response, however, was not consistently found in subsequent experiments. Toxicity was apparent in CHO cells, both with and without metabolic activation, although it was more evident in assays conducted without S9. GV-treated cells, especially AS52 cells, became tenaciously attached to the plates such that it required longer trypsinization times to dissociate them during subculture. When observed with an inverted microscope, the exposed cells appeared larger and more spindle-shaped than control cells. The test chemical did not induce gene mutation in Chinese hamster ovary (CHO) cell strain CHO-K1-BH₄and CHO-AS52 in the presence and absence of S9 metabolic activation system and hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Based on the data summarized, Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the data summarized, Reaction mass of 3H-Indolium, 2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]ethenyl]-1,3,3-trimethyl- & acetate did not induce gene mutation in the Salmonella typhimurium strains used in the presence and absence of S9 metabolic activation system and hence it is not likely to be mutagenic in vitro.