2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium 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 2-({4-[ethyl(2-hydroxyethyl)amino]phenyl}diazenyl)-6-methoxy-3-methyl-1,3-benzothiazol-3-ium acetate(84051-87-6) 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 2-({4-[ethyl(2-hydroxyethyl)amino]phenyl}diazenyl)-6-methoxy-3-methyl-1,3-benzothiazol-3-ium acetate(84051-87-6) 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

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: 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 data
3.
- 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 modification

DURATION
- 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 available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION 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 available

OTHER: 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 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 . 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, test chemical 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 report is based on three gene mutation in vitro toxicity studies as-
1,The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical
2,The test chemical was tested for its ability to induce chromosomal aberrations (ABs) in cultured Chinese hamster ovary (CHO) cells using a standard protocol with and without exogenous metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

1. Thymidine kinase (TK) locus
2. No data

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:

2

Details on mammalian cell type (if applicable):

- Type and identity of media: Stocks of CHO cells were maintained at 37°C in McCoy's 5A (modified) medium buffered with 20 mM HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 pg/ml streptomycin (Gibco. Grand Island,
NY).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, Cells were tested regularly for mycoplasma contamination using 4' ,6-diamidin0-2-phenyl-indole (DAPI) fluorescence and were found to be free of mycoplasma
- Periodically checked for karyotype stability: Yes, To ensure karyotypic stability, cells were not used beyond the fifteenth passage after cloning.
- 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/mL
2. Without S9:
Trial 1: 0.0000, 5.0100, 15.0000, 50.1000 µg/mL
Trial 2: 0.0, 76.0000, 99.8000, 160.000 µg/mL

With S9:
Trial 1: 0.0000, 150.0000, 501.0000, 1500.0000 µg/mL
Trial 2: 0.0000, 134.0000, 267.0000, 446.0000, 668.0000 µg/mL

Vehicle / solvent:

1./2.
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

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:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

2

Details on test system and experimental conditions:

1, METHOD OF APPLICATION: in medium

Cells at start: 6000000 cells

DURATION
- 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 available

SELECTION AGENT (mutation assays): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/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 cultures

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

OTHER: No data available

2. METHOD OF APPLICATION: in medium

Cells seeded: 1.75 X 106 cells/75 cm2 flask

DURATION
- Preincubation period: No data
- Exposure duration: Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100-200 cells/dose

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

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

OTHER: No data

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. Selection of cells for scoring was based on well-spread chromosomes with good morphology and a chromosome number of 21 ± 2. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis.

A positive response at a single dose was designated “ + W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘ + W*”. A test was designated “ + ” if at least two doses gave significantly increased responses.

Statistics:

1. Not specified
2. A binomial sampling assumption as described by Margolin et al. was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. Only the “total” percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.

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:

2

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:

1. No data
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five
concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical 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 test chemical . 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.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 10⁶cells/75 cm²flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data summarized, test chemical 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

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical2-({4-[ethyl(2-hydroxyethyl)amino]phenyl}diazenyl)-6-methoxy-3-methyl-1,3-benzothiazol-3-ium acetate(84051-87-6) . The studies are as mentioned below:

Ames test:

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.

 

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 106cells/75 cm2flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Based on the data summarized, 2-({4-[ethyl(2-hydroxyethyl)amino]phenyl}diazenyl)-6-methoxy-3-methyl-1,3-benzothiazol-3-ium acetate(84051-87-6)  does not induce gene mutation in the Salmonella typhimurium strains and the 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.

Justification for classification or non-classification

Based on the data summarized, 2-({4-[ethyl(2-hydroxyethyl)amino]phenyl}diazenyl)-6-methoxy-3-methyl-1,3-benzothiazol-3-ium acetate(84051-87-6)  did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.