2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity of the test item was assessed in an in vitro testing battery under GLP (OECD 471, OECD 473 and OECD 476). The target substance was not genotoxic in an OECD 473 in vitro chromosome aberration test, but was tested positive in a bacterial reverse mutation assay (OECD 471). In an in vitro mammalian mutagenicity test (OECD 476) a read-across partner substance was tested negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1998-09-14 to 1999-01-28

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)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro chromosome aberration

Target gene:

n.a.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED:
MEM + 10% FCS (complete medium)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test concentration were chosen based on the results from a pre-test.
Main study:
without S9: 0.625, 1.25, 2.5, 5.0 and 10.0 µg/mL
with S9: 3.125, 6.25, 12.5, 18.75 and 25.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group. In each chamber 44000 cells/slide were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT:
EXPOSURE PERIOD 4 hours (with S9 mix): The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing different concentrations of the test article and 50 µL/mL S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
EXPOSURE PERIOD 18 hours (without S9 mix): The culture medium of exponentially growing cell cultures was replaced with complete medium (10 % FCS) containing different concentrations of the test article without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37° C in a humidified atmosphere with 4.5 % C02 (95.5 % air).

PREPARATION OF THE CULTURES:
16 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa. Additionally, two cultures V79 cells per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the solvent control.

ANALYSIS OF METAPHASE CELLS:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Rationale for test conditions:

according to OECD test guideline 473

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.
A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration in the pre-test on toxicity (1600 µg/ml) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test concentrations between 12.5 and 1600 µg/ml (with and without S9 mix) were applied for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers below 50 % of the corresponding control were observed after treatment with 12.5 µg/ml (21 % of control) and above. In the presence of S9 mix after 4 h treatment reduced cell numbers were determined at 25.0 µg/ml (22 % of control) and above. Precipitation of the test article in culture medium was observed in the absence of S9 mix after treatment with 100 µg/ml and at 200 µg/ml in the presence of S9 mix. No influence of the test article on the pH value or osmolarity was observed (solvent control: 280 mOsm, pH 7.3 versus 278 mOsm and pH 7.3 at 1600 µg/ml).

MAIN EXPERIMENT:
In the main experiment the mitotic indices of the evaluated concentrations in the presence and absence of S9 were reduced to 39.1 % (2.5 µg/mL) and 26.5 % (12.5 µg/mL) of control. However, the corresponding cell numbers were not reduced. The evaluation of cultures after treatment with the next higher concentrations was not feasible, since no or not enough mitotic cells were found. Thus, only the concentrations groups 2.5 µg/mL (without S9) and 12.5 µg/mL (with S9) were chosen for further evaluation for structural chromosome aberrations. Neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (0.5 % and 1.0 %, excluding gaps) were near to the range of the solvent control values (1.0 % - 2.0 %) and within the range of our historical control data: 0.0 % - 4.0 %. EMS (600 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Table 1: Pre-test: Determination of Toxicity
Concentration µg/mL		mean number of cells		% of solvent control
without S9
solvent control		870		100
12.5		185		21
25		189		22
50		167		19
100		149		17
200		204		24
400		185		21
800		141		16
1600		172		20
with S9 mix
solvent control		554		100
12.5		385		69
25		123		22
50		176		32
100		81		15
200		227		41
400		147		27
800		196		35
1600		not evaluated		not evaluated

Table 2: Main experiment: Determination of Toxicity
Concentration µg/mL		mean number of cells		% of solvent control
without S9
solvent control		441		100
0.625		655		149
1.25		462		105
2.5		268		61
5.0		421		96
10.0		82		19
with S9 mix
solvent control		676		100
3.125		332		49
6.25		386		57
12.5		343		51
18.75		203		30
25		321		48

Table 3: Summary of the main study results
Exposure period	Concentration of the test item in µg/mL		Polyploid cells in %		Mitotic index in % of control		Aberrant cells in %
							incl. gaps	excl. gaps*	exchanges
without S9
18 h	negative control		4.7		100**		1.5	1.5	0
	solvent control		2.7		100***		3.5	2	0.5
	positive control		3.5		94.2		18 (s)	18 (s)	9.5
	2.5		3.5		39.1		0.5	0.5	0
with S9 mix
4h	negative control		3.8		100**		1.5	0.5	0
	solvent control		4.5		100***		2	1	0
	positive control		4		86.9		27	25.5 (s)	11
	12.5		4.3		26.5		1.5	1	0

*= inclusive cells carrying exchanges

**= for the positive control groups, the relative values of the mitotic index are related to the negative controls

***= for the test item treatment groups the values are related to the solvent controls

(s)= Aberration frequency statistically significant higher than corresponding solvent control values

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Executive summary:

In an in vitro chromosome aberration test (in accordance to OECD 473), V79 Chinese hamster lung cell cultures were exposed to the test item (90% purity), solved in deionised water, at concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10.0 µg/mL without metabolic activation and with metabolic activation of concentrations of 0, 3.125, 6.25, 12.5, 18.75 and 25.0 µg/mL. The test item was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of structural chromosome aberrations induced over background. This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The source substance is the methyl sulphate salt of the target substance. Bothe substances have the same chromophore, which is responsible for any posssible toxic effects. It is therefore feasible to use the results of the study with Basic Blue 41 methyl sulphate for Basic Blue 41 Chloride.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

The study was performed in two independent main experiments, using identical procedures, with and without liver microsomal activation. Strong toxic effects occurred at concentrations of 30 µg/mL and above without and 10 µg/mL and above with metabolic activation. In the second experiment several closely spaced concentrations of the test article were chosen to cover the threshold of toxicity. No precipitation of the test article occurred up to the maximal concentration. In the presence and absence of metabolic activation the cloning efficiency of the cells was reduced down to almost zero at the highest concentrations tested. In the mass-cultures of the larger flasks these toxic effects were less striking. The range of the controls was not exceeded in any of the test groups with or without metabolic activation. Furthermore, there was no indication of any concentration dependent increase of the number of colonies below the threshold of biological relevance. In both experiments of this study (with and without S9 mix) the range of the negative controls was from 5.0 up to 17.5 mutant colonies per 10^6 cells; the range of the groups treated with the test article was from 3.0 up to 15.2 mutant colonies per 10^6 cells. EMS (0.6 mg/mL) and DMBA (3.85 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Table 1: Pre-test: Determination of Toxicity
Concentration µg/mL		extinction mean plus standard deviation		% of control
without S9
Blanc		0.163 +/- 0.021		/
Negative control		1.067 +/- 0.045		102.92
Solvent control		1.041 +/-0.063		100
30		0.853 +/- 0.055		78.52
50		0.604 +/- 0.085		50.21
100		0.439 +/- 0.029		31.39
300		0.307 +/- 0.021		16.34
500		0.210 +/- 0.009		5.31
1000		0.191 +/- 0.005		3.22
3000		0.193 +/- 0.006		3.35
5000		0.194 +/- 0.004		3.52
with S9 mix
Blanc		0.140 +/- 0.034		/
Negative control		1.177 +/- 0.043		101.87
Solvent control		1.158 +/- 0.042		100
30		1.129 +/- 0.063		97.12
50		0.903 +/- 0.175		74.95
100		0.322 +/- 0.018		17.85
300		0.538 +/- 0.015		39.12
500		0.464 +/- 0.049		31.84
1000		0.206 +/- 0.024		6.49
3000		0.187 +/- 0.004		4.66
5000		0.187 +/- 0.008		4.62

Conclusions:

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

In a mammalian cell HPRT gene mutation assay, V79 cells cultured in vitro were exposed to the test item in aqua bidest at concentrations of 1.0, 3.0, 10.0, 30.0, 60.0 and 100.0 µg/mL in the absence of mammalian metabolic activation and at concentrations of 1.0, 3.0, 10.0, 30.0, 100.0 and 300 µg/mL in the presence of S9 (experiment I). Strong toxic effects occurred at concentrations of 30.0 µg/mL and above without and at 10.0 µg/mL and above with metabolic activation. Thus, in a second experiment several closely spaced concentrations of the test article were chosen to cover the threshold of toxicity (1.0, 3.0, 10.0, 30.0, 40.0 and 50.0 µg/mL (without S9) and 1.0, 3.0, 10.0, 20.0, 30.0 and 40.0 (with S9)). The following concentrations were evaluated for mutagenicity: 1.0, 3.0, 10.0 and 30.0 µg/mL in the presence and absence of mammalian metabolic activation (experiment I) and for experiment II concentrations of 1.0, 10.0, 40.0 and 50.0 µg/mL (without S9) and 1.0, 10.0, 30.0 and 40.0 µg/mL with metabolic activation. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-06-14 to 1995-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Nutrient medium: 8 g Merck Nutrient Broth and 5 g NaCl per liter,
Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal agar was used as selective agar,
Overlay Agar: 6.0 g Merck Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine, 12.2 mg Biotin per liter.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

According to the results of the pre-experiment the concentrations applied in the main experiments were chosen:
Experiment I: 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate
Experiment II: 33.3, 66.6, 100, 166.6, 333.3 and 1000 µg/plate
Experiment IIa (strains TA 1535 and TA 1537): 3.3, 10.0, 33.3, 66.6, 100.0 and 166.6 µg/plate

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in aqua bidest. The solvent was chosen because of its solubility properties.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA 1535 and TA 100, 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Remarks:

without S9, TA 1537 and TA 98, 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, all strains, 2.5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level

DETERMINATION OF CYTOTOXICITY: Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

ACCEPTED CONDITIONS FOR EVALUATION:
- corresponding background growth on both negative and test plates
- normal range of spontaneous reversion rates

Rationale for test conditions:

N.A.

Evaluation criteria:

A test article is considered positive if either a dose related and/or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test article producing neither a dose related nor reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strain TA 100 or thrice on TA 1535, TA 1537, and TA 98. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Distinct toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. All plates incubated with the test article showed reduced background growth starting at concentrations as low as 33.3 µg/plate in some of the strains. Substantial increases in revertant colony numbers were observed following treatment with the test item with and without metabolic activation in all strains used. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Table 1: Summary of Results without S9 mix
Revertants/plate
mean from three plates
Concentration  µg/plate		TA 1535		TA 1537		TA 98		TA 100		TA 1535		TA 1537
		I	II	I	II	I	II	I	II	IIa		Iia
Negative Control		17	17	5	13	44	30	141	125	16		10
Solvent Control		15	14	7	14	43	41	149	126	15		11
Positive Control		360	843	37	47	279	146	870	767	537		50
3.3		/	/	/	/	/	/	/	/	19		8
10		/	/	/	/	/	/	/	/	43		15
33.3		21	34	16	30	35	53	718	775	28		27
66.6		/	17	/	31	/	173	/	1083	9		12
100		7	9	26	23	72	161	303	1086	6		20
166.6		/	6	/	12	/	92	/	245	6		12
333.3		0	3	6	19	54	195	895	272	/		/
1000		0	3	0	3	38	127	230	42	/		/
2500		0	/	0	/	26	/	76	/	/		/
5000		10	/	0	/	30	/	65	/	/		/

/= not performed

Table 2: Summary of Results with S9 mix
Revertants/plate
mean from three plates
Concentration  µg/plate		TA 1535		TA 1537		TA 98		TA 100		TA 1535		TA 1537
		I	II	I	II	I	II	I	II	IIa		Iia
Negative Control		16	19	6	21	48	43	147	149	12		16
Solvent Control		20	16	7	18	50	46	155	168	15		19
Positive Control		221	372	261	149	629	849	899	1209	206		126
3.3		/	/	/	/	/	/	/	/	17		19
10		/	/	/	/	/	/	/	/	20		21
33.3		47	70	26	35	51	158	192	209	74		38
66.6		/	63	/	37	/	129	/	318	17		39
100		12	13	15	38	39	176	308	428	12		41
166.6		/	7	/	13	/	280	/	620	8		17
333.3		0	4	7	11	52	284	472	444	/		/
1000		0	1	0	10	44	311	293	164	/		/
2500		0	/	0	/	23	/	176	/	/		/
5000		0	/	0	/	26	/	181	/	/		/

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA
100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

 In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (90% purity) in water at concentrations of 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate (experiment I) and at concentrations of 33.3, 66.6, 100, 166.6, 333.3 and 1000 µg/plate (experiment II) in the presence and absence of mammalian metabolic activation. Strains TA 1535 and TA 1537 were additionally tested in at concentrations of 3.3, 10, 33.3, 66.6, 100 and 166.6 µg/plate (experiment IIa). The test item was tested up to the limit concentration (5000 µg/plate). Due to overlapping toxic effects, the number of revertants was reduced at the higher concentrations in the strains TA 1535, TA 1537, and TA 100 in experiments I, II and IIa. The positive controls induced the appropriate responses in the corresponding strains. Substantial increases in revertant colony numbers were observed following treatment with the test item with and without metabolic activation in all strains used. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay. This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genotoxicity of the test item was assessed in an in vitro testing battery under GLP (OECD 471, OECD 473 and OECD 476). The target substance was not genotoxic in an OECD 473 in vitro chromosome aberration test, but was tested positive in a bacterial reverse mutation assay (OECD 471). In an in vitro mammalian mutagenicity test (OECD 476) a read-across partner substance was tested negative.

Based on the result of the higher tier mammalian cell assay for mutagenicity (OECD 476) and the overall evaluation of the available data in a weight-of-evidence approach the target substance is considered to be non-mutagenic. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Justification for classification or non-classification

Based on the available data, the target substance does not warrant classification for mutagenicity.