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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

248-053-0

EC Name:

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

Cas Number:

26850-47-5

Molecular formula:

C19H23ClN4O2S

IUPAC Name:

2-[(E)-2-{4-[ethyl(2-hydroxyethyl)amino]phenyl}diazen-1-yl]-6-methoxy-3-methyl-1,3-benzothiazol-3-ium chloride

Test material form:

solid

Details on test material:

see below

Method

Target gene:

n.a.

Species / strain

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

Controls

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

Results and discussion

Test results

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).

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.