Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-04-24 until 2012-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 476

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activationsecond experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

EC Number:

223-989-2

EC Name:

Sodium p-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate

Cas Number:

4156-21-2

Molecular formula:

C9H6Cl2N4O3S.Na

IUPAC Name:

sodium 4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulfonate

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:without metabolic activation: 18.8; 37.5; 75.0; 150.0; 225.0; 300.0 µg/mLwith metabolic activation: 9.4; 18.8; 37.5; 75.0; 112.5; 150.0 µg/mLExperiment II:without metabolic activation: 75.0; 150.0; 300.0; 600.0; 800.0; 1000.0; 1200.0 µg/mLwith metabolic activation: 9.4; 18.8; 37.5; 75.0; 93.8; 112.5 µg/mL In both experiments without metabolic activation the lowest concentration of 18.8 or 75.0 µg/mL was not continued since only four analysable concentrations are required. The concentration of 150 µg/mL in the first experiment with metabolic activation was not con-tinued due to exceedingly severe cytotoxicity. In the second experiment the maximum concentrations of 112.5 µg/mL with and 1200 µg/mL without metabolic activation were not continued for the same reason.

Vehicle / solvent:

Solvent used: deionised water- Justification for choice of solvent/vehicle: solubility properties

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation- Expression time (cells in growth medium): 72 hours - Selection time (if incubation with a selection agent): 10 daysSELECTION AGENT (mutation assays): 6-Thioguanine NUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: >1,5x10exp. 6DETERMINATION OF CYTOTOXICITY - Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.A positive response is described as follows:A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen¬trations in the experiment.The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.However, in a case by case evaluation this decision depends on the level of the corre-spon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: Not increased (pH 7.36 in the solvent control, pH 7.22 at 4900 µg/mL (measured in the max. concentration of the pre-experiment)- Effects of osmolality: Not effected (273 in the solvent control, 299 at 4900 mg/mL (measured in the max. concentration of the pre-experiment)- Evaporation from medium: Not examined- Water solubility: Not indicated- Precipitation: Only in the pre-experiment precipitation occurred at 4900 µg/mL in the presence of metabolic activation after 4 hours treatment.- Other confounding effects: NoneRANGE-FINDING/SCREENING STUDIES: According to the current OECD Guideline for Cell Gene Mutation Tests at least four ana-lysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the ana-lysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evalu-ated at the end of treatment by the unaided eye. The highest concentration used in the pre-test was 4900 µg/mL equal to a molar concen-tration of about 10 mM adjusted to the purity of the test item. The test item was dissolved in deionised water. Test item concentrations between 38.3 µg/mL and 4900µg/mL used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative sus-pension growth below 50 % were observed at 306.3 µg/mL and above without metabolic activation following 4 h treatment, at 153.1 µg/mL and above with metabolic activation following 4 h treatment, and at 1225 µg/mL and above without metabolic activation follow-ing 24 h treatmentThe test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Precipitation occurred at 4900 µg/mL in the presence of metabolic activation (4 hours treatment).There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.COMPARISON WITH HISTORICAL CONTROL DATA: CompliesADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects, indicated by a relative cloning efficiency I of less than 50% compared to the corresponding solvent control in both parallel cultures, occurred in experiment I at 112.5 µg/mL and above with and at 225.0 µg/mL and above without metabolic activation. In experiment II cytotoxic effects were noted at 75.0 µg/mL and above with and at 800 µg/mL and above without metabolic activation.

Remarks on result:

other: See table below

Any other information on results incl. tables

Summary Table

			relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
			%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	12.5	1.0	100.0	100.0	100.0	9.5	1.0
Positive control (EMS)	150.0	-	113.4	115.8	130.1	124.5	10.0	92.9	109.1	88.7	90.0	9.4
Test item	18.8	-	106.8	culture was not continued^#				114.0	culture was not continued^#
Test item	37.5	-	113.8	129.8	136.7	20.8	1.7	113.9	115.6	88.6	6.7	0.7
Test item	75.0	-	88.9	100.2	148.7	8.9	0.7	85.2	101.0	108.9	4.3	0.5
Test item	150.0	-	67.9	177.3	119.0	9.1	0.7	44.8	162.5	104.1	9.5	1.0
Test item	225.0	-	8.6	125.3	145.6	12.7	1.0	4.6	135.1	94.9	16.6	1.7
Test item	300.0	-	0.0	125.1	147.4	4.9	0.4	0.0	121.9	106.9	6.8	0.7
Solvent control with water		+	100.0	100.0	100.0	9.8	1.0	100.0	100.0	100.0	9.8	1.0
Positive control (DMBA)	1.1	+	58.4	81.8	96.1	694.1	70.9	52.6	72.6	90.4	633.9	64.7
Test item	9.4	+	99.0	116.3	94.0	7.3	0.7	98.1	99.5	83.4	12.6	1.3
Test item	18.8	+	98.3	108.0	95.7	11.7	1.2	99.3	91.1	90.8	10.4	1.1
Test item	37.5	+	101.6	107.4	92.2	7.0	0.7	100.1	121.1	94.4	3.8	0.4
Test item	75.0	+	59.2	114.3	93.2	4.5	0.5	49.7	102.5	88.0	6.1	0.6
Test item	112.5	+	0.0	90.9	98.4	4.5	0.5	3.6	99.4	80.0	7.6	0.8
Test item	150.0	+	0.0	culture was not continued^##				0.0	culture was not continued^##
Experiment II / 24 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	7.2	1.0	100.0	100.0	100.0	15.7	1.0
Positive control (EMS)	150.0	-	92.1	105.2	99.0	263.1	36.7	113.6	74.8	81.1	368.4	23.5
Test item	75.0	-	103.7	culture was not continued^#				116.9	culture was not continued^#
Test item	150.0	-	102.5	104.8	90.3	5.2	0.7	109.3	112.2	112.2	6.3	0.4
Test item	300.0	-	104.4	107.1	96.8	8.1	1.1	114.8	85.6	95.5	25.6	1.6
Test item	600.0	-	97.6	107.9	88.5	10.3	1.4	100.1	86.3	84.3	20.0	1.3
Test item	800.0	-	28.2	137.8	87.3	2.3	0.3	27.1	103.8	66.7	18.1	1.2
Test item	1000.0	-	0.3	91.6	46.1	39.7	5.5	0.0	64.5	44.1	17.5	1.1
Test item	1200.0	-	0.0	culture was not continued^##				0.0	culture was not continued^##
Experiment II / 4 h treatment
Solvent control with water		+	100.0	100.0	100.0	11.1	1.0	100.0	100.0	100.0	23.4	1.0
Positive control (DMBA)	1.1	+	60.5	62.4	74.6	1193.8	107.4	79.9	61.5	77.6	624.0	26.7
Test item	9.4	+	95.6	61.9	104.5	17.7	1.6	100.8	68.6	79.8	11.5	0.5
Test item	18.8	+	97.1	70.0	102.2	21.9	2.0	95.6	62.9	112.2	20.7	0.9
Test item	37.5	+	74.7	66.6	105.1	28.2	2.5	76.7	50.8	112.1	23.7	1.0
Test item	75.0	+	20.0	23.0	87.1	10.7	1.0	23.8	28.0	54.9	24.4	1.0
Test item	93.8	+	0.0	11.8	79.7	17.6	1.6	0.6	10.2	45.0	19.9	0.9
Test item	112.5	+	0.0	culture was not continued^##				0.0	culture was not continued^##

^# culture was not continued since a minimum of only four concentrations is required
^## culture was not continued due to exceedingly serve cytotoxic effects

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeIn conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Sodium p[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item Sodium p[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzenesulphonate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration in the main experiments was limited by cytotoxicity of the test item. Precipitation at the end of treatment, visible to the unaided eye was not observed.

Cytotoxic effects, indicated by a relative cloning efficiency I of less than 50% compared to the corresponding solvent control in both parallel cultures, occurred in experiment I at 112.5 µg/mL and above with and at 225.0 µg/mL and above without metabolic activation. In experiment II cytotoxic effects were noted at 75.0 µg/mL and above with and at 800 µg/mL and above without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the second experiment without metabolic activation at 1000.0 µg/mL. This isolated increase was judged as biologically irrelevant however, as it was not reproduced in the parallel culture under identical experimental conditions and there was no dose dependent increase as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.2 up to 23.4 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.3 to 39.7 mutants per 10⁶cells.

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase of induced mutant colonies.