Tetrasodium 4-amino-6-[[2,5-dimethoxy-4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-5-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2014-04-23 until 2014-07-08

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:

2014

Report date:

2014

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 activation
second 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:

Structural Analogue 03

IUPAC Name:

Structural Analogue 03

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

see below

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: 525.0; 1050; 2100; 3150; 4200 µg/mL
with metabolic activation: 1050; 2100; 4200; 6300; 8400 µg/mL
Experiment II:
without metabolic activation: 134.2; 268.4; 536.9; 805.3; 1073.8 µg/mL
with metabolic activation: 268.4; 536.9; 1073.6; 2147.5; 4295.0 µg/mL

Vehicle / solvent:

deionised water

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 medium


DURATION
- 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 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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 to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency 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.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. 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 and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.32 measured in the solvent control versus pH 7.36 measured at 8590 µg/mL)
- Effects of osmolality: no relevant increase (287 measured in the solvent control versus 329 measured at 8590 µg/mL)
- Evaporation from medium: Not examined
- Water solubility:123g/L (without correction for purity)
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable 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 analysed 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 evaluated at the beginning and at the end of treatment by the unaided eye.

The range finding pre-experiment was performed using a concentration range of 67.1 to 8590 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (8590 µg/ml) was equal to 5 mg/mL of the pure substance.

Relevant toxic effects occurred after 4 hours treatment at 4295 µg/mL and above with metabolic activation. Following 4 hours treatment without metabolic activation toxic effects were noted at 2147.5 µg/mL and above. Another low value of the cloning efficiency was noted at 536.9 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next higher concentration. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 536.9 µg/mL. At all higher concentrations the cell growth was completely inhibited.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.

There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.

To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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	10.1	1.0	100.0	100.0	100.0	12.4	1.0
Positive control (EMS)	150.0	-	90.6	77.6	99.9	188.3	18.7	97.8	55.3	87.1	221.4	17.8
Test item	131.3	-	55.9	culture was not continued^#				79.5	culture was not continued^#
Test item	262.5	-	63.4	culture was not continued^#				68.7	culture was not continued^#
Test item	525.0	-	49.9	93.0	111.9	8.3	0.8	67.6	69.6	105.6	4.9	0.4
Test item	1050.0	-	29.6	93.0	104.6	5.9	0.6	38.2	81.5	93.4	12.2	1.0
Test item	2100.0	-	32.2	95.4	90.8	21.6	2.1	31.2	91.8	100.1	19.9	1.6
Test item	3150.0	-	9.1	83.8	96.4	4.6	0.5	16.8	76.9	96.5	5.6	0.4
Test item	4200.0	-	8.1	76.5	94.8	17.6	1.7	10.0	66.3	85.7	21.6	1.7
Solvent control with water		+	100.0	100.0	100.0	16.3	1.0	100.0	100.0	100.0	7.3	1.0
Positive control (DMBA)	1.1	+	99.7	99.9	93.8	131.5	8.1	89.2	100.6	95.2	144.6	19.7
Test item	262.5	+	82.5	culture was not continued^#				100.4	culture was not continued^#
Test item	525.0	+	92.9	culture was not continued^#				69.5	culture was not continued^#
Test item	1050.0	+	81.4	120.2	76.2	4.5	0.3	63.6	130.1	50.6	6.9	0.9
Test item	2100.0	+	89.2	109.1	76.6	18.1	1.1	78.3	107.1	93.6	19.2	2.6
Test item	4200.0	+	89.0	121.4	107.7	9.9	0.6	59.1	93.5	125.9	16.6	2.3
Test item	6300.0	+	69.2	107.4	120.2	16.1	1.0	60.1	97.4	129.3	13.3	1.8
Test item	8400.0	+	62.0	94.6	100.0	9.4	0.6	47.5	88.4	99.1	11.2	1.5
Experiment II / 24 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	5.5	1.0	100.0	100.0	100.0	23.4	1.0
Positive control (EMS)	150.0	-	95.1	84.4	98.3	450.7	82.4	96.1	83.9	78.3	639.7	27.3
Test item	33.5	-	90.6	culture was not continued^#				95.7	culture was not continued^#
Test item	67.1	-	93.1	culture was not continued^#				94.9	culture was not continued^#
Test item	134.2	-	93.1	63.8	100.8	13.6	2.5	92.5	69.5	91.5	21.9	0.9
Test item	268.4	-	95.8	67.7	108.6	8.4	1.5	84.2	50.9	89.5	9.9	0.4
Test item	536.9	-	88.4	56.9	96.8	7.9	1.5	83.2	59.8	87.3	35.4	1.5
Test item	805.3	-	10.3	51.9	101.3	10.0	1.8	8.8	40.7	88.4	31.4	1.3
Test item	1073.8	-	0.0	42.0	101.5	10.8	2.0	0.0	48.8	93.2	16.5	0.7
Experiment II / 4 h treatment
Solvent control with water		+	100.0	100.0	100.0	17.8	1.0	100.0	100.0	100.0	20.1	1.0
Positive control (DMBA)	2.2	+	98.9	99.6	105.1	184.1	10.3	100.3	58.2	93.6	264.7	13.2
Test item	268.4	+	87.9	86.9	96.3	19.4	1.1	105.6	104.8	99.0	20.5	1.0
Test item	536.9	+	91.2	124.3	93.6	15.4	0.9	99.9	107.7	100.5	15.5	0.8
Test item	1073.8	+	99.7	116.2	93.6	14.9	0.8	95.2	100.3	100.8	8.9	0.4
Test item	2147.5	+	96.6	93.0	95.9	11.6	0.7	84.2	97.1	97.4	23.1	1.1
Test item	4295.0	+	16.5	36.5	95.6	35.0	2.0	15.4	42.4	96.0	8.0	0.4
Test item	6442.5	+	0.0	3.8	culture was not continued^##			0.0	4.5	culture was not continued^##
Test item	8590.0	+	0.0	culture was not continued^##				0.0	culture was not continued^##

#       culture was not continued since a minimum of only four analysable concentrations is required

##     culture was not continued due to exceedingly severe cytotoxic effects

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In 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.

Executive summary:

The test item 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 main experiments were evaluated at the following concentrations:

 

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	525.0	1050	2100	3150	4200
4 hours	+	1050	2100	4200	6300	8400
		Experiment II
24 hours	-	134.2	268.4	536.9	805.3	1073.8
4 hours	+	268.4	536.9	1073.6	2147.5	4295.0

 

No precipitation of the test item was observed up to the maximum concentration in any of the experiments.

 

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

 

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. 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 5.5 up to 23.4 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 10⁶ cells.

 

EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.