Trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The hazard of gene mutation was determined in mammalian cells (V79) in vitro with the following result: not mutagenic. The finding is supported by an Ames test on an analogous substance according to the pre-incubation method for azo-dyes using the salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without Syrian hamster liver microsomal activation. The test article was tested at concentrations up to 5000 µg/plate. Positive as well as negative controls were valid. The test item did not induce gene mutations in 4 strains of Salmonella typhimurium with and without metabolic activation by Syrian hamster liver S9.mix. Findings were confirmed in two standard Ames tests with negative results. Additionally a study of chromosomal aberrations in human lymphocytes did not show a mutagenic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-09 until 2013-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: according to OECD 476

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

HPRT

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 (4 h treatment): 175; 350; 700; 1400; 2100; 2800 µg/mL
with metabolic activation (4 h treatment): 350; 700; 1400; 2800; 4200; 5600 µg/mL
Experiment II:
without metabolic activation (24 h treatment): 87.5; 175; 350; 700; 1400; 2100 µg/mL
with metabolic activation (4 h treatment): 350; 700; 1400; 2800; 4200; 5600 µg/mL
In both experiments with and without metabolic activation the lowest concentration was not continued as a minimum of only four analysable concentrations is required.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

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 using SYSTAT(R)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.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.33 in the solvent control versus pH 7.36 measured at 5600 µg/mL, pre-experiment)
- Effects of osmolality: No relevant increase (mOsm 269 in the solvent control versus 301 measured at 5600 µg/mL, pre-experiment)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation: Pre-experiment: The 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. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activa-tion after 4 and 24 hours treatment.
Main experiments: No precipitation observed

- Other confounding effects: None


RANGE-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 test item concentration used in the pre-test was 5600 µg/mL equal to ap-proximately 5000 µg/mL of the pure substance. The test item was dissolved in deionised water. Test item concentrations between 43.8 µg/mL and 5600 µg/mL were used to evalu-ate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treat-ment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50% were observed at 1400 µg/mL and above without metabolic activation following 4 and 24 hours treatment, and at 2800 µg/mL and above with metabolic activa-tion (4 h treatment).
The 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. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activa-tion after 4 and 24 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: Complies

ADDITIONAL 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 1400 µg/mL and above without and at 4200 µg/mL and above with metabolic activation. In experiment II cytotoxic effects were noted at 1400 µg/mL and above without and at 5600 µg/mL and above with metabolic activation.

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	19.2	1.0	100.0	100.0	100.0	16.8	1.0
Positive control (EMS)	150.0	-	89.0	89.8	142.9	61.7	3.2	86.3	80.5	107.3	65.3	3.9
Test item	175.0	-	83.9	culture was not continued^#				94.7	culture was not continued^#
Test item	350.0	-	80.4	81.3	74.6	21.8	1.1	94.6	79.2	63.2	27.9	1.7
Test item	700.0	-	88.2	77.6	124.6	15.7	0.8	97.6	89.2	106.1	10.3	0.6
Test item	1400.0	-	31.8	71.4	112.3	17.3	0.9	36.9	76.1	92.9	17.4	1.0
Test item	2100.0	-	13.1	63.6	104.6	11.9	0.6	15.3	68.0	103.2	11.0	0.7
Test item	2800.0	-	2.4	65.5	82.5	22.1	1.2	4.1	60.4	97.7	14.8	0.9
Solvent control with water		+	100.0	100.0	100.0	28.3	1.0	100.0	100.0	100.0	25.1	1.0
Positive control (DMBA)	1.1	+	88.5	76.8	139.5	221.4	7.8	85.3	78.2	106.4	147.0	5.9
Test item	350.0	+	107.8	culture was not continued^#				96.8	culture was not continued^#
Test item	700.0	+	92.4	74.0	173.3	14.3	0.5	93.4	84.0	84.3	22.5	0.9
Test item	1400.0	+	95.7	81.0	155.4	21.6	0.8	86.2	77.1	96.2	26.3	1.1
Test item	2800.0	+	78.4	75.6	129.5	10.2	0.4	73.6	80.4	118.8	13.7	0.5
Test item	4200.0	+	40.8	74.0	118.8	31.6	1.1	40.3	74.5	128.2	29.4	1.2
Test item	5600.0	+	24.6	48.5	177.0	8.1	0.3	27.7	64.9	143.4	14.1	0.6
Experiment II / 24 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	31.6	1.0	100.0	100.0	100.0	35.5	1.0
Positive control (EMS)	150.0	-	77.5	170.7	97.3	491.8	15.6	92.1	108.0	93.9	521.9	14.7
Test item	87.5	-	99.5	culture was not continued^#				106.4	culture was not continued^#
Test item	175.0	-	113.1	116.2	94.2	16.5	0.5	100.3	105.5	93.1	26.4	0.7
Test item	350.0	-	85.9	74.3	99.5	36.7	1.2	87.7	84.7	106.1	35.2	1.0
Test item	700.0	-	80.0	124.2	98.6	11.9	0.4	77.5	75.7	75.6	40.6	1.1
Test item	1400.0	-	36.9	84.7	104.1	19.2	0.6	26.1	68.0	107.1	21.0	0.6
Test item	2100.0	-	1.3	93.4	108.2	8.6	0.3	0.9	41.7	120.4	23.8	0.7
Experiment II / 4 h treatment
Solvent control with water		+	100.0	100.0	100.0	20.5	1.0	100.0	100.0	100.0	24.4	1.0
Positive control (DMBA)	1.1	+	76.7	68.7	51.7	1105.4	54.0	90.6	81.7	76.5	911.6	37.3
Test item	350.0	+	96.4	culture was not continued^#				102.4	culture was not continued^#
Test item	700.0	+	83.6	85.0	78.5	41.9	2.0	89.7	87.3	83.8	20.4	0.8
Test item	1400.0	+	76.0	98.7	95.0	23.8	1.2	85.7	72.2	113.7	16.4	0.7
Test item	2800.0	+	61.8	71.7	97.6	23.0	1.1	64.6	75.5	104.5	9.8	0.4
Test item	4200.0	+	59.8	61.1	72.7	26.6	1.3	51.9	56.1	101.6	27.2	1.1
Test item	5600.0	+	30.8	46.1	63.2	61.0	3.0	27.9	62.9	101.9	38.9	1.6

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

Conclusions:

Conclusion:

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. Therefore, Acid Brown 425 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item Acid Brown 425 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 cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	350	700	1400	2100	2800
4 hours	+	700	1400	2800	4200	5600
		Experiment II
24 hours	-	175	350	700	1400	2100
4 hours	+	700	1400	2800	4200	5600

 

The maximum concentration in the experimental parts without metabolic activation 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 1400 µg/mL and above without and at 4200 µg/mL and above with metabolic activation. In experiment II cytotoxic effects were noted at 1400 µg/mL and above without and at 5600 µg/mL and above with 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 reached in the first culture of the second experiment with metabolic activation at 5600 µg/mL. The range of the historical solvent control data (3.4 - 36.6 mutant colonies/10⁶cells) was also exceeded at this concentration (61.0 mutant colonies/10⁶cells). This isolated increase was judged as biologically irrelevant however, as the threshold was not reached in the parallel culture under identical experimental conditions and there was no dose dependent increase as indicated by the lacking statistical significance.

In the second experiment the mutation frequency slightly exceeded the historical range of solvent controls at 700 µg/mL in culture I with metabolic activation (41.9 mutant colonies/10⁶cells). However, the induction factor did not exceed the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increase was judged as biologically irrelevant.

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 16.8 up to 35.5 mutants per 10⁶cells; the range of the groups treated with the test item was from 8.1 to 61.0 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The hazard of gene mutation was determined in mammalian cells (V79) in vitro with the following result: not mutagenic. The study included several repeats with and without metabolic activation.

The test item did not induce gene mutations in 2 standard Ames tests in salmonella typhimurium or escherichia coli. An analogous substance did not induce gene mutations in Salmonella typhimurium with and without metabolic activation by syrian hamster liver S9-mix.

Chromosomal aberrations were determined after in vitro incubation of human lymphocytes. No hazard was identified.

In conclusion the test item is considered not mutagenic in in-vitro test systems.

Justification for selection of genetic toxicity endpoint
Mammalian cells are considered the most relevant available model for gene mutation in humans.

Justification for classification or non-classification

No classification.

The test item did not induce gene mutations in bacteria and mammalian cells and did not provoke chromosomal aberrations in human lymphocytes.