7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-09-12 until 2013-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

EC Number:

287-574-8

EC Name:

7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

Cas Number:

85536-87-4

Molecular formula:

C20H19N3O11S3.xNa C20H(19-x)N3NaxO11S3

IUPAC Name:

7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Identification: Reactive Orange 72-78

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: 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II:
without metabolic activation: 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
with metabolic activation: 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL

Vehicle / solvent:

Deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).

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 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.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

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 7.42 in the solvent control versus pH 7.43 at 9100 µg/mL) measured in the range finding pre-experiment
- Effects of osmolality: No relevant increase (281 mOsm in the solvent control versus 340 mOsm at 9100 µg/mL, and 320 mOsm at 4550 µg/mL) measured in the pre-experiment
- Evaporation from medium: Not examined
- Water solubility: Soluble
- 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:
The range finding pre-experiment was performed using a concentration range of 71.1 to 9100 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 (9100 µg/ml) was equal to 5 mg/mL of the pure substance.

Relevant toxic effect occurred after the 4-hour treatment at 4550 µg/mL and above without metabolic activation. Following the 24-hour treatment without metabolic activation toxic effects were noted at 568.8 µg/mL and above. Another low value of the cloning efficiency was noted at 568.8 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 two higher concentrations.

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 pH of the medium even at the maximum concentration of the test item. The osmolarity was increased from 283 to 340 mOsm at 9100 µg/mL and 320 mOsm at 4550 µg/mL. Both levels of osmolarity are sufficiently close to the physiological level of approximately 300 mOsm.
The dose range of the first experiment was set according to the cytotoxicity 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.

Table 1 Doses applied in the gene mutation assay with Reactive Orange 72-78
Experiment I
4 hours treatment without S9 mix: 142.2; 284.4; 568.8; 1137.5; 2275.0; 4550.0; 6825.0 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL
Experiment II
24 hours treatment without S9 mix: 17.8; 35.6; 71.1; 142.2; 284.4; 568.8; 716.2 µg/mL
4 hours treatment with S9 mix: 284.4; 568.8; 1137.5; 2275.0; 4550.0; 9100.0 µg/mL

In experiment I and II the cultures at the two lowest concentrations without metabolic activation and the culture at the lowest concentration with metabolic activation were not continued since a minimum of only four concentrations is required by the guidelines.

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 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 µg/mL and above without metabolic activation.

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 contro with water		-	100.0	100.0	100.0	17.2	1.0	100.0	100.0	100.0	15.4	1.0
Positive control (EMS)	150.0	-	96.4	58.2	108.2	167.6	9.8	97.5	108.8	98.1	168.3	10.9
Test item	142.2	-	99.3	culture was not continued^#				85.4	culture was not continued^#
Test item	284.4	-	82.0	culture was not continued^#				83.1	culture was not continued^#
Test item	568.8	-	91.2	69.4	111.4	14.5	0.8	95.2	135.9	102.2	11.5	0.7
Test item	1137.5	-	100.7	53.2	100.5	19.8	1.2	91.1	117.4	101.3	21.3	1.4
Test item	2275.0	-	80.4	57.1	99.0	13.2	0.8	79.9	112.1	100.6	13.1	0.8
Test item	4550.0	-	32.0	56.3	98.2	20.2	1.2	28.3	108.2	95.6	20.9	1.4
Test item	6825.0	-	4.9	30.4	97.6	21.7	1.3	4.1	73.2	103.6	19.1	1.2
Solvent contro with water		+	100.0	100.0	100.0	6.3	1.0	100.0	100.0	100.0	7.5	1.0
Positive control (DMBA)	1.1	+	99.2	80.3	103.2	159.0	25.4	93.6	108.4	75.9	214.1	28.6
Test item	284.4	+	100.0	culture was not continued^#				98.1	culture was not continued^#
Test item	568.8	+	103.9	90.9	76.8	14.9	2.4	94.4	109.5	66.3	11.8	1.6
Test item	1137.5	+	96.4	82.5	93.8	12.2	1.9	91.7	133.5	90.2	9.6	1.3
Test item	2275.0	+	97.5	80.9	77.0	6.6	1.1	103.1	110.7	81.4	11.9	1.6
Test item	4550.0	+	83.2	73.4	80.2	10.4	1.7	83.9	114.1	81.9	19.5	2.6
Test item	9100.0	+	52.9	79.8	80.6	15.3	2.4	48.9	86.9	87.7	9.2	1.2
Experiment II / 24 h treatment			culture I					culture II
Solvent contro with water		-	100.0	100.0	100.0	15.5	1.0	100.0	100.0	100.0	19.8	1.0
Positive control (EMS)	150.0	-	94.5	89.8	89.8	269.9	17.4	89.0	85.3	97.5	318.3	16.1
Test item	17.8	-	101.8	culture was not continued^#				99.9	culture was not continued^#
Test item	35.6	-	100.3	culture was not continued^#				99.1	culture was not continued^#
Test item	71.1	-	103.6	117.2	89.3	21.2	1.4	97.9	73.6	101.7	11.7	0.6
Test item	142.2	-	97.3	74.7	92.3	18.9	1.2	98.7	89.4	102.8	15.0	0.8
Test item	284.4	-	102.6	89.5	113.9	13.1	0.9	96.1	110.4	104.7	18.4	0.9
Test item	568.8	-	21.7	92.0	98.3	12.0	0.8	19.0	131.0	103.9	15.8	0.8
Test item	716.2	-	0.8	102.2	77.0	10.9	0.7	3.3	107.0	104.6	31.7	1.6
Experiment II / 4 h treatment			culture I					culture II
Solvent contro with water		+	100.0	100.0	100.0	11.8	1.0	100.0	100.0	100.0	14.2	1.0
Positive control (DMBA)	1.1	+	88.1	91.0	95.8	77.7	6.6	92.7	147.4	89.7	92.5	6.5
Test item	284.4	+	83.7	culture was not continued^#				112.5	culture was not continued^#
Test item	568.8	+	78.5	132.2	122.4	14.0	1.2	91.9	129.3	92.5	14.5	1.0
Test item	1137.5	+	76.2	167.0	110.2	12.2	1.0	101.5	142.1	93.8	16.2	1.1
Test item	2275.0	+	80.9	166.2	117.0	13.9	1.2	94.1	127.8	92.8	15.5	1.1
Test item	4550.0	+	95.5	182.7	102.7	28.8	2.4	103.4	159.2	82.4	17.8	1.3
Test item	9100.0	+	72.2	78.8	121.8	2.9	0.2	94.9	133.2	91.8	6.9	0.5

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

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Reactive Orange 72/78 is considered to be non-mutagenic in this HPRT assay.

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	-	568.8	1137.5	2275.0	4550.0	6825.0
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0
		Experiment II
24 hours	-	71.1	142.2	284.4	568.8	716.2
4 hours	+	568.8	1137.5	2275.0	4550.0	9100.0

 

No precipitation of the test item was observed up to the maximum concentration in all 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 4550 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 568.8 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 mutation frequency did not exceed the historical range of solvent controls.

 

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 6.3 up to 19.9 mutants per 10⁶cells; the range of the groups treated with the test item was from 2.9 up to 31.7 mutants per 10⁶cells.

 

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. The positive control with DMBA in the first culture of the second experiment with metabolic activation did not quite reach the historical range of positive controls (77.7 versus 91.4 - 2666.3 colonies per 10⁶cells). The positive control was valid however, as the induction factor was 6.6 and the positive control of the parallel culture remained within the historical range of positive controls.

 

 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, the test item is considered to be non-mutagenic in this HPRT assay.