Disodium 8-[[4-[[(4-methylphenyl)sulphonyl]oxy]phenyl]azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]naphthalene-1-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September from 10th to 29th, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Only four out of five strains reccomanded into the updated OECD guideline were tested

Justification for type of information:

Justification for read-across is detailed in the report attached to the IUCLID section 13.

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

September from 10th to 29th, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Only four out of five strains reccomanded into the updated OECD guideline were tested

Justification for type of information:

Justification for read-across is detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Preculture: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

MAIN TEST (exp. I and II): 33, 100, 333, 1000, 2500 and 5000 μg/plate
PRE-EXPERIMENT FOR TOXICITY: 3, 10.0, 133, 100, 333, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

- Solvent: on the day of the experiment, the test article was dissolved in DMSO.
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test article occurred up to the highest investigated dose.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Method: experiment I plate incorporation test; experiment II pre-incubation test.
- Incubation: the bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Overlay Agar content per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H20, 12.2 mg Biotin.
- Sterilisations: performed at 121 °C in an autoclave.

REPLICATES: for each strain and dose level, including the controls three plates were used.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 μl Bacteria suspension (cf. test system, pre-culture of the strains);
2000 μl Overlay agar
- Pre-incubation assay: 100 μl test solution, 500 μl S9 mix / S9 mix substitution buffer and 100 μl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Post pre-incubation: after pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Incubation: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

PRE-EXPERIMENT FOR TOXICITY
- Strains: pre-experiment was performed with strains TA 98 and TA 100.
- Concentrations: 8 concentrations were tested.
- Replicate: mutatation induction with each 3 plates.
- Test conditions: the experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
- Toxicity definition: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Anima source: the S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in deionised water and ß-Naphthoflavone orally dissolved in corn oil on three subsequent days.
- Liver collection: after cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The livers were prepared 24 hours after the last treatment.
- Homogenate: it was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4° C.
- Storage of stock solution: a stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use.
- Protein content: the protein content was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 24.7 mg/ml.
- S9 mix preparation: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5mM Glucose-6-phosphate, 5mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix preparation was performed according to Ames et al.
- Storage: during the experiment the S9 mix was stored in an ice bath.

ACCEPTED CONDITIONS
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies: TA 1535 10-29; TA 1537 5 - 28; TA 98 15 - 57; TA 100 77 - 189.

Evaluation criteria:

A test article is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
a test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any concentration level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Summary of revertants/plate, mean from three plates - without S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	12	28	15	9	17	25	95	101
Solvent control	15	25	12	9	17	20	96	97
Positive control*	927	997	135	114	522	668	700	924
33	16	25	12	15	12	19	109	105
100	17	21	10	12	11	22	101	106
333	18	24	12	14	12	25	97	116
1000	16	28	9	12	12	22	96	121
2500	14	21	8	10	13	21	89	102
5000	18	16	8	11	15	30	86	107

*Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

Summary of revertants/plate, mean from three plates - with S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	17	20	13	26	18	30	116	131
Solvent control*	19	23	19	21	18	28	118	110
Positive control	194	191	83	86	426	445	594	453
33	22	19	17	22	19	33	114	132
100	16	20	14	18	17	30	132	123
333	22	22	17	18	15	35	114	131
1000	15	21	14	25	19	28	118	138
2500	16	19	20	18	17	39	119	125
5000	14	21	18	20	15	33	108	133

*2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

Pre-Experiment for Toxicity

Substance	Concentration μg/plate	TA 98		TA 100
		-	+	-	+
Negative control	-	17	18	95	116
Solvent control	-	17	18	96	118
4-NOPD	10.0	522	/	/	/
NaN3	10.0	/	/	700	/
2-AA	2.5	/	426	/	594
Test item	3	17	21	90	112
	10.0	16	17	99	116
	133	12	19	109	114
	100	11	17	101	132
	333	12	15	97	114
	1000	12	19	96	118
	2500	13	17	89	119
	5000	15	15	86	108

Conclusions:

The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01 of Acid Orange 051:1

IUPAC Name:

Similar substance 01 of Acid Orange 051:1

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Preculture: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

MAIN TEST (exp. I and II): 33, 100, 333, 1000, 2500 and 5000 μg/plate
PRE-EXPERIMENT FOR TOXICITY: 3, 10.0, 133, 100, 333, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

- Solvent: on the day of the experiment, the test article was dissolved in DMSO.
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test article occurred up to the highest investigated dose.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Method: experiment I plate incorporation test; experiment II pre-incubation test.
- Incubation: the bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Overlay Agar content per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H20, 12.2 mg Biotin.
- Sterilisations: performed at 121 °C in an autoclave.

REPLICATES: for each strain and dose level, including the controls three plates were used.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 μl Bacteria suspension (cf. test system, pre-culture of the strains);
2000 μl Overlay agar
- Pre-incubation assay: 100 μl test solution, 500 μl S9 mix / S9 mix substitution buffer and 100 μl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Post pre-incubation: after pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Incubation: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

PRE-EXPERIMENT FOR TOXICITY
- Strains: pre-experiment was performed with strains TA 98 and TA 100.
- Concentrations: 8 concentrations were tested.
- Replicate: mutatation induction with each 3 plates.
- Test conditions: the experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
- Toxicity definition: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Anima source: the S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in deionised water and ß-Naphthoflavone orally dissolved in corn oil on three subsequent days.
- Liver collection: after cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The livers were prepared 24 hours after the last treatment.
- Homogenate: it was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4° C.
- Storage of stock solution: a stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use.
- Protein content: the protein content was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 24.7 mg/ml.
- S9 mix preparation: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5mM Glucose-6-phosphate, 5mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix preparation was performed according to Ames et al.
- Storage: during the experiment the S9 mix was stored in an ice bath.

ACCEPTED CONDITIONS
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies: TA 1535 10-29; TA 1537 5 - 28; TA 98 15 - 57; TA 100 77 - 189.

Evaluation criteria:

A test article is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
a test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any concentration level, neither in the
presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Any other information on results incl. tables

Summary of revertants/plate, mean from three plates - without S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	12	28	15	9	17	25	95	101
Solvent control	15	25	12	9	17	20	96	97
Positive control*	927	997	135	114	522	668	700	924
33	16	25	12	15	12	19	109	105
100	17	21	10	12	11	22	101	106
333	18	24	12	14	12	25	97	116
1000	16	28	9	12	12	22	96	121
2500	14	21	8	10	13	21	89	102
5000	18	16	8	11	15	30	86	107

*Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

Summary of revertants/plate, mean from three plates - with S9

Concentration μg/plate	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control	17	20	13	26	18	30	116	131
Solvent control*	19	23	19	21	18	28	118	110
Positive control	194	191	83	86	426	445	594	453
33	22	19	17	22	19	33	114	132
100	16	20	14	18	17	30	132	123
333	22	22	17	18	15	35	114	131
1000	15	21	14	25	19	28	118	138
2500	16	19	20	18	17	39	119	125
5000	14	21	18	20	15	33	108	133

*2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

Pre-Experiment for Toxicity

Substance	Concentration μg/plate	TA 98		TA 100
		-	+	-	+
Negative control	-	17	18	95	116
Solvent control	-	17	18	96	118
4-NOPD	10.0	522	/	/	/
NaN3	10.0	/	/	700	/
2-AA	2.5	/	426	/	594
Test item	3	17	21	90	112
	10.0	16	17	99	116
	133	12	19	109	114
	100	11	17	101	132
	333	12	15	97	114
	1000	12	19	96	118
	2500	13	17	89	119
	5000	15	15	86	108

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.