Reaction mass of trisodium 4-amino-3-[[4-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and trisodium 4-amino-3-[[4-[[4-[(4-amino-2-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

November 16th to 29th, 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The test was conducted by means of Read Across approach. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168:1 - Similar Substance 01

IUPAC Name:

Direct Black 168:1 - Similar Substance 01

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):

- Bacteria growth: several days before the beginning of the study, 4 tials of the bacterial strains were defrosted and grown on Petri dishes with nutrient agar in order to obtain pure cultures.
- Storage: the plates were stored at 4 °C until required.
- Preparation: 16 hours before the study began, an inoculum of each of the four bacterial strains was prepared in 20 ml of nutrient broth and incubated at 37 °C. - Periodically checked: the histidine requirements, the presence or absence of plasmide pKM 101, the rfa factor (sensitivity to crystal violet) and the uvrβ factor (sensitivity to ultraviolet rays) were checked in the strains used to prepare the inocula.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254, at 10 % in standard cofactors

Test concentrations with justification for top dose:

31.25, 625, 1250, 2500, 5000 and 10000 μg/plate, for both experiments.

Vehicle / solvent:

- Solvents used: sterile, redistilled water (for the test substance and sodium azide reference substance solution) and DMSO (for 9 aminoacridine, 4-nitro-o-phenylendiamin and 2-aminoanthracen reference solutions).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene // 4-nitro-o-phenylendiamin

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The tHo experiments Here carried out on di f ferent days.

METHOD OF APPLICATION: preincubation and plate incorporation.

NUMBER OF REPLICATIONS: three replicates per concentration.

SETTING UP OF THE TEST PLATES
- Plate preparation: 0.1 ml of bacterial culture and 0.1 ml of the diluted test substance were added to 0.5 ml of phosphate buffer (without metabolic activation) or 0.5 ml of S-9 (with metabolic activation). For the controls, 0.1 ml of the corresponding solvents were added to each product.
- First incubation: plates were incubated for 20 minutes at 37 °C and added to 2 ml of top agar melted at 45 °C containing biotin and histidine.
- Incubation: the test tubes were rapidly mixed in a rotamixer, and poured on to plates containing a base layer of minimal agar. The agar added was left to solidify and the plates were incubated at 30 - 35 °C for 48 hours.

Statistics:

The results from the standard products and the control group were compared using the Student's t test. In each group, the statistical comparison between the different concentrations of the test substance was carried out using one-way analysis of the variance.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.
In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.
During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

POSITIVE CONTROLS
In both experiments I and II, all the bacterial strains responded positively and the metabolic activation system responded as expected.
Aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strain TA1537 with the addition of S9, in the experiment I. In the experiment II, aminoanthracen, at a concentration of 10 µg/ plate, showed toxicity in strains TA1535 and TA1537 with the addition of S9.

Any other information on results incl. tables

Summary of results obtained in experiment 1

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	6.3	6.0	5.7	5.3	7.7	5.3	6.0
TA-1537	0	10.7	9.7	9.0	10.0	9.7	9.3	10.0
TA-98	0	14.0	12.0	14.3	12.3	14.0	14.3	14.0
TA-100	0	121.0	119.0	119.7	121.7	124.0	122.0	119.0
TA-1535	10	7.7	8.0	8.0	8.3	6.7	8.0	6.7
TA-1537	10	10.7	10.3	10.7	12.0	10.7	9.0	8.3
TA-98	10	21.7	22.7	22.3	25.7	28.0	29.0	20.3
TA-100	10	131.7	132.0	130.7	131.7	132.0	131.3	131.7

Summary of results obtained in experiment 2

Bacterial  strain	% S-9	Mean no. of revertants per plate (μg/plate)
		0	31.25	62.5	125	250	500	1000
TA-1535	0	13.7	12.0	14.0	13.7	13.3	12.7	13.3
TA-1537	0	12.0	13.3	11.3	12.0	13.0	10.7	12.3
TA-98	0	28.0	27.3	27.3	27.0	26.3	26.7	26.3
TA-100	0	150.0	151.0	147.3	148.0	146.0	149.0	148.0
TA-1535	10	13.7	13.0	13.7	13.3	14.0	14.3	14.3
TA-1537	10	18.0	17.0	16.7	18.3	17.7	19.7	16.7
TA-98	10	33.3	30.7	33.3	31.3	31.0	32.7	28.7
TA-100	10	150.7	148.3	151.0	150.0	150.0	147.3	150.3

Summary of statistical analysis for each strain

Experiment 1
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.99	6	14	N.S.
TA-1537	0	0.58	6	14	N.S.
TA-98	0	1.67	6	14	N.S.
TA-100	0	2.23	6	14	N.S.
TA-1535	10	0.37	6	14	N.S.
TA-1537	10	2.44	6	14	N.S.
TA-98	10	4.53	6	14	V.S.
TA-100	10	0.10	6	14	N.S.
Experiment 2
Bacterial  strain	% S-9	Statistic F	Degrees of freedom		Significance of F (p)
			N	D
TA-1535	0	0.72	6	14	N.S.
TA-1537	0	0.41	6	14	N.S.
TA-98	0	0.23	6	14	N.S.
TA-100	0	0.90	6	14	N.S.
TA-1535	10	0.10	6	14	N.S.
TA-1537	10	1.18	6	14	N.S.
TA-98	10	0.92	6	14	N.S.
TA-100	10	0.36	6	14	N.S.

N.S. = Non-significant.

V.S = Significant for p < 0.01.

Applicant's summary and conclusion

Conclusions:

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.

Executive summary:

The mutagenic potential of the substance, using four strains of Salmonella typhimurium (TA 98, TA100, TA1535, TA1537), with and without the addition of a metabolic activation system was assessed according to OECD Guideline 471 and EU Method B.14. For this reason six concentrations of the substance (1000 - 500 - 250 - 125 - 62.5 and 31.25 μg/plate) were inoculated with each of the strains for 48 hours at 30 -35 °C in two experiments. Concurrent positive controls were used in order to assess the metabolic activation system, while solvent controls also run in parallel.

The substance showed no toxicity for any of the strains at any of the concentrations tested, in both experiments I and II.

In experiment I, no mutagenic response was observed for any of the strains tested, with or without the addition of S9; strain TA98 showed a statistically significant response but it can not be considered to be mutagenic as the highest revertants count never doubled that of the control.

During the experiment II, no mutagenic response was observed for any of the strains tested, with or without the addition of S9.

Conclusion

The substance is non mutagenic to Salmonella strains, with and without metabolic activation.