2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-, coupled with 3-aminophenol, diazotized 5-amino-2-[(4-aminophenyl)amino]benzenesulfonic acid and diazotized benzenamine, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 17 to December 23, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26th, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.14

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168

IUPAC Name:

Direct Black 168

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Periodically checked regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory according to Ames et al.
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre: 8 g Difco Nutrient Broth, 5 g NaCl.
- Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37° C.

Metabolic activation:

with and without

Metabolic activation system:

homogenate of rat liver (S9), induced by Arochlor 1254

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate in both experiments

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121° C in an autoclave.
- Overlay Agar: the overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O, 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.

NUMBER OF REPLICATIONS: for each strain and dose level, including the controls, a minimum of three plates were used.

SETTING UP THE PLATES: 0.1 ml of test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control) and 0.5 ml of S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation) and 0.1 ml of bacteria suspension and 2 ml of overlay agar were mixed in a test tube and poured onto the selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

PRE EXPERIMENT FOR TOXICITY AND DOSE SELECTION: prestudy was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates in the same conditions as for the main experiment. Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DATA RECORDING: the colonies were counted using the BIOTRAN III counter. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

PREPARATION OF MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9: the S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1:3 in KCl was centrifuged cold at 9.000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at - 70 °C. Small numbers of the ampoules were kept at - 20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.
- S9 mix: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7. The composition of the cofactor solution was concentrated to yield the concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Evaluation criteria:

For the evaluation of the results the corresponding background growth on both negative vontrol and test plates and the normal range of spontaneous reversion rates are used.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows: a test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent 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 above described enhancement factors or not.

Statistics:

No evaluated statistical procedure was used.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in TA1535, at 100.0 μg/plate (exp 1) without metabolic activation and in TA98 at 1000 and 5000 μg/plate with metabolic activation (exp. I & exp.II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxic effects evidenced by a reduction in the number of spontaneous revertants occurred in strain TA 1535 at 100.0 µg/plate (exp. I), and in strain TA 1537 at 1000.0 µg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 µg/plate with metabolic activation in both experiments.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
A slight increase in revertant colony numbers was observed in strain TA 100 (exp. I) at 333.3 µg/plate. This effect was not reproduced in the independent experiment and therefore it is considered not to be relevant.
In strain TA 1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 µg/plate. This increase was less significant in exp. II. To prove the effects of exp. I and exp. II a third experiment was performed. In this additional experiment no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA 1537 cannot be excluded, however the effect was not reproduced in all three experiments.
In strain TA 1538 (with S9 mix) in both experiments the numbers of revertants were distinctly enhanced at concentrations higher than 10.0 µg/plate. In exp. I a dose-dependent increase was obtained up to 333.3 ug/plate and in exp. II up to 1000.0 µg/plate.
At higher concentrations a decrease in the number of revertants was obtained indicating beginning of toxicity.
Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in the strains TA 1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

Any other information on results incl. tables

The following table presents the revertants/plate mean for the three experiments for each strain.

	Revertants/plate mean from three plates, without S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	9	7	5	4	6	6	15	14	20	17	114	77
Solv. Control	9	6	8	4	4	6	15	13	18	16	110	71
10.0	7	6	9	5	5	5	14	18	18	20	112	60
100.0	4	8	9	4	5	4	14	12	21	22	113	61
333.3	7	6	7	6	5	4	12	18	25	18	100	69
1000.0	5	4	5	4	4	3	14	23	25	16	86	64
5000.0	6	4	5	7	4	6	17	18	21	16	98	57
Sodium azide (10 μg/plate)	697	905	579
4-Nitropophenylene- diamine (50 μg/plate)				171	155	149	1126	1148	1895	1230
	Revertants/plate mean from three plates, with S9 mix
Dose μg/plate I/II/III	TA1535			TA1537			TA1538		TA98		TA100
	I	II	III	I	II	III	I	II	I	II	I	II
Neg. Control	5	7	5	4	4	5	18	17	44	19	116	84
Solv. Control	5	8	7	4	5	6	14	13	44	14	100	75
10.0	7	10	5	5	5	4	23	16	43	17	105	65
100.0	7	11	8	4	7	10	39	37	48	20	123	92
333.3	8	8	10	8	8	7	45	35	47	19	164	98
1000.0	6	9	8	10	5	5	25	55	28	8	101	75
5000.0	7	12	7	12	10	9	22	26	22	7	121	66
2-aminoanthracene (10 μg/plate)	82	97	540	179	319	175	1867	1563	2455	2231	2285	1496

Applicant's summary and conclusion

Conclusions:

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.

Executive summary:

The substance was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation. According to the results of these experiments, a third experiment (III) was performed only with TA1535 and TA1537 with and without metabolic activation. Five concentrations (10.0, 100.0, 333.3, 1000.0 and 5000.0 μg/plate) including the control were inoculated with each of the strains for 72 hours at 37 °C. Each concentration was tested in triplicate.

Toxic effects occurred in strain TA1535 at 100.0 μg/plate (exp. I) and in strain TA1537 at 1000.0 μg/plate (exp. Ill) both without S9 mix. Also in strain TA 98 at 5000.0 μg/plate with metabolic activation in both experiments. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. In strain TA1537 (with S9 mix) a dose-dependent increase in the number of revertants was observed in the concentration range of 333.3 up to 5000.0 ug/plate. This increase was less significant in exp. II. In the experiment III no relevant increase in the number of revertants was observed. Based on these results a mutagenic potential of the test article in strain TA1537 cannot be excluded, but this the effect was not reproduced in all three experiments. In strain TA1538 (with S9 mix) a dose-dependent increase was obtained up to 333.3 μg/plate (exp. I) and up to 1000.0 μg/plate (exp. ΙI). No indication of mutagenic response occured in strains TA1535, TA 98, and TA 100 as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists, with and without metabolic activation. The substance induced point mutations by frameshifts in the genome of the strain TA1538 and there is an indication of a possible mutagenic response in strain TA1537.

Conclusion

Under the tested conditions, the test article induced point mutations by frameshifts in the genome of strain TA1538. There was also an indication of a possible mutagenic response in strain TA1537.