Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

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:

From November 28th to December 15th, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Principles of method if other than guideline:

The experiments were performed using similar methods to those described in HRC protocol MCB 104 adopting modifications found in 'On concrete techniques for the mutagenicity test using micro-organisms' issued by the Japanese Ministry of Labour.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Black 222

IUPAC Name:

Acid Black 222

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Prof. B.N. Ames, University of California, Berkeley, California, U.S.A.
- Storage temperature: - 80 °C
- Storage conditions: amploules containing 0.8 ml of bacterial suspension and 0.07 ml of dimethylsulphoxide.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: rof. National Collection of lndustrial Bacteria, Aberdeen, Scotland.
- Storage temperature: - 80 °C
- Storage conditions: amploules containing 0.8 ml of bacterial suspension and 0.07 ml of dimethylsulphoxide.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Range findig experiment: 0, 100, 1000 and 10000 µg/plate
Main experiment: 0, 50, 100, 500, 1000, 5000 and 10000 µg/plate

Vehicle / solvent:

Solvent: dimethylsulphoxide

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

methylmethanesulfonate

other: 4-notro-o-phanylenediamine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: the compound was tested using the poured plate method.

TEST CONDITIONS: 0.1 ml of bacterial suspension; 0.1 ml of test solution, 0.5 ml of sodium phosphate buffer or S9 mix, 2.0 ml top agar.

INCUBATION: 72 hours at 37 °C

PREPARATION OF S9-MIX
- Storage temperature: -80 °C
- Animals: rat (Crl: COBS CD (SD) BR); males, weighting ca 200 grams and approximately 8 weeks old.
- Induction material: Araclor 1254 (a polychlorinoted biphenyl mixture).
- Induction administration: single interperitoneal injection.
- Induction dose level: 500 mg/kg bw
- Induction period: 5 days.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

With all five tester strains of S. typhimurium, test item, both in the presence and absence of liver microsomal fraction, provoked large dose related increases in revertant colony numbers.
With tester strain E. coli WP2 uvr, test item did not produce any substantial increases in revertant colony numbers.

Any other information on results incl. tables

Main test - Mean revertant colony counts obtained per plate using S. typhimurium strains TA 1535, TA 1597, TA 1538, TA 98 and TA 100 and E. coli strain WP2 uwA

Test concentration µ/plate	S9 mix	Reverse mutation (number of colonies/plate)
		Base pair exchange type			Frame shift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537	TA 1538
Solvent control	-	71	10	25	24	6	14
Test item, 10000	-	307 p	6p	20 p	61 p	3 p	0 p
Test item, 5000	-	259	7	23	67	19	6
Test item, 1000	-	359	17	25	124	21	185
Test item, 500	-	242	20	21	99	23	167
Test item, 100	-	160	-	11	68	13	86
Test item, 50	-	117	11	17	44	12	50
Solvent control	+	88	13	12	22	9	12
Test item, 10000	+	120 p	80 p	14 p	70 p	20 p	30 p
Test item, 5000	+	321	56	22	108	47	76
Test item, 1000	+	369	33	27	129	35	196
Test item, 500	+	283	75	22	107	28	125
Test item, 100	+	162	-	16	58	19	58
Test item, 50	+	112	17	13	40	10	35
Methyl methane sulphonate, 500	-	894
N-ethyl-N'-nitro-N-nitrosoguanidine, 10	-		164
N-ethyl-N'-nitro-N-nitrosoguanidine, 5	-			1636
2-nitrofluorene, 2	-				2737
9-amino-acridine, 10	-					92
4-nitro-o-phenylenediamine, 10	-						861
2-amino-anthracene, 0.5	+	1164			726		51
2-amino-anthracene, 1	+		80			53
2-amino-anthracene, 40	+			224

Dose range finding test - Revertant colony counts obtained

Test concentration µ/plate	S9 mix	Reverse mutation (number of colonies/plate)
		Bose pair exchange type		Frame shift type
		TA 100	TA 1535	TA 98	TA 1537	TA 1538
Solvent control	-	58	5	18	9	10
Test item, 10000	-	218	19	49	3	13
Test item, 1000	-	271	17	102	29	173
Test item, 100	-	87	6	48	9	65
Test item, 10	-	79	14	28	9	21
Solvent control	+	72	4	9	5	9
Test item, 10000	+	203	5	127	38	35
Test item, 1000	+	297	23	93	32	178
Test item, 100	+	132	16	61	17	99
Test item, 10	+	71	6	15	10	26

WP2 uvrA was not tested because strain failed to grow on day of test

Applicant's summary and conclusion

Conclusions:

Test item showed evidence of strong mutagenic potential when tested in the bacterial system.

Executive summary:

Microbial metabolic activation test was conducted to assess the potential mutagenic effect of the test item, using Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 100 and TA 98 strains and Escherichia coli WP2 uvrA strain. The experiments were performed using similar methods to those described in HRC protocol MCB 104 adopting modifications found in 'On concrete techniques for the mutagenicity test using micro-organisms' issued by the Japanese Ministry of Labour.

Dimethylsulphoxide was used as solvent. The substance was tested at concentrations of 0 (solvent), 50, 100, 500, 1000, 5000 and 10000 µg/plate. Adequate positive controls were also assayed.

With all five tester strains of S. typhimurium, test item, both in the presence and absence of liver microsomal fraction, provoked large dose related increases in revertant colony numbers.

With tester strain E. coli WP2 uvrA, test item did not produce any substantial increases in revertant colony numbers.

Conclusion

Test item showed evidence of strong mutagenic potential when tested in the bacterial system.