Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Published in O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

EC Number:

602-814-7

Cas Number:

12269-90-8

IUPAC Name:

Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %

4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %

4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %

4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder

Method

Target gene:

gene for histidine or tryptophan synthesis

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg per plate

Vehicle / solvent:

water for injection

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8); N-methyl-N´-nitro-N-nitrosoguanidine (CAS: 70-25-7);

Details on test system and experimental conditions:

CHEMICALS AND MEDIA
Solvent:
- water for injection
metabolic activator:
- S9: prepared in-house
Positive controls:
- sodium azide (AS) (CAS: 26647-22-8)
- 4-nitro-2-phenylenediamine (NPD) (CAS: 99-56-9)
- 2-aminofluorene (2-AF) (CAS: 153-78-6)
- 2-aminoanthracene (2-AA) (CAS: 613-13-8)
- N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (CAS: 70-25-7)
- 9-aminoacridine hydrochloride monohydrate (9-AAc) (CAS: 52417-22-8)
Media:
- Nutrient Broth for microbiology
- Nutrient agar
- Agar-agar

TEST SYSTEM
Bacterial strains:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno. Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of strains:
Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70°C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 30 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7 or 19%). In experiments without metabolic activation only buffer was added to the top agar.
Controls:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

PLATE INCORPORATION TEST
Test procedure:
100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 10^8-10^9 CFU/mL, 0.5 mL relevant buffer and 30, 50 or 100 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3°C. After shaking the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 48 - 72 h at 37±1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.
Selection of doses/toxicity:
The test substance was dissolved in water for injection till the maximum recommended concentration 5000 μg per 0.1 mL. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. Although no particles could be observed in higher concentrations due to dark colour of solutions, no particles were observable in top agar and Petri dishes.
The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
No toxicity or precipitation was observed in any dose. The concentration of 5000 µg per 0.1 mL-1 was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2· √10. Mutagenicity occurred in some cases in higher doses so lower concentrations were omitted and generally higher concentrations were used in the second mutagenicity experiments. For increase of sensibility, the second experiments were performed with pre-incubation for 30 minutes at 37±1°C and shaking. Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.

Evaluation criteria:

EVALUATION OF RESULTS
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Statistics:

1. Maron D. M., Ames B. N., Revised methods for the Salmonella mutagenicity test, Mutation Research, 113, p. 173 - 215 (1983);
2. Dunkel V. C., Chu K.C., Evaluation of methods for analysis of microbial mutagenicity assays, The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, p. 231 - 417 (1980);

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: The effect of the test substance

Doses µg/plate	S9 µL			revertants per plate							mean ±sd	Rt/Rc	S9 µL		revertants per plate				mean ±sd	Rt/Rc
Escherichia coli WP2 uvrA
	experiment I
Sp.rev.	0			35		39			31		35± 3	-	100		33	45		29	36± 7	-
water	0			34		28			35		32± 3	-	100		34	35		32	34± 1	-
50	0			34		32			35		34± 1	1.0	100		28	28		39	32± 5	0.9
150	0			30		37			36		34± 3	1.1	100		35	32		33	33± 1	1.0
500	0			33		33			41		36± 4	1.1	100		35	33		37	35± 2	1.0
1500	0			68		62			50		60± 7	1.9	100		39	26		44	36± 8	1.1
5000	0			114		140			131		128±11	4.0	100		96	105		71	91±14	2.7
MNNG/2-AA	0			1038		945			NT		992±47	30.7	100		147	153		NT	150± 3	4.5
	experiment II
Sp. rev.	0			40		38			32		37± 3	-	100		36	46		49	44± 6	-
water	0			38		44			32		38± 5	-	100		43	42		39	41± 2	-
1000	0			88		58			67		71±13	1.9	100		40	42		36	39± 2	1.0
2000	0			85		90			104		93± 8	2.4	100		47	52		52	50± 2	1.2
3000	0			133		113			100		115±14	3.0	100		64	66		47	59± 9	1.4
4000	0			126		139			169		145±18	3.8	100		99	81		114	98±13	2.4
5000	0			172		176			170		173± 2	4.5	100		126	133		138	132± 5	3.2
MNNG/2-AA	0			1028		1022			NT		1025±3	27.0	100		207	196		NT	202± 6	4.9
Salmonella typhimurium TA 1537
	experiment I
Sp.rev.	0		12		7			7			9± 2	-	30		15	16	11		14± 2	-
water	0		9		11			11			10± 1	-	30		16	8	14		13± 3	-
50	0		10		18			10			13± 4	1.2	30		19	15	18		17± 2	1.4
150	0		10		9			18			12± 4	1.2	30		16	15	21		17± 3	1.4
500	0		16		11			11			13± 2	1.2	30		18	14	15		16± 2	1.2
1500	0		23		25			21			23± 2	2.2	30		28	25	17		23± 5	1.8
5000	0		71		52			60			61± 8	5.9	30		37	45	44		42± 4	3.3
9-AAc/2-AA	0		980		1109			NT			1045±65	101.1	20		100	117	NT		109± 9	8.6
	experiment II
Sp. rev.	0		8		13			8			10± 2	-	30		11	18	0		15± 4	-
water	0		10		11			12			11± 1	-	30		10	18	14		14± 3	-
1000	0		18		23			18			20± 2	1.8	30		12	17	17		15± 2	1.1
2000	0		29		29			29			29± 0	2.6	30		21	19	22		21± 1	1.5
3000	0		36		42			28			35± 6	3.2	30		30	41	35		35± 4	2.5
4000	0		48		44			40			44± 3	4.0	30		49	50	47		49± 1	3.5
5000	0		39		41			55			45± 7	4.1	30		66	43	47		52± 10	3.7
9-AAc/2-AA	0		1291		1314			NT			1303±12	118.4	20		201	201	NT		201±0	14.4
Salmonella typhimurium TA 100
	experiment I
Sp.rev.	0	85					89			111	95±11	-	30		104	105		112	107± 4	-
water	0	89					121			102	104±13	-	30		111	96		118	108± 9	-
50	0	112					111			109	111± 1	1.1	30		108	c		107	108± 1	1.0
150	0	98					87			95	93± 5	0.9	30		88	96		111	98±10	0.9
500	0	101					95			106	101± 4	1.0	30		105	95		127	109±13	1.0
1500	0	114					124			102	113± 9	1.1	30		127	128		121	125± 3	1.2
5000	0	140					128			158	142±12	1.4	30		135	131		184	150±24	1.4
AS/2-AF	0	481					511			NT	496±15	4.8	20		1315	1328		NT	1322± 7	12.2
	experiment II
Sp. rev.	0	83					78			75	79± 3	-	30		84	108		91	94±10	-
water	0	84					106			82	91±11	-	30		97	89		105	97± 7	-
1000	0	105					113			127	115± 9	1.3	30		90	96		86	91± 4	0.9
2000	0	139					135			132	135± 3	1.5	30		110	107		106	108± 2	1.1
3000	0	138					161			136	145±11	1.6	30		110	131		120	120± 9	1.2
4000	0	129					134			140	134± 4	1.5	30		132	122		140	131± 7	1.4
5000	0	180					183			171	178± 5	2.0	30		134	154		135	141± 9	1.5
AS/2-AF	0	492					492			NT	492± 0	5.4	20		950	1034		NT	992±42	10.2
Salmonella typhimurium TA 1535
	experiment I
Sp.rev.	0	21					17			25	21± 3	-	30		18	23		16	19± 3	-
water	0	19					20			25	21± 3	-	30		12	19		12	14± 3	-
50	0	22					27			29	26± 3	1.2	30		14	15		12	14± 1	1.0
150	0	20					24			21	22± 2	1.0	30		16	16		12	15± 2	1.0
500	0	15					28			27	23± 6	1.1	30		18	12		11	14± 3	1.0
1500	0	19					24			24	22± 2	1.0	30		18	15		10	14± 3	1.0
5000	0	27					20			23	23± 3	1.1	30		25	14		24	21± 5	1.5
AS/2-AA	0	410					419			NT	415± 5	19.4	20		184	150		NT	167±17	11.7
	experiment II
Sp. rev.	0	28					18			18	21± 5	-	30		17	22		19	19± 2	-
water	0	23					21			24	23± 1	-	30		20	15		24	20± 4	-
1000	0	21					17			16	18± 2	0.8	30		15	24		19	19± 4	1.0
2000	0	18					24			19	20± 3	0.9	30		k	15		21	18± 3	0.9
3000	0	21					27			20	23± 3	1.0	30		15	18		24	19± 4	1.0
4000	0	25					20			22	22± 2	1.0	30		25	19		17	20± 3	1.0
5000	0	24					24			19	22± 2	1.0	30		21	25		32	26± 5	1.3
AS/2-AA	0	414					444			NT	429±15	18.9	20		191	196		NT	194±3	9.8
Salmonella typhimurium TA 98
	experiment I
Sp.rev.	0	31					25			31	29± 3	-	30	30		39		39	36± 4	-
water	0	25					30			25	27± 2	-	30	40		35		36	37± 2	-
50	0	26					29			29	28± 1	1.1	30	35		40		25	33± 6	0.9
150	0	26					22			23	24± 2	0.9	30	38		40		29	36± 5	1.0
500	0	27					29			30	29± 1	1.1	30	38		33		26	32± 5	0.9
1500	0	24					27			30	27± 2	1.0	30	32		25		23	27± 4	0.7
5000	0	31					36			30	32± 3	1.2	30	39		36		34	36± 2	1.0
NPD/2-AF	0	664					817			NT	741±77	27.8	20	1796		1871		NT	1834±38	49.6
	experiment II
Sp.rev.	0	32					31			24	29± 4	-	30	35		48		38	40± 6	-
water	0	30					33			34	32± 2	-	30	38		44		41	41± 2	-
1000	0	38					46			32	39± 6	1.2	30	41		39		34	38± 3	0.9
2000	0	35					31			34	33± 2	1.0	30	37		33		28	33± 4	0.8
3000	0	43					38			37	39± 3	1.2	30	42		37		36	38± 3	0.9
4000	0	56					115			36	69±34	2.1	30	36		34		38	36± 2	0.9
5000	0	55					75			77	69±10	2.1	30	56		56		43	52± 6	1.3
NPD/2-AF	0	615					658			NT	637±22	19.7	20	1571		1578		NT	1575± 4	38.4

- S9        without metabolic activation

+ S9       with metabolic activation

CFU        colony forming units, number of live bacteria in suspension

sd          standard deviation

Rt/Rc    ratio of number of revertants at tested dose to number of revertants in negative control

S9          amount of supernatant of rat liver homogenate per plate

Sp.rev. spontaneous reversion (untreated control)

AS         sodium azide

NPD      4-nitro-2-phenylenediamine

2-AF     2-aminofluorene

2-AA     2-aminoanthracene

9-AAc   9-aminoacridine hydrochloride monohydrate

MNNG   N-methyl-N´-nitro-N-nitrosoguanidine

NT          not tested

c             contamination 

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance, Acid Brown 235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.
The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.

Executive summary:

The test substance, Acid Brown 235, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and one indicator, Escherichia coliWP2 uvrA strain, were used. The test substance was diluted in water for injection and assayed in doses of 50 - 5000 mg per plate, which were applied to plates in volume of 0.1 mL. The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactorsby the plate in corporation test with a dose range of 50 – 5000mg per plate. Some signs of a positive response were observed in some tester strains, so, to increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation and concentrations were shifted towards expected mutagenic doses to obtain a dose-response.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the test conditions, the test substance, Acid Brown235, was mutagenic for the Salmonella typhimurium TA 1537, Salmonella typhimurium TA 100 and Escherichia coli WP2uvrA in experiments with as well as without metabolic activation.

The test substance was non-mutagenic for Salmonella typhimurium TA 98 and TA 1535 in experiments with as well as without metabolic activation.