Acid Brown 432:2

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

From 01. Feb. to 11. Feb. 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

4 strains used in previous guideline instead of the current standard 5 strains

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-320-4

EC Name:

-

Cas Number:

94933-05-8

Molecular formula:

not applicable

IUPAC Name:

Reaction products of diazotised anthranilic acid coupled with resorcinol and diazotised 5-aminonaphthalene-1-sulphonic acid, metallised with Chromium (III) basic sulphate, sodium salts

Test material form:

solid: particulate/powder

Method

Target gene:

Salmonella typhimurium strain LT2

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: Dr. Heinz Träger, Knoll AG, 6700 Ludwigshafen, DE
The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations. However, the bacteria most commonly used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback, an exogenous metabolic system is added in the form of mammalian microsome enzyme activation mixture.
In spite of great differences between bacterial and eukaryotic cells with respect to structure and function, there is an association between mutagenicity in bacteria and carinogenicity in mammals described in literature.
Reverse mutation assays determine the frequency at which an agent abolishes or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversions of becteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxothrophy to prototrophy) is the most widely used marker.
The S. typhimurium histidine (his) reversion system measures his- to his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535 and TA 100) and frameshift (TA 1537 and TA 98) mutations.

MEDIA USED
- Selective agar: 20 ml of nutrient medium (2.0 % Vogel-Bonner-Glucose-Minimal-Agar) in each petri dish
- Overlay agar: 6 g/l Merck Agar Agar, 6 g/l NaCl, 10.5 mg/l L-histidine x HCl x H20 and 12.2 mg/l biotin

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal fraction S9

Test concentrations with justification for top dose:

- Concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate
- Justification: according to a preliminary experiment for toxicity performed with strains TA 98 and TA 100 to evaluate toxicity, the maximum concentration selected was 5000 µ/plate and the concentration range covered two decadic logarithms

Vehicle / solvent:

- Vehicle: water bidest
- Justification: the vehicle was chosen for its solubility properties

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

water bidest.

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

water bidest.

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF PRECULTURED STRAINS
- The strain cultures were stored as stock cultures in ampoules with nutrient broth and 5 % DMSO in liquid nitrogen.
- From thawed ampoules of the strains, 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium (8 g/l Merck nutrient broth; 5 g/l NaCl).
- The bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.

PREPARATION OF S9 MIX FROM MAMMALIAN MICROSOMAL FRACTION
- The S9 liver microsomal fraction was obtained from the liver of 7 to 8 week old Syrian golden hamsters (SAVO-Ivanovas, med. Versuchstierzuchten GMBH, 7964 Kisslegg, DE).
- After cervical dislocation, the livers of the animals were removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidistilled water, and homogenised.
- The homogenate, diluted to 1:3 in sodium phosphate buffer, was centrifuged cold at 9000 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 are kept at -20 °C for only several weeks before use.
- The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories (8000 München, DE: Bio-Rad protein assay, Catalogue 500 000 6). The protein concentration in the S9 preparation was 29.4 mg/ml.
- Before the experiment, an appropriate quantity of S9 supernatant was thawed and mixed to a ration of 3:7 in co-factor solution to yield final concentrations in 100 mM sodium-ortho-phosphate-buffer, pH 7.4, of 33 mM KCl, 8 mM MgCl2, 20 mM glucose 6-phosphate, 2.8 units/ml glucose 6-phosphate dehydrogenase, 4 mM NADP, 2 mM NADH, and 2 mM FMN.
- During the experiment, the S9 mix was stored in an ice bath.

METHOD
For each strain and dose level, including the controls, a minimum of three plates were evaluated. The following materials were mixed in a test tube and gently shaken at 30 °C for 30 minutes:
- 100 µl test item concentrate at each dose level, or solvent control, negative control or reverence positive control
- 500 µl S9 mix, or S9 substitution-buffer
- 100 µl bacteria suspension (pre-cultured strains)
After pre-incubation, 2.0 ml of molten 45 °C overlay agar was added to each test tube. The mixture was poured on minimal agar plates, and after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Rationale for test conditions:

Due to mutations in the histidine locus of the S. typhimurium strain LT2, these strains are histidine dependent. Additionally, due to the "deep rough" (rfa-) mutation, they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named uvrB-. In the strains TA 98 and TA 100, the R-factor plasmid pKM 101 carries the ampicillin resistance marker. A summary of the mutations of the bacterial strains used in this study can be seen in Table 1 of "any other information on materials and methods". Regular checking of the properties of the Salmonella typhimurium strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates was performed.

Evaluation criteria:

A test item 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 item 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 not mutagenic in this system.
A significant response is described as follows: a test item is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate, as specified in Table 2 of "any other information on materials and methods". Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless of whether the highest dose induced these described enhancement factors or not.

Statistics:

Due to international guidelines, a statistical evaluation of the results is recommended, however, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

water bidest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

water bidest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

water bidest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Remarks:

water bidest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

water bidest.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Toxicity: toxic effects were observed in strain TA 98 at 5000 µg/plate, in the presence of metabolic activation only, as evidenced by a reduction in the number of revertants.
- Background growth: plates incubated with the test item showed normal background growth up to 5000 µg/plate, both with and without metabolic activation, in all four strains.
- Revertants: no significant or reproducible increases in number of revertants were found in any strain, compared to solvent control.
- Dose-dependent findings: no concentration-dependent enhancement of revertants was shown
- Metabolic activation: presence of S9 mix did not influence the findings.
- Irrelevant findings: a weak increase in reventant colony numbers in strains TA 1535 (experiment 1, with S9, 5000 µg/plate) and TA 100 (experiment 1, with S9, 1000 and 5000 µg/plate) were considered irrelevant as they were not reproducible in the second experiment.
- Positive controls: appropriate reference mutagens were used as positive controls and showed a distinct, predictable increase in induced revertant colonies.

Any other information on results incl. tables

Table 1: Results for the preliminary toxicity experiment.

Test group	Concentration (µg/plate)	Revertants per plate
		TA 98		TA 100
presence of S9 mix:		-	+	-	+
negative control	-	32	31	105	91
solvent control: 4-NOPD	-	34	32	103	83
sodium azide	50.0	2286	-	-	-
congo red	10.0	-	-	973	-
test item	500.0	-	333	-	265
	1.0	30	34	94	89
	3.3	28	31	109	101
	10.0	33	25	113	95
	33.3	28	37	101	112
	100.0	39	33	102	86
	333.3	30	36	109	114
	1000.0	28	25	112	133
	5000.0	32	20	98	131

- = not performed

Table 2: Experiment I results: number of revertants per plate.

	TA1535						TA1537						TA98						TA100
Dose (µg/plate)	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+
	1	2	3	mean	s.d.		1	2	3	mean	s.d.		1	2	3	mean	s.d.		1	2	3	mean	s.d.
Without S9 mix
negative control	9	7	12	9	2.5		4	5	4	4	0.6		31	29	35	32	3.1		95	124	95	105	16.7
solvent control	8	7	11	9	2.1	1.0	4	5	5	5	0.6	1.0	37	32	33	34	2.6	1.0	104	114	90	103	12.1	1.0
10.0	8	5	11	8	3.0	0.9	5	9	4	6	2.6	1.3	32	40	27	33	6.6	1.0	112	108	119	113	5.6	1.1
100.0	9	6	9	8	1.7	0.9	4	3	4	4	0.6	0.8	42	34	41	39	4.4	1.1	107	105	94	102	7.0	1.0
333.3	7	11	13	10	3.1	1.2	3	4	4	4	0.6	0.8	29	24	38	30	7.1	0.9	118	100	109	109	9.0	1.1
1000.0	10	5	9	8	2.6	0.9	3	8	6	6	2.5	1.2	25	20	40	28	10.4	0.8	110	109	118	112	4.9	1.1
5000.0	9	9	11	10	1.2	1.1	5	4	4	4	0.6	0.9	34	28	34	32	3.5	0.9	90	105	100	98	7.6	1.0
positive control*	921	956	958	945	20.8	109.0	230	219	178	209	27.4	44.8	2455	2205	2199	2286	146.1	67.2	982	995	941	973	28.2	9.5
With S9 mix
negative control	13	13	10	12	1.7		4	5	6	5	1.0		23	23	31	31	7.5		90	102	81	91	10.5
solvent control	6	5	6	6	0.6	1.0	5	5	6	5	0.6	1.0	28	28	31	32	4.6	1.0	92	83	75	83	8.5	1.0
10.0	5	6	14	8	4.9	1.5	3	3	4	3	0.6	0.6	25	25	29	25	3.5	0.8	95	104	86	95	9.0	1.1
100.0	10	4	9	8	3.2	1.4	4	4	5	4	0.6	0.8	30	30	38	33	4.4	1.0	90	90	79	86	6.4	1.0
333.3	5	5	7	6	1.2	1.0	6	4	5	5	1.0	0.9	41	41	32	36	4.7	1.1	79	143	120	114	32.4	1.4
1000.0	6	6	9	7	1.7	1.2	4	3	4	4	0.6	0.7	25	25	19	25	6.00	0.8	122	150	126	133	15.1	1.6
5000.0	7	13	14	11	3.8	2.0	4	4	2	3	1.2	0.6	19	19	24	20	3.6	0.6	110	144	139	131	18.4	1.6
positive control**	245	315	369	310	62.2	54.6	382	417	369	389	24.8	73.0	299	299	385	333	45.7	10.4	267	225	304	265	39.5	3.2

+ enhancement factor = sum of revertants/concentration of test item / sum of revertants/solvent control

*  sodium azide (10 µg/plate) or 4-Nitro-o-phenylene-diamine (50 µg/plate)

**  congo red (500 µg/plate)

Table 3: Experiment II results: number of revertants per plate.

	TA1535						TA1537						TA98						TA100
Dose (µg/plate)	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+	Plate			Revertants/plate		factor+
	1	2	3	mean	s.d.		1	2	3	mean	s.d.		1	2	3	mean	s.d.		1	2	3	mean	s.d.
Without S9 mix
negative control	17	10	10	12	4.0		7	8	5	7	1.5		33	31	26	30	3.6		120	103	97	107	11.9
solvent control	10	16	11	12	3.2	1.0	6	7	6	6	0.6	1.0	21	23	15	20	4.2	1.0	117	111	112	113	3.2	1.0
10.0	11	20	13	15	4.7	1.2	5	5	4	5	0.6	0.7	20	23	23	22	1.7	1.1	95	91	96	94	2.6	0.8
100.0	23	12	22	19	6.1	1.5	6	5	4	5	1.0	0.8	32	19	35	29	8.5	1.5	98	69	69	79	16.7	0.7
333.3	16	15	13	15	1.5	1.2	3	8	14	8	5.5	1.3	30	24	31	28	3.8	1.4	99	94	85	93	7.1	0.8
1000.0	16	14	12	14	2.0	1.1	6	13	11	10	3.6	1.6	28	38	27	31	6.1	1.6	83	74	97	85	11.6	0.7
5000.0	11	16	15	14	2.6	1.1	10	10	12	11	1.2	1.7	32	28	25	28	3.5	1.4	71	84	78	78	6.5	0.7
positive control*	1264	958	1017	1080	162.3	87.5	260	205	256	240	30.7	37.9	2196	1900	2131	2076	155.6	105.5	1147	938	1426	1170	244.8	10.3
With S9 mix
negative control	10	16	17	14	3.8		4	5	4	4	0.6		50	--	39	45	7.8		96	78	105	93	13.7
solvent control	14	11	11	12	1.7	1.0	4	5	5	5	0.6	1.0	47	37	46	43	5.5	1.0	96	100	103	100	3.5	1.0
10.0	14	13	18	15	2.6	1.3	3	3	4	3	0.6	0.7	50	43	44	46	3.8	1.1	97	106	89	97	8.5	1.0
100.0	17	11	9	12	4.2	1.0	3	2	5	3	1.5	0.7	37	50	39	42	7.0	1.0	93	81	107	94	13.0	0.9
333.3	20	10	16	15	5.0	1.3	4	2	3	3	1.0	0.6	51	51	46	49	2.9	1.1	111	131	119	120	10.1	1.2
1000.0	17	10	14	14	3.5	1.1	2	5	4	4	1.5	0.8	48	47	45	47	1.50	1.1	128	126	142	132	8.7	1.3
5000.0	12	22	13	16	5.5	1.3	3	10	7	7	3.5	1.4	23	19	25	22	3.1	0.5	130	144	114	129	15.0	1.3
positive control**	361	291	379	344	46.5	28.6	407	378	363	383	22.4	82.0	486	477	503	489	13.2	11.3	354	324	336	338	15.1	3.4

+ enhancement factor = sum of revertants/concentration of test item / sum of revertants/solvent control

-- = contaminated by fungi

* sodium azide (10 µg/plate) or 4-Nitro-o-phenylene-diamine (50 µg/plate)

**  congo red (500 µg/plate)

Table 4: Summary of results.

Without S9 mix
Dose (µg/plate)	Mean revertants/plate
	TA1535		TA1537		TA98		TA100
	I	II	I	II	I	II	I	II
negative control	9	12	4	7	32	30	105	107
solvent control	9	12	5	6	34	20	103	113
10.0	8	15	6	5	33	22	113	94
100.0	8	19	4	5	39	29	102	79
333.3	10	15	4	8	30	28	109	93
1000.0	8	14	6	10	28	31	112	85
5000.0	10	14	4	11	32	28	98	78
sodium azide (10 µg/plate)	945	1080	-	-	-	-	973	1170
4-Nitro-o-phenylene-diamine (50 µg/plate)	-	-	209	240	2286	2076	-	-
With S9 mix
Dose (µg/plate)	Mean revertants/plate
	TA1535		TA1537		TA98		TA100
	I	II	I	II	I	II	I	II
negative control	12	14	5	4	31	45	91	93
solvent control	6	12	5	5	32	43	83	100
10.0	8	15	3	3	25	46	95	97
100.0	8	12	4	3	33	42	86	94
333.3	6	15	5	3	36	49	114	120
1000.0	7	14	4	4	25	47	133	132
5000.0	11	16	3	7	20	22	131	129
congo red (500 µg/plate)	310	344	389	383	333	489	265	338

Applicant's summary and conclusion

Conclusions:

The test item did not induce point mutations by frameshifts or base pair substitutions under the test conditions.

Executive summary:

The potential of the test item to induce point mutations by base pair substitutions or frameshifts was evaluated in an experimental study according to the OECD Guideline 471 (1983) and the EU Method B.14 (1984): a reverse mutation assay utilising the pre-incubation test (formulated specifically for azo dyes) using Salmonella typhimurium strains TA 97, TA 98, TA 100 and TA 1535. A preliminary test was conducted for toxicity and dose selection. The test item (10, 100, 333.3, 1000 and 5000 µg/plate) was evaluated for its ability to induce frameshift mutations (TA 1537 and TA 98) and base pair substitution mutations (TA 100 and TA 1535), both with and without the presence of mammalian microsomal fraction (S9 metabolic activation). 100 µl of pre-cultured bacterial strain, 100 µl test item concentrate and 500 µl of S9 mix (or stock solution, if without S9) were mixed thoroughly in test tubes and pre-incubated at 30 °C for 30 minutes. 2.0 ml of molten (45 °C) overlay agar was added and the mix was plated onto Minimal agar plates. After solidification, plates were incubated upside down at 37 °C in the dark for 48 hours. Reverse mutations were then quantified by counting colonies using a BIOTRAN III counter. A negative control, a solvent control (water bidest.) and positive controls (without metabolic activation: sodium azide (TA 1535 and TA 100) and 4 -nitro-o-phenylene-diamine (TA 1537 and TA 98); with metabolic activation: Congo Red) were tested in parallel to the test item. Each test concentration and control was plated in triplicate, and the experiment was repeated twice. A result was considered positive (mutagenic) if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration was induced. A significant increase is characterised by a mean reversion count of two (TA 100) or three (TA 1535, TA 1537 and TA 98) times higher than the known spontaneous reversion rates.

Toxic effects were observed in strain TA 98 at 5000 µg/plate, in the presence of metabolic activation only, as evidenced by a reduction in the number of revertants. Plates incubated with the test item showed normal background growth up to 5000 µg/plate, both with and without metabolic activation, in all four strains. No significant or reproducible increase in number of revertants was found in any strain, compared to the solvent control. No dose-dependent enhancement of revertants was observed. Presence of S9 mix did not influence these findings. Appropriate reference mutagens used as positive controls showed a distinct, predictable increase in induced revertant colonies. A weak increase in reventant colony numbers in strains TA 1535 (experiment 1, with S9, 5000 µg/plate) and TA 100 (experiment 1, with S9, 1000 and 5000 µg/plate) was considered irrelevant as these findings were not reproducible in the second experiment. Based on these findings, the test item did not induce point mutations by base pair substitution or frameshifts in S. typhimurium strains TA 98, TA 100, TA 1535 or TA 1537, therefore, it is not considered to be mutagenic.