Chromate(2-), [4-[(5-chloro-2-hydroxy-3-nitrophenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

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:

20 July, 1993 to 22 Sptember, 1993.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium[4-{[5-chloro-2-(hydroxy-O)-3-nitrophenyl]diazenyl-N1}-3-methyl-1-phenyl-1H-pyrazol-5-olato(2-)-O][3-{[1-(3-chlorophenyl)-5-(hydroxy-O)-3-methyl-1H-pyrazol-4-yl]diazenyl-N1}-4-(hydroxy-kO)-5-nitrobenzenesulfonato(3-)]chromate(2-)

Cas Number:

72017-66-4

Molecular formula:

C32H19Na2Cl2CrN10O11S

IUPAC Name:

Disodium[4-{[5-chloro-2-(hydroxy-O)-3-nitrophenyl]diazenyl-N1}-3-methyl-1-phenyl-1H-pyrazol-5-olato(2-)-O][3-{[1-(3-chlorophenyl)-5-(hydroxy-O)-3-methyl-1H-pyrazol-4-yl]diazenyl-N1}-4-(hydroxy-kO)-5-nitrobenzenesulfonato(3-)]chromate(2-)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: FAT 20'202/E
Batch No.: Op. 161
Aggregate State at RT: solid
Colour:light red
Purity: 70.1 (active ingredient)
Analysis: cf. information in sponsor's file
Stability: Pure: until March, 1998 In solvent: if necessary will be performed by the sponsor at a later date.
Storage: room temperature
Expiration Date: March, 1998

Method

Target gene:

Histidine auxotrophs

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: The Salmonella typhimurium histidine (his) reversion system measures his- —> his+ reversions.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535 and TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation - TA 1537 and TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activaton - TA 102

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation - TA 1535, TA 1537, TA 98, TA 100, TA 102

Details on test system and experimental conditions:

Without metabolic activation
TA 1535, TA 100, TA 102 - double distilled water
TA 15377, TA 98 - DMSO


With metabolic activation
TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO

THE TEST SYSTEM
Characterisation of the Salmonella typhimurium Strains.The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally due to the "deep rough" (rfa-minus) 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-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin resistance marker. The strain TA 102 does not contain the uvrB"-mutation. Additionally TA 102 contains the multicopy plasmid pAQl, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) 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 Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

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. Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar*
6.0 g NaCl*
10.5 mg L-histidine x HCl x H20*
12.2 mg biotin*

Sterilisations were performed at 121 °C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation by C CR) The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (Charles River Wiga GmbH, D-97633 Sulzfeld, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) 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, 3 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, D-80939 München:Bio-Rad protein assay, Catalogue 500 000 6 (6).The protein concentration in the S9 preparation was 43.8 mg/ml
(lot 010293) in the pre-experiment as well as in experiment I, and 45.0 mg/ml (lot 190793) in experiment II.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cof actor solution. The amount of S9 supernatant was 15 % v/v. The composition of the cofactor solution was concentrated to yield the following 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. The
S9 mix preparation was performed according to Ames et al.(2).


PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy was performed with strains TA 9 8 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). 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.


* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.

Rationale for test conditions:

None

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
corresponding background growth on both negative control and test plates normal range of spontaneous reversion rates.

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.

A test article is considered as positive if either a dose related and reproducible 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 dose related and reproducible 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 mutagenic if in strain TA 100 and TA 102 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.

Also, a dose-dependent and reproducible 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 appropriate statistical method is available.

Results and discussion

Test results

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

None

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from: rat liver (Batch R 010293)

Test strain: TA 1535

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		8	14	13	12	3.2
Solvent Control		22	19	10	17	6.2	1
	33.3	12	13	14	13	1	0.8
	100	8	10	16	11	4.2	0.7
	333.3	17	12	13	14	2.6	0.8
	1000	10	7	19	12	6.2	0.7
	2500	3	1	2	2	1	0.1
	5000	0	0	0	0	0	0
Positive Control Sodium azide (10µg/plate)		1066	1051	1069	1062	9.6	62.5
with S9 mix
Negative Control		20	10	20	17	5.8
Solvent Control		24	15	21	20	4.6	1
	33.3	7	12	7	9	2.9	0.4
	100	14	17	21	17	3.5	0.9
	333.3	26	24	35	28	2.9	1.4
	1000	14	15	18	16	2.1	0.8
	2500	12	19	17	16	3.6	0.8
	5000	11	12	7	10	2.6	0.5
Positive Control 2-Aminoanthracene (2.5µg/plate)		139	151	343	211	114.5	10.6

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from: rat liver       

Test strain: TA 1537

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		9	16	13	13	3.5
Solvent Control		12	10	6	9	3.1	1
	33.3	5	10	10	8	2.9	0.9
	100	13	11	15	13	2	1.4
	333.3	18	8	11	12	5.1	1.3
	1000	8	12	12	11	2.3	1.1
	2500	9	11	11	10	1.2	1.1
	5000	4	3	3	3	0.6	0.4
Positive Control 4-nitor-o-phenylene diamine (10µg/plate)		92	91	85	89	3.8	9.6
with S9 mix
Negative Control		18	14	19	17	2.6
Solvent Control		26	17	20	21	4.6	1
	33.3	23	21	21	22	1.2	1
	100	19	17	27	24	4.6	1.2
	333.3	23	20	32	25	6.2	1.2
	1000	29	23	21	24	4.2	1.2
	2500	24	17	22	21	3.6	1
	5000	25	15	27	22	6.4	1.1
Positive Control 2-Aminoanthracene (2.5µg/plate)		95	91	93	93	2	4.4

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 010293)

Test strain : TA 98

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		26	27	36	30	5.5
Solvent Control		23	19	28	23	4.5	1
	33.3	21	32	38	30	8.6	1.3
	100	13	27	21	20	7	0.9
	333.3	20	19	20	20	0.6	0.8
	1000	15	12	17	15	2.5	0.6
	2500	7	2	7	5	2.9	0.2
	5000	10	3	10	8	4	0.3
Positive Control      4-nitorphenylene diamine (10µg/plate)		817	807	736	787	44.2	33.7
with S9 mix
Negative Control		41	53	36	43	8.7
Solvent Control		39	41	55	45	8.7	1
	33.3	46	54	39	46	7.5	1
	100	40	44	49	44	4.5	1
	333.3	39	33	32	35	3.8	0.8
	1000	24	29	26	26	2.5	0.6
	2500	20	31	23	25	5.7	0.5
	5000	10	16	17	14	3.8	0.3
Positive Control 2-Aminoanthracene (2.5µg/plate)		583	502	498	528	48	11.7

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 010293)

Test strain : TA 100

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		101	117	96	105	11
Solvent Control		115	97	120	111	12.1	1
	33.3	71	107	88	89	18	0.8
	100	110	101	99	103	5.9	0.9
	333.3	85	78	99	87	10.7	0.8
	1000	82	81	79	81	1.5	0.7
	2500	97	116	86	100	15.2	0.9
	5000	99	98	100	99	1	0.9
Positive Control      sodium azide (10µg/plate)		755	737	666	719	47.1	6.5
with S9 mix
Negative Control		183	146	154	161	19.5
Solvent Control		159	163	166	163	3.5	1
	33.3	185	175	178	179	5.1	1.1
	100	174	167	159	167	7.5	1
	333.3	164	179	167	170	7.9	1
	1000	180	194	197	190	9.1	1.2
	2500	195	203	186	195	8.5	1.2
	5000	228	199	192	206	19.1	1.3
Positive Control     2-Aminoanthracene (2.5µg/plate)		1007	884	1090	994	103.6	6.1

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 010293)

Test strain : TA 102

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		156	143	163	154	10.7
Solvent Control		161	138	136	145	13.9	1
	33.3	153	153	176	161	13.3	1.1
	100	172	178	122	157	30.7	1.1
	333.3	202	195	194	197	4.4	1.4
	1000	178	174	179	177	2.6	1.2
	2500	107	87	109	101	12.2	0.7
	5000	14	14	14	14	0	0.1
Positive Control   methyl methane sulfonate                (1.0µl/plate)		1066	1040	1154	1087	59.7	7.5
with S9 mix
Negative Control		239	267	215	240	26
Solvent Control		266	280	268	271	7.6	1
	33.3	288	313	299	300	12.5	1.1
	100	213	231	205	216	13.3	0.8
	333.3	295	288	321	301	17.4	1.1
	1000	250	294	237	260	29.9	1
	2500	226	285	329	280	51.7	1
	5000	277	376	346	333	50.8	1.2
Positive Control      2-Aminoanthracene (10µg/plate)		893	868	834	865	29.6	3.2

 

 

PRE-INCUBATION TEST

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 190793)

Test strain : TA 1535

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		14	6	13	11	4.4
Solvent Control		5	12	13	10	4.4	1
	33.3	13	11	6	10	3.6	1
	100	3	4	7	5	2.1	0.5
	333.3	7	11	5	8	3.1	0.8
	1000	10	9	11	10	1	1
	2500	6	7	4	6	1.5	0.6
	5000	0	0	0	0	0	0
Positive Control Sodium azide (10µg/plate)		526	494	0	340	294.9	34
with S9 mix
Negative Control		22	21	24	22	1.5
Solvent Control		22	33	38	31	8.2	1
	33.3	13	25	29	22	8.3	0.7
	100	21	18	29	23	5.7	0.7
	333.3	12	25	17	18	6.6	0.6
	1000	26	21	33	27	6	0.9
	2500	23	18	26	22	4	0.7
	5000	14	14	17	15	1.7	0.5
Positive Control 2-Aminoanthracene (2.5µg/plate)		412	426	414	417	7.6	13.5

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 190793)

Test strain : TA 1537

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		17	11	9	12	4.2
Solvent Control		14	10	16	13	3.1	1
	33.3	10	5	7	7	2.5	0.6
	100	9	9	14	11	2.9	0.8
	333.3	8	14	11	11	3	0.8
	1000	13	11	21	15	5.3	1.1
	2500	3	3	4	3	0.6	0.3
	5000	0	0	0	0	0	0
Positive Control 4-nitor-o-phenylene diamine (10µg/plate)		109	103	111	108	4.2	8.1
with S9 mix
Negative Control		11	10	8	10	1.5
Solvent Control		14	11	13	13	1.5	1
	33.3	6	13	19	13	6.5	1
	100	17	11	10	13	3.8	1
	333.3	15	9	11	12	3.1	0.9
	1000	12	11	14	12	1.5	1
	2500	13	12	15	13	1.5	1.1
	5000	9	13	16	13	3.5	1
Positive Control 2-Aminoanthracene (2.5µg/plate)		74	75	87	79	7.2	6.2

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 190793)

Test strain : TA 98

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		26	28	21	25	3.6
Solvent Control		17	24	17	19	4	1
	33.3	24	25	16	22	4.9	1.1
	100	13	19	25	19	6	1
	333.3	22	30	34	29	6.1	1.5
	1000	11	18	22	17	5.6	0.9
	2500	10	10	9	10	0.6	0.5
	5000	0	0	0	0	0	0
Positive Control      4-nitorphenylene diamine (10µg/plate)		557	516	516	530	23.7	27.4
with S9 mix
Negative Control		38	46	36	40	5.3
Solvent Control		45	44	30	40	8.4	1
	33.3	44	58	47	50	7.4	1.3
	100	54	59	47	53	6	1.3
	333.3	47	50	53	50	3	1.3
	1000	37	63	58	53	13.8	1.3
	2500	29	25	33	29	4	0.7
	5000	22	27	15	21	6	0.5
Positive Control 2-Aminoanthracene (2.5µg/plate)		420	486	409	438	41.6	11.1

 

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 190793)

Test strain : TA 100

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		85	94	77	85	8.5
Solvent Control		103	89	112	101	11.6	1
	33.3	75	103	83	87	14.4	0.9
	100	64	74	76	71	6.4	0.7
	333.3	69	48	80	76	5.9	0.7
	1000	101	81	75	86	13.6	0.8
	2500	60	73	97	77	18.8	0.8
	5000	16	59	81	52	33.1	0.5
Positive Control      sodium azide (10µg/plate)		407	430	474	437	34	4.3
with S9 mix
Negative Control		173	172	186	177	7.8
Solvent Control		158	153	160	157	3.6	1
	33.3	178	152	136	155	21.2	1
	100	168	151	151	157	9.8	1
	333.3	184	186	169	180	9.3	1.1
	1000	165	177	170	171	6	1.1
	2500	179	171	121	157	31.4	1
	5000	126	131	131	129	2.9	0.8
Positive Control     2-Aminoanthracene (2.5µg/plate)		869	937	935	914	38.7	5.8

 

Test article: FAT 20202/E Lanacron Rot S-3G roh feucht

S9 mix from : rat liver (Batch R 190793)

Test strain : TA 102

 

 

Doseµg/plate (active ingredient)		PLATE			Revertants/plate
		1	2	3	mean	S.D.	Factor+
without S9 mix
Negative Control		159	157	156	157	1.5
Solvent Control		152	182	173	169	15.4	1
	33.3	142	154	171	156	14.6	0.9
	100	135	135	130	133	2.9	0.8
	333.3	179	173	206	186	17.6	1.1
	1000	179	178	186	181	4.4	1.1
	2500	26	53	41	40	13.5	0.2
	5000	0	0	0	0	0	0
Positive Control   methyl methane sulfonate                (1.0µl/plate)		967	924	881	924	43	5.5
with S9 mix
Negative Control		251	271	269	264	11
Solvent Control		236	200	236	224	20.8	1
	33.3	281	265	279	275	8.7	1.2
	100	238	217	261	239	22	1.1
	333.3	213	202	257	224	29.1	1
	1000	210	272	271	251	35.5	1.1
	2500	258	205	203	222	31.2	1
	5000	254	230	220	235	17.5	1
Positive Control      2-Aminoanthracene (10µg/plate)		631	639	693	654	33.7	2.9

Applicant's summary and conclusion

Conclusions:

FAT 20202/E was determined to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

FAT 20202/E was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 1535, TA 1537, TA 98, TA 100, and TA 102. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In both experiments toxic effects occurred in the strains TA 1535 and TA 98 with and without S9 mix, as well as in the strains TA 1537 and TA 102 without S9 mix all in experiment I and II at higher concentrations. The plates incubated with the test article showed normal background growth up to 5000 ug/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 20202/E; Lanacron Rot S-3G roh feucht at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 20202/E; Lanacron Rot S-3G roh feucht is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.