Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27-02-2018 - 26-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate (CAS no. 2783-94-0) to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	110	26	124	28
	R2	106	24	122	26
	R3	104	25	126	24
T1 (0.002)	R1	91	14	86	16
	R2	84	13	82	18
	R3	87	13	88	16
T2 (0.005)	R1	86	14	90	19
	R2	92	17	88	21
	R3	85	18	86	17
T3 (0.016)	R1	90	16	102	19
	R2	96	14	98	20
	R3	84	16	106	21
T4 (0.050)	R1	90	18	106	21
	R2	88	16	98	23
	R3	98	16	90	19
T5 (0.158)	R1	95	18	104	20
	R2	88	19	100	18
	R3	96	18	108	22
T6 (0.501)	R1	94	20	108	18
	R2	101	21	110	20
	R3	98	19	114	24
T7 (1.582)	R1	96	20	112	18
	R2	104	23	116	22
	R3	98	20	118	24
T8 (5)	R1	102	23	118	23
	R2	98	25	114	25
	R3	100	24	120	24
PC	R1	1232	896	1458	1242
	R2	1280	944	1432	1180
	R3	1296	936	1488	1208

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	8	15	28	124	284
	R2	8	14	26	122	278
	R3	6	16	24	126	284
T1 (0.005)	R1	6	10	19	90	228
	R2	5	11	21	88	238
	R3	5	12	17	86	232
T2 (0.016)	R1	7	12	19	102	227
	R2	5	11	20	98	244
	R3	6	12	21	106	236
T3 (0.050)	R1	6	13	21	106	242
	R2	7	12	23	98	254
	R3	6	12	19	90	236
T4 (0.158)	R1	6	14	20	104	254
	R2	5	13	18	100	260
	R3	5	12	22	108	262
T5 (0.501)	R1	7	14	18	108	268
	R2	5	15	20	110	274
	R3	6	15	24	114	270
PC	R1	172	398	1242	1458	1336
	R2	186	408	1180	1432	1384
	R3	190	440	1208	1488	1344

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	16	26	110	272
	R2	6	15	24	106	296
	R3	8	14	25	104	268
T1 (0.005)	R1	4	11	14	86	226
	R2	5	10	17	92	231
	R3	5	11	18	85	218
T2 (0.016)	R1	5	13	16	90	226
	R2	6	12	14	96	234
	R3	5	10	16	84	232
T3 (0.050)	R1	6	14	18	90	242
	R2	6	13	16	88	238
	R3	6	11	16	98	250
T4 (0.158)	R1	6	14	18	95	244
	R2	6	15	19	88	252
	R3	5	13	18	96	260
T5 (0.501)	R1	7	14	20	94	268
	R2	7	16	21	101	274
	R3	6	14	19	98	284
PC	R1	168	1218	896	1232	1624
	R2	183	1230	944	1280	1688
	R3	175	1170	936	1296	1680

NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                  2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                        Sodium azide [10μg/plate]: TA 1535, TA 100                                                 

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]   Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	8	17	28	121	278
	R2	6	16	27	119	262
	R3	7	16	27	120	272
T1 (0.005)	R1	5	12	19	104	234
	R2	5	12	21	98	240
	R3	4	11	22	102	230
T2 (0.016)	R1	5	13	23	106	240
	R2	6	13	22	110	236
	R3	5	12	24	104	242
T3 (0.050)	R1	5	12	20	112	250
	R2	6	15	21	108	240
	R3	4	13	23	112	258
T4 (0.158)	R1	5	14	23	114	266
	R2	6	13	23	108	260
	R3	6	14	24	112	258
T5 (0.501)	R1	7	15	26	117	260
	R2	7	14	25	115	272
	R3	6	14	23	118	258
PC	R1	179	398	1360	1518	1736
	R2	158	418	1384	1448	1704
	R3	184	376	1328	1480	1712

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	`7	16	28	114	264
	R2	7	15	26	112	256
	R3	6	13	26	118	274
T1 (0.005)	R1	5	10	20	102	229
	R2	4	10	22	108	237
	R3	4	11	18	110	240
T2 (0.016)	R1	6	12	21	104	242
	R2	4	11	23	110	246
	R3	5	14	22	108	256
T3 (0.050)	R1	5	13	19	106	260
	R2	4	13	23	110	252
	R3	5	12	22	111	254
T4 (0.158)	R1	5	11	25	116	262
	R2	5	12	20	112	250
	R3	6	13	23	108	260
T5 (0.501)	R1	6	13	26	116	266
	R2	6	14	23	112	252
	R3	7	12	21	114	258
PC	R1	187	1208	928	1086	1608
	R2	178	1160	894	1164	1656
	R3	182	1224	952	1128	1568

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	1.15	15.00	1.00	26.00	2.00	124.00	2.00	282.00	3.46
T1 (0.005)	5.33	0.58	11.00	1.00	19.00	2.00	88.00	2.00	232.67	5.03
T2 (0.016)	6.00	1.00	11.67	0.58	20.00	1.00	102.00	4.00	235.67	8.50
T3 (0.050)	6.33	0.58	12.33	0.58	21.00	2.00	98.00	8.00	244.00	9.17
T4 (0.158)	5.33	0.58	13.00	1.00	20.00	2.00	104.00	4.00	258.67	4.16
T5 (0501)	6.00	1.00	14.67	0.58	20.67	3.06	110.67	3.06	270.67	3.06
PC	182.67	9.45	415.33	21.94	1210.00	31.05	1459.33	28.02	1354.67	25.72

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	15.00	1.00	25.00	1.00	106.67	3.06	278.67	15.14
T1 (0.005)	4.67	0.58	10.67	0.58	16.33	2.08	87.67	3.79	225.00	6.56
T2 (0.016)	5.33	0.58	11.67	1.53	15.33	1.15	90.00	6.00	230.67	4.16
T3 (0.050)	6.00	0.00	12.67	1.53	16.67	1.15	92.00	5.29	243.33	6.11
T4 (0.158)	5.67	0.58	14.00	1.00	18.33	0.58	93.00	4.36	252.00	8.00
T5 (0501)	6.67	0.58	14.67	1.15	20.00	1.00	97.67	3.51	275.33	8.08
PC	175.33	7.51	1206.00	31.75	925.33	25.72	1269.33	33.31	1664.00	34.87

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100 Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                  

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	16.33	0.58	27.33	0.58	120.00	1.00	270.67	8.08
T1 (0.005)	4.67	0.58	11.67	0.58	20.67	1.53	101.33	3.06	234.67	5.03
T2 (0.016)	5.33	0.58	12.67	0.58	23.00	1.00	106.67	3.06	239.33	3.06
T3 (0.050)	5.00	1.00	13.33	1.53	21.33	1.53	110.67	2.31	249.33	9.02
T4 (0.158)	5.67	0.58	13.67	0.58	23.33	0.58	111.33	3.06	261.33	4.16
T5 (0501)	6.67	0.58	14.33	0.58	24.67	1.53	116.67	1.53	263.33	7.57
PC	173.67	13.80	397.33	21.01	1357.33	28.10	1482.00	35.04	1717.33	16.65

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.67	0.58	14.67	1.53	26.67	1.15	114.67	3.06	264.67	9.02
T1 (0.005)	4.33	0.58	10.33	0.58	20.00	2.00	106.67	4.16	235.33	5.69
T2 (0.016)	5.00	1.00	12.33	1.53	22.00	1.00	107.33	3.06	248.00	7.21
T3 (0.050)	4.67	0.58	12.67	0.58	21.33	2.08	109.00	2.65	255.33	4.16
T4 (0.158)	5.33	0.58	12.00	1.00	22.67	2.52	112.00	4.00	257.33	6.43
T5 (0501)	6.33	0.58	13.00	1.00	23.33	2.52	114.00	2.00	258.67	7.02
PC	182.33	4.51	1197.33	33.31	924.67	29.14	1126.00	39.04	1610.67	44.06

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using th e Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the 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 biological relevance.

The spontaneous reversion rates in the negative, positive controls are within the range of our historical data.

The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.  

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Including Prival modification

Principles of method if other than guideline:

Ames Salmonella typhimurium mutagenicity test including the Prival modification was conducted to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Liver S9 homogenate was prepared from Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix
- source of S9 : Liver S9 homogenate was prepared from Syrian golden hamsters
- method of preparation of S9 mix : The postmitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : Uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate),
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0, 10, 33, 100, 333, 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

congo red

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30 mins without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced.
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was checked in preliminary dose range finding study

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Mutagenic response of the test chemical:

Prival Modification	Doses ug/plate	TA98	TA100
		Negative	Negative
	DMSO	24 ± 6	139 ± 24
	10ug	33 ± 7	142 ± 19
	33ug	24 ± 4	166 ± 52
	100ug	39 ± 13	124 ± 19
	333ug	31 ± 4	161 ± 13
	1000ug	27 ± 4	193± 9
	Positive 1	190 ± 24	368 ± 50
	Positive 2	522 ± 67	1150 ± 55

Conclusions:

The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Executive summary:

Ames mutagenicity test with Prival modification was conducted as per OECD 471 for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000 µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonells strains and Tryptophan for E. coli strains

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (TA1537, TA98, -S9); 2-aminoanthracene (TA1535, TA1537, TA98, TA100, WP2 uvrA + S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added : in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for the an increase in the number of revertants

Statistics:

No data

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

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:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with strains TA1535, TA1537, TA98, TA100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. 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 results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen. According to the dose selection criteria the test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The results of the pre-experiment ate included are reported as a part of the main experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Ames test:
- Signs of toxicity : The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
- Individual plate counts : No data
- Mean number of revertant colonies per plate and standard deviation : No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and the Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test chemical at any dose level, neither in the presence nor in the 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 biological relevance. Appropriate reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Data source

Reference

Reference Type:

publication

Title:

WoE of gene mutation in vitro toxicity study for CAS no 73507-17-2

Author:

Sustainability Support Services (Europe) AB

Year:

2019

Bibliographic source:

WoE report, Sustainability Support Services (Europe) AB, 2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Including Prival modification / 3

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

EC Number:

277-492-0

EC Name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Cas Number:

73507-17-2

Molecular formula:

C52H28Cl2CrN14O18S44Na

IUPAC Name:

Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)

Details on test material:

- Name of the test chemical: Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
- Molecular formula: C52H28Cl2CrN14O18S4.H.4Na
- Molecular weight: 2693.61 g/mol
- Substance type: Organic

Method

Target gene:

Histidine for Salmonella strains and Tryptophan for E. coli strains

Species / strain

Species / strain 1

Species / strain / cell type:

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

Remarks:

2

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA98, TA100

Remarks:

3 / 5

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

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

Remarks:

4

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

4

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

2. Aroclor 1254 induced S9 metabolic activation system

3/5. Type and composition of metabolic activation system: Liver S9 homogenate was prepared from Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 m L/mL of mix
- source of S9 : Liver S9 homogenate was prepared from Syrian golden hamsters
- method of preparation of S9 mix : The postmitochondrial (microsomal) enzyme fractions were prepar ed as described by Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : Uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

4. The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats

Test concentrations with justification for top dose:

2. 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
3. 0, 10, 33, 100, 333, 1000 µg/plate
4. 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate
5. 0, 10, 33, 100, 333, 1000, 3333 or 10000 µg/plate

Vehicle / solvent:

2. - Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water

3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

4. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

5. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Remarks:

2

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

congo red

Remarks:

3

Controls 3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (TA1537, TA98, -S9); 2-aminoanthracene (TA1535, TA1537, TA98, TA100, WP2 uvrA + S9)

Remarks:

4

Controls 4

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

congo red

Remarks:

5

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

3/5. METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30 mins without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was checked in preliminary dose range finding study

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

4. NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added : in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data

Rationale for test conditions:

No data

Evaluation criteria:

2. A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

3. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100) in the mean number of revertants per plate of at least on e tester strain. This increase in the mean revertants per plate had to be accompanied by a dose res ponse to increasing concentrations of the test chemical.

4. The plates were observed for the an increase in the number of revertants

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

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

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA98, TA100

Remarks:

3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Remarks:

4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Remarks:

4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

3. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxici ty observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the ch emical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result
other: No mutagenic potential

4. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with strains TA1535, TA1537, TA98, TA100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. 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 results in a reduction in the number of spontaneous revertants or a clearing of the bacterial ba ckground lawn. Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen. According to the dose selection criteria the test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The results of the pre-expe riment ate included are reported as a part of the main experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Ames test:
- Signs of toxicity : The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
- Individual plate counts : No data
- Mean number of revertant colonies per plate and standard deviation : No data

Remarks on result other: No mutagenic potential

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The summaries are as mentioned below:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using th e Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the 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 biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.  

Ames mutagenicity test with Prival modifications was also conducted as per the OECD 471 guideline for the test chemical to evaluate its genetoxic effects. In the FMN-modified assay, the test chemical was exposed toSalmonella typhimuriumstrains TA98 and TA100 at dose concentrations of 0, 10, 33, 100, 333, 1000  µg/plate. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control used for the experiment was Congo red. All plates were counted with an Artek automated colony counter or Mini-count colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

In another study, Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test chemical at any dose level, neither in the presence nor in the 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 biological relevance. Appropriate reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation inSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In the same study as mentioned above, Ames mutagenicity test with Prival modification was conducted as per OECD 471 guideline for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000, 3333 or 10000  µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.