Tetrasodium {5-[(4-chloro-6-{[4-(alkenylsulfonyl)phenyl]amino}-heteromonocycl-2-yl)amino]-4-(hydroxy-κO)-3-{[2-(hydroxy-κO)phenyl]diazenyl-κN}naphthalene-disulfonato(4-)}[3-(hydroxy-κO)-4-{[2-(hydroxy-κO)naphthalen-1-yl]diazenyl-κN}naphthalene-sulfonato(3-)]chromate(4-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 5th February 2014 Experimental completion date: 1st April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA OCSPP harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 20341/A TE
Batch: BOP 02-12 (Navy PLK 241, BS)
Purity: 80.1% (w/w)
Physical State/Appearance: Black powder, solid at 20 °C
Expiry Date: 21 November 2017
Storage Conditions: Room temperature in the dark

Method

Target gene:

Histidine operon for Salmonella.

Species / strain

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: PLEASE REFER TO SECTION "ANY OTHER INFORMATION ON MATERIALS AND METHOD'

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

A pre-test for toxicity was conducted. The maximum concentration was 5000 µg/plate. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Test for Mutagenicity Range-finding Test (Experiemnt 1): 50, 150, 500, 1500 and 5000 µg/plate

Test for Mutagencity Main Test (Experiment 2): 500, 1000, 1500, 3000, 4000 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix (0.5 µg/plate for TA102)

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix (3 µg/plate for TA 100, 5 µg/plate for TA 1535)

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix (80 µg/plate for TA 1537)

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix (0.2 µg/plate for TA 98)

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9 mix (1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537) )

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraqiuinone (DAN)

Remarks:

With S9 mix (10 µg/plate for TA 102)

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix (5 µg/plate for TA 98)

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutuation rates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

Used in conjunction with the uninduced hamster S9 (50 µg/plate for TA 98 and TA 100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The Prival-Mitchell Modification to the Ames Test
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.

Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification

NUMBER OF REPLICATIONS: Three

All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 500 µg/plate because of test item induced colouration.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and all of the S9-mixes used in both experiments were shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An intense test item coloration was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in two separate experiments.

In Experiment 1 (range-finding test), there were small but statistically significant increases in revertant colony frequency in tester strains TA100 and TA98, in the absence of S9 only, at and above 1500 µg/plate. Although these increases were between 1.3 and 1.6 fold above the concurrent solvent controls, they were only at or below the maximum limit of the historical range (depending on the strain). There was also only moderate evidence of a dose response relationship. Therefore, the dose range used in Experiment 2 (main test) was amended in an attempt to replicate and magnify the response observed in Experiment 1. No increases in colony frequency, at any dose of the test item, either with or without metabolic activation were observed. Therefore, the response in the range-finding test was considered to have no biological or toxicological relevance because of the lack of reproducibility, the response was either below or at the maximum limit of the historical range and lacked a convincing dose-response relationship.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1     Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
91		13		328		18		19
102	(89)	13	(12)	299	(285)	13	(19)	8	(12)
75		9		227		25		8

 

Main Test (Experiment 2)

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
99		9		279		23		9
82	(96)	13	(13)	311	(284)	19	(21)	7	(8)
108		18		261		21		9

 

Table2     Test Results: Range-finding Test (Experiment 1) – Without Metabolic Activation

Test Period		From: 28 February 2014					To: 03 March 2014
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98			TA1537
	Solvent Control (Water)	106 103 122	(110) 10.2#	22 27 15	(21) 6.0	323 295 252		(290) 35.8	18 25 16		(20) 4.7	13 10 11	(11) 1.5
	50 µg	91 103 104	(99) 7.2	15 16 10	(14) 3.2	283 283 200		(255) 47.9	14 18 18		(17) 2.3	12 7 10	(10) 2.5
	150 µg	81 93 99	(91) 9.2	9 16 12	(12) 3.5	227 224 205		(219) 11.9	19 27 26		(24) 4.4	12 10 10	(11) 1.2
	500 µg	97 I 88 I 108 I	(98) 10.0	21 I 8 I 24 I	(18) 8.5	223 I 239 I 214 I		(225) 12.7	27 I 14 I 30 I		(24) 8.5	10 I 11 I 8 I	(10) 1.5
	1500 µg	144 I 149 I 136 I	(143) 6.6	18 I 14 I 11 I	(14) 3.5	192 I 248 I 228 I		(223) 28.4	34 I 33 I 30 I		(32) 2.1	12 I 8 I 7 I	(9) 2.6
	5000 µg	172 I 163 I 176 I	(170) 6.7	14 I 14 I 13 I	(14) 0.6	256 I 236 I 228 I		(240) 14.4	35 I 29 I 33 I		(32) 3.1	9 I 8 I 9 I	(9) 0.6
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		MMC			4NQO			9AA
		3 µg		5 µg		0.5 µg			0.2 µg			80 µg
		903 1074 833	(937) 124.0	891 967 1013	(957) 61.6	2077 1882 1695		(1885) 191.0	243 251 252	(249) 4.9		621 540 705	(622) 82.5

  Table 3     Test Results: Range-finding Test (Experiment 1) – With Metabolic Activation

Test Period		From: 28 February 2014					To: 03 March 2014
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		TA102			TA98		TA1537
	Solvent Control (Water)	103 103 108	(105) 2.9#	17 13 18	(16) 2.6	321 304 327		(317) 11.9	36 32 27	(32) 4.5	23 16 20	(20) 3.5
	50 µg	96 95 96	(96) 0.6	13 8 11	(11) 2.5	309 324 301		(311) 11.7	29 31 34	(31) 2.5	21 15 15	(17) 3.5
	150 µg	104 97 110	(104) 6.5	12 11 13	(12) 1.0	319 229 313		(287) 50.3	33 30 23	(29) 5.1	16 16 19	(17) 1.7
	500 µg	95 I 111 I 114 I	(107) 10.2	13 I 20 I 15 I	(16) 3.6	371 I 353 I 253 I		(326) 63.6	25 I 26 I 26 I	(26) 0.6	16 I 16 I 14 I	(15) 1.2
	1500 µg	111 I 100 I 102 I	(104) 5.9	18 I 12 I 12 I	(14) 3.5	274 I 360 I 284 I		(306) 47.0	27 I 19 I 33 I	(26) 7.0	9 I 13 I 12 I	(11) 2.1
	5000 µg	116 I 101 I 119 I	(112) 9.6	16 I 17 I 20 I	(18) 2.1	264 I 228 I 288 I		(260) 30.2	26 I 21 I 28 I	(25) 3.6	12 I 9 I 13 I	(11) 2.1
Positive controls Rat S9-Mix (+)	Name	2AA		2AA		DAN			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	522 528 521	(524) 3.8	257 266 245	(256) 10.5	1455 1302 1388		(1382) 76.7	108 120 136	(121) 14.0	294 267 200	(254) 48.4
Positive controls Hamster S9-Mix (+)	Name	CR							CR
	Dose Level	50 µg							50 µg
	No. of Revertants	231 587 665	(494) 231.4						146 209 234	(196) 45.3

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

 

Methods…

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 

·     OECD Guidelines for Testing of Chemicals No. 471 (1997) "Bacterial Reverse Mutation Test".

·     Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.

·     USA, EPA OCSPP harmonized guidelines.

·     Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. 

….

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames plate pre-incubation method (Prival and Mitchell modification) at up to six dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for the range-finding test (Experiment 1) was determined in a Preliminary Toxicity Test and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was based on the results of the range-finding test and was 500, 1000, 1500, 3000, 4000 and 5000 µg/plate.

 

 

Results…….

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver S9-mix and the uninduced hamster liver S9-mix were validated.

 

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate. An intense test item coloration was noted at and above 500 mg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no toxicologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in two separate experiments. 

 

In Experiment 1 (range-finding test), there were small but statistically significant increases in revertant colony frequency in tester strains TA100 and TA98, in the absence of S9 only, at and above 1500 µg/plate. Although these increases were between 1.3 and 1.6 fold above the concurrent solvent controls, they were only at or below the maximum limit of the historical range (depending on the strain). There was also only moderate evidence of a dose response relationship. Therefore, the dose range used in Experiment 2 (main test) was amended in an attempt to replicate and magnify the response observed in Experiment 1. No increases in colony frequency, at any dose of the test item, either with or without metabolic activation were observed. Therefore, the response in the range-finding test was considered to have no biological or toxicological relevance because of the lack of reproducibility, the response was either below or at the maximum limit of the historical range and lacked a convincing dose-response relationship. 

 

 

Conclusion

FAT 20341/A TEwas considered to be non-mutagenic under the conditions of this test.