Reaction mass of Potassium sodium 3-{[4-({3-(substitutedamino)-4-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]phenyl}amino)-6-chloro-heteromonocyl-2-yl]amino}-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (1:3:1) and Potassium sodium 3-[(4-{[3-(subsitutedamino)-4-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}phenyl]amino}-6-chloro-heteromonocy-2-yl)amino]-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (3:9:3)

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:

21 February 2014 to 26 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

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

Guideline:

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

Deviations:

no

Test guideline 3

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011

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 40863/A TE
Batch: BOP 02-13 (BS-ROE1670)
Appearance: Brownish powder, solid at 20°C
Purity: 82.1 %, dose calculation not adjusted to purity
Stability in solvent: Stable during this short time study in a range of pH 5 to 8
Retest date: 05 June 2018
Storage Conditions: At room temperature

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in deionised water
- Justification for choice of solvent/vehicle: best suitable solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene, congo red

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding 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 and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: soluble to 50 mg/mL
- Precipitation:
No precipitation of the test item occurred up to the highest investigated dose.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed no deviation
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

   Table1     Summary of Experiment I

Study Name: 1610201	Study Code: Harlan CCR 1610201
Experiment: 1610201 VV Plate	Date Plated: 21/02/2014
Assay Conditions:	Date Counted: 24/02/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			21 ± 2	10 ± 2	26 ± 4	96 ± 5	53 ± 9
	Untreated			18 ± 2	7 ± 2	25 ± 5	113 ± 9	50 ± 4
	FAT 40863/A TE	3 µg		20 ± 7	13 ± 4	28 ± 5	114 ± 10	58 ± 9
		10 µg		17 ± 2	9 ± 3	23 ± 3	116 ± 15	56 ± 11
		33 µg		22 ± 2	11 ± 2	27 ± 8	100 ± 8	58 ± 8
		100 µg		19 ± 3	11 ± 2	29 ± 6	116 ± 11	59 ± 11
		333 µg		16 ± 1	10 ± 5	29 ± 5	106 ± 6	54 ± 0
		1000 µg		16 ± 3^D	10 ± 5^D	23 ± 2^D	103 ± 5^D	63 ± 5^D
		2500 µg		14 ± 8^D	10 ± 3^D	26 ± 4^D	109 ± 4^D	60 ± 7^D
		5000 µg		15 ± 3^D	7 ± 2^D	22 ± 4^D	92 ± 7^D	51 ± 7^D
	NaN3	10 µg		3219 ± 204			2409 ± 58
	4-NOPD	10 µg				311 ± 16
	4-NOPD	50 µg			65 ± 6
	MMS	2.0 µL						1268 ± 64
With Activation	Deionised water			15 ± 3	12 ± 3	43 ± 10	125 ± 14	63 ± 8
	Untreated			17 ± 8	14 ± 2	50 ± 2	133 ± 14	69 ± 3
	FAT 40863/A TE	3 µg		13 ± 2	12 ± 2	41 ± 12	130 ± 18	69 ± 3
		10 µg		14 ± 2	11 ± 4	32 ± 4	129 ± 12	70 ± 5
		33 µg		14 ± 3	14 ± 3	33 ± 3	123 ± 5	64 ± 5
		100 µg		14 ± 2	16 ± 1	41 ± 6	130 ± 15	70 ± 5
		333 µg		13 ± 3	15 ± 3	41 ± 7	147 ± 11	71 ± 12
		1000 µg		9 ± 1^D	9 ± 3^D	38 ± 2^D	126 ± 6^D	74 ± 9^D
		2500 µg		10 ± 5^D	15 ± 2^D	27 ± 5^D	127 ± 20^D	69 ± 2^D
		5000 µg		13 ± 3^D	13 ± 6^D	29 ± 1^D	136 ± 9^D	75 ± 3^D
	2-AA	2.5 µg		597 ± 23	404 ± 47	3911 ± 185	4642 ± 84
	2-AA	10.0 µg						321 ± 22

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	D	Densely coloured plate

 

 

Table2     Summary of Experiment II

Study Name: 1610201	Study Code: Harlan CCR 1610201
Experiment: 1610201 HV2 Pre	Date Plated: 20/03/2014
Assay Conditions:	Date Counted: 26/03/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			18 ± 5	8 ± 1	22 ± 6	97 ± 11	59 ± 5
	Untreated			14 ± 3	9 ± 3	30 ± 5	104 ± 11	52 ± 10
	FAT 40863/A TE	10 µg		15 ± 4	11 ± 4	22 ± 7	105 ± 3	49 ± 1
		33 µg		18 ± 8	10 ± 3	20 ± 4	90 ± 3	50 ± 2
		100 µg		16 ± 1	10 ± 4	22 ± 3	97 ± 8	53 ± 9
		333 µg		15 ± 2	9 ± 3	23 ± 5	93 ± 20	48 ± 2
		1000 µg		14 ± 5^D	9 ± 3^D	23 ± 6^D	99 ± 9^D	63 ± 3^D
		2500 µg		16 ± 4^D	7 ± 3^D	18 ± 2^D	94 ± 13^D	55 ± 2^D
		5000 µg		16 ± 7^D	5 ± 2^D	22 ± 2^D	92 ± 12^D	43 ± 2^D
	NaN3	10 µg		2866 ± 197			1905 ± 137
	4-NOPD	10 µg				271 ± 16
	4-NOPD	50 µg			57 ± 12
	MMS	2 µL						757 ± 36
With Activation	Deionised water			20 ± 8	26 ± 4	50 ± 6	103 ± 11	55 ± 0
	Untreated			24 ± 9	25 ± 7	50 ± 2	105 ± 10	69 ± 9
	FAT 40863/A TE	10 µg		18 ± 9	30 ± 15	50 ± 6	101 ± 3	58 ± 6
		33 µg		18 ± 2	31 ± 6	51 ± 12	112 ± 9	66 ± 5
		100 µg		20 ± 8	30 ± 3	45 ± 4	122 ± 13	61 ± 6
		333 µg		21 ± 4	31 ± 12	44 ± 4	110 ± 11	70 ± 3
		1000 µg		16 ± 3^D	22 ± 7^D	50 ± 7^D	112 ± 13^D	65 ± 4^D
		2500 µg		20 ± 3^D	27 ± 5^D	52 ± 5^D	118 ± 6^D	53 ± 3^D
		5000 µg		20 ± 4^D	24 ± 3^D	47 ± 3^D	123 ± 4^D	61 ± 11^D
	2-AA	2.5 µg					1426 ± 120
	2-AA	2.5 µg		421 ± 83	194 ± 11
	2-AA	10 µg						888 ± 81
	Congo red	500 µg				350 ± 55

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	D	Densely coloured plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without rat and hamster S9

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without rat and hamster S9.

Executive summary:

This study was performed to investigate the potential of FAT 40863/A TE to induce gene muta­tions according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), 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 FAT 40863/A TE at any dose level, neither in the presence nor 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 were used as positive controls and showed a distinct in­crease of induced revertant colonies.