Reaction mass of pentasodium 2-{[4-chloro-6-(ethyl{3-[(2-sulfonatoethyl)sulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate and tetrasodium 2-[(4-chloro-6-{ethyl[3-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate

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:

16 December 2005 to 10 Febuary 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

Deviations:

not specified

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: Jananese guideline: Kanpoan No. 287 - Environment Protection Agency;Eisei No. 127 - Ministry of Health & Welfare; Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

Deviations:

not specified

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

Details on test material:

-Batch No.: ROE 805 BOP 04/05
-Colour: Dark red
-Solubility in water: >100g/L at 20 °C
-Solubility in vehicle: Miscible
-Stability in solvent: 7 days in Water, Saline, Polyethylene Glycol, Carboxymethylcellulose, and 1 day in Vaseline and FCA at room temperature.
-Storage: At room temperature, in the desicator
-Expiration Date: October 01, 2010
-Purity: Approx. 82 % organic part (Na-salt),
all coloured components = 80.3 %;
Main component 1: 36.2 %,
Main component 2: 27.5 %, Oligomers: 10 %

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1 : approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Method

Target gene:

histidine dependent S. typhimurium and Escherichia coli strains

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

s9 mix

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Deionised water

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:

for TA 1535, TA 100 without S9

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, 4-NOPD

Remarks:

for TA 1537, TA 98 without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

for WP2 uvrA without S9

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:

for all strains with S9

Details on test system and experimental conditions:

-Precultures:
From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCI (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

-S9Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8mM MgCI2
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revenants 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.

Statistics:

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

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

HISTORICAL CONTROL DATA

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	19	6	9	35	21	7	7	41
	Negative control	18	5	10	30	21	6	9	38
	Positive control	1681	789	1003	4900	387	126	172	695
TA 1537	Solvent control	12	3	4	29	18	6	6	36
	Negative control	11	3	5	29	19	6	8	33
	Positive control	87	18	52	191	337	191	94	746
TA 98	Solvent control	26	6	14	•58	39	9	21	57
	Negative control	26	6	15	€0	41	9	17	64
	Positive control	361	204	176	1818	2386	1195	296	4854
TA 100	Solvent control	131	24	91	198	147	25	109	281
	Negative control	140	21	101	189	154	23	103	254
	Positive control	2030	340	1178	2872	2629	1326	546	•5230
WP2uvrA	Solvent control	52	8	31	67	55	10	34	75
	Negative control	50	8	36	64	52	8	33	•64
	Positive control	998	515	320	1976	342	134	221	930

 

Mean = mean value of revertants/plate; SD s standard deviation; Min = minimal value; Max = maximal value

Applicant's summary and conclusion

Conclusions:

FAT 40824/A did induce gene mutations by frameshifts in the genome of strain TA 1537 in the absence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA with and without liver microsomal activation. 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate were administered in Pre-Experiment/Experiment I; 33; 100; 333; 1000; 2500; and 5000 μg/plate were administrated in Experiment II and VVa test. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. A minor but dose dependent increase in revertant colony numbers was observed following treatment with test substance in strain TA 1537 in the absence of metabolic activation (S9 mix). The number of colonies reached the threshold of thrice (strain TA 1537) the number of the corresponding solvent control at 5000 μg/plate in the pre-experiment. Since the threshold was just reached only at 5000 μg/plate and the values were still in the range of the laboratories historical data, the plate incorporation assay was repeated with strain TA 1537 without metabolic activation (reported as VVa) in parallel to the second experiment (pre-incubation). Both experiments showed a substantial and dose dependent increase in revertant colony numbers. The number of colonies exceeded the threshold of thrice the number of the corresponding solvent control at 1000 μg/plate and above in the pre- incubation assay and at 2500 μg/plate and above in the repeated plate incorporation. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. Thus, it can be concluded that the test item can induce gene mutations by frameshifts in the genome of strain TA 1537 in the absence of metabolic activation.