Disodium 2-[4-[[2-cyano-3-[4-[methyl(2-sulphonatoethyl)amino]phenyl]-1-oxoallyl]amino]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the test item was determined in an Ames test according to OECD guideline 471. No mutagenic potential was found.

Link to relevant study records

Reference

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:

2015-06-26 to 2015-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenates (S9) from male Wistar rats induced with phenobarbital and β-naphthoflavone (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany)

Test concentrations with justification for top dose:

33, 100, 333, 1000, 3650, 7300 µg/plate (with and without metabolic activation)

Vehicle / solvent:

Vehicle used: Water, DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

Without S9 mix: TA1535, TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Without S9 mix: TA 98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine (AAC)

Remarks:

Without S9 mix: TA 1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix: E.coli WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water (The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With S9 mix: All strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), preincubation

DURATION
- Preincubation period: At 37°C for the duration of about 20 minutes using a shaker
- Exposure duration: 48-72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (decrease in the number of his+) was observed in the standard plate test without metabolic activation only in tester strain TA 98 from 3650 μg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
According to the results of the present study the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). However, the slight increase in the number of his+ revertants observed in the standard plate test with tester strains TA 100 and TA 98 after the addition of S9 mix did not exceeded the threshold of factor 2, and so these findings have to be regarded as biological irrelevant.
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. However, in the preincubation test (4th Experiment) with TA 98 the revertant rates of the positive control 2-AA were below the range of our recent historical control. But, the number of his+ revertants of the positive control exceeding the treshold of factor 3 and, therefore it has to be considered that this deviation has no detrimental impact on the validity of this experimental part.

Toxicity: A weak bacteriotoxic effect (decrease in the number of his+) was observed in the standard plate test without metabolic activation only in tester strain TA 98 from 3650 μg/plate onward. In the preincubation assay no bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed up to the highest required concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Experiment 1 (SPT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
Water	10.7	90.7	6.7	19.7	17.3
Positive control	4584.7	4176.0	1095.3	343.3	1253.0
33	5.7	96.7	7.3	11.3	21.3
100	7.0	92.0	6.7	15.0	20.3
333	9.0	105.3	6.3	14.0	24.7
1000	11.7	93.7	5.3	13.7	17.7
3650	9.3	83.3	4.3	8.0	13.3
7300	12.0	90.0	5.3	8.7	21.7
	Results with S9
Water	8.3	102.0	5.7	25.3	29.0
Positive control	234.7	1842.7	116.3	1564.0	111.0
33	9.3	105.0	8.0	25.3	16.0
100	8.0	100.3	7.7	21.3	20.7
333	10.0	112.3	5.7	28.0	23.3
1000	11.0	116.0	9.0	31.3	20.3
3650	9.0	142.3	6.3	42.7	20.0
7300	8.7	194.3	6.7	49.0	26.0
Experiment 2 (PIT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
Water	7.3	80.7	5.0	17.3	20.0
Positive control	2417.3	1636.7	675.3	349.7	453.3
33	8.0	90.3	6.3	12.7	17.3
100	9.0	102.7	5.7	19.0	18.3
333	5.7	96.0	6.0	16.7	23.7
1000	10.7	99.0	8.0	17.3	22.0
3650	8.0	87.3	3.7	15.0	17.0
7300	10.3	83.3	5.3	20.3	21.0
	Results with S9
Water	9.0	104.0	6.0	25.7	20.7
Positive control	194.0	1550.7	106.7	508.7	69.0
33	9.7	107.7	7.3	22.3	29.0
100	6.7	98.0	8.7	25.0	27.3
333	8.3	94.3	10.7	27.7	28.0
1000	8.0	118.3	4.7	28.0	28.0
3650	7.7	140.3	6.7	42.0	26.3
7300	8.7	184.3	6.3	37.0	24.3

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test:

Key

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD guideline 471. The following strains were used: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a dose range of 33 μg - 7300 μg/plate . Standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were used to assess the mutagenic potential. No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was observed only in the standard plate test without S9 mix on tester strain TA 98 from 3650 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Disregarded

In another study (BASF, 1998) the mutagenic potenial of an aqueous solution of the test item (10.8 g/100 g active ingredient) was assessed based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, TA 100, TA1537 and TA98) and Escherichia coli (e. coli WP2 uvrA), in a reverse mutation assay (OECD 471). The test concentrations was 180 µg - 45000 µg/plate (all test strains) and 180 µg - 50000 µg/plate with TA 98. No precipitation of the test substance was found. No bacteriotoxic effect was observed. A weakly positive reaction with S-9 mix was observed in TA 100 only at 45000 µg/plate (factor 1.7). Depending on the experiment mutagenicity was observed with metabolic activation in TA 98 from about 10.000 µg - 22.500 µg/plate onward with a maximum increase in the number of his+ revertants by a factor of 8.1 at 30,000 µg/plate. According to the results of the present study, the test material was mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions chosen. However, as impurities in the test substance preparation are suspected as cause of the mutagenic effect this study is disregarded. The study was also rated as disregarded study as an aqueous preparation containing only 10.8 g/100 g active gradient was tested.

Justification for selection of genetic toxicity endpoint
GLP and guideline study

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008.