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

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:

2015-06-26 to 2015-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-694-3

EC Name:

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

Cas Number:

2498-95-5

Molecular formula:

C27H24N4O7S3.2Na

IUPAC Name:

disodium 2-{4-[(2-cyano-3-{4-[methyl(2-sulfonatoethyl)amino]phenyl}acryloyl)amino]phenyl}-6-methyl-1,3-benzothiazole-7-sulfonate

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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