2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from July 7 to August 26, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Basic Red 014 acetate

IUPAC Name:

Basic Red 014 acetate

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Standard plate test
1) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 60, 300, 1500, 7500, 15000 µg/plate.
2) TA 100: 0, 200, 400, 600, 800, 1000 µg/plate
3) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 1000, 2000, 3000, 4000 and 5000 µg/plate.

Preincubation test
4) TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA: 0, 30, 100, 300, 1000, 3000 µg/plate.
5) TA 100: 0, 500, 1000, 1500, 2000, 2500 µg/plate.

Vehicle / solvent:

- Vehicle: water.
- Justification for choice of solvent/vehicle: good solubility of test substance in water.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene, 4-nitro-o-phenylendiamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method

Test tubes containing 2-mI portions of soft agar (overlay agar, i.e. 100 ml agar (0.8 % agar + 0.6 % NaCI) and 10 ml amino acid solution (minimal aminoacid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within ca. 30 seconds.

Composition of the minimal glucose agar for Salmonella Typhimurium:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Composition of the minimal glucose agar for Escherichia coli:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

METHOD OF APPLICATION: preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S9 mix or phosphate buffer are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within ca. 30 seconds.
After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

The test substance is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either with or without S9 mix, is noted.

A test substance is generally considered nonmutagenic in this test if the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in standard plate test (from 3000 µg/plate), in preincubation test (from 1000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation: no test substance precipitation was found. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is selected and analysed (if not toxic) as the maximum dose.

COMPARISON WITH HISTORICAL CONTROL DATA: at non cytotoxic levels, test results are in the range of historical control data, except for TA 100 strain with metabolic activation. Further investigation on ths specific strain did not confirm this results, which was then considered as accidental.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- bacteriotoxic effect (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was observed in the standard plate test depending on the strain and test conditions from about 3000 to 5000 µg/plate onward.
- in preincubation assay, batteriotoxicity (reduced his- and trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in titer) was obserevd depending on the strain and test conditions at doses ≥ 1000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Standard plate test

historical data	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
without S9-mix	11-25	82-155	6-16	17-44	25-51
with S9-mix	12-25	88-156	6-19	20-49	25-56

µg/plate	TA 1535		TA 100		TA 1537		TA 98		WP2 uvrA
	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix
0	18	17	111	112	12	13	28	43	34	40
60	18	16	106	168	13	8	28	45	36	46
300	18	16	105	212	11	15	26	44	40	43
1500	15	18	103	170	9	15	30	35	40	44
7500	2	2	13	11	0	2	4	13	14	18
15000	0	0	0	0	0	0	0	1	3	5

µg/plate	TA 100
	with S9-mix
0	112
200	152
400	138
600	150
800	149
1000	119

µg/plate	TA 1535		TA 100		TA 1537		TA 98		WP2 uvrA
	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix
0	18	19	103	124	10	12	31	38	36	38
1000	17	20	111	130	11	9	28	38	32	41
2000	17	21	105	147	11	8	26	36	34	39
3000	13	22	62	78	4	6	23	39	35	33
4000	7	19	14	30	1	2	17	22	29	26
5000	1	8	9	10	0	1	8	12	21	18

Preincubation test

historical data	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
without S9-mix	13 -25	90 -160	5 -19	18 -44	25-54
with S9-mix	11-25	92 -159	6 -20	17 -50	25-59

µg/plate	TA 1535		TA 100		TA 1537		TA 98		WP2 uvrA
	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix	without S9-mix	with S9-mix
0	17	17	118	111	10	13	29	36	35	43
30	18	17	110	145	12	10	24	30	47	36
100	16	17	100	144	8	9	24	41	40	35
300	15	14	118	137	9	11	31	28	42	35
1000	9	20	29	189	3	4	13	30	22	42
3000	0	10	0	56	0	3	0	20	7	23

µg/plate	TA 100
	with S9-mix
0	111
500	136
1000	131
1500	110
2000	99
2500	88

Applicant's summary and conclusion

Conclusions:

Negative.
Under these experimental conditions, the substance is not a mutagenic agent in bacterial reverse mutation assay with Salmonella typhimurium and Escherichia coli strains. The occasionally observed slight increase in the number of revertant colonies using TA 100 with S9 mix both in the standard plate test and in the preincubation assay could not be confirmed in additional studies and is therefore regarded as incidental and not as indication of a point mutagenic activity of test substance.

Executive summary:

Method

Bacterial reverse mutation assay (Ames test) with Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) and Escherichia coli (WP2 uvrA) without or with metabolic activation (S9 mix). Two methods were used for the assays: standard plate and preincubation methods. Water was used as vehicle due to the high solubility of test substance in water. In standard plate test, doses ranged between 60 and 15000 µg/plate; in preincubation test, doses were between 30 and 3000 µg/plate. Solvent controls, sterility controls and positive controls were used.

Results

Based on negative responses under test conditions, the substance is considered as non mutagenic. Indeed the number of revertant colonies was found to be within historical control ranges. Depending on strain and test conditions, from ca. 3000 - 5000 µg/plate onward the substance was cytotoxic in the standard plate test, while at doses ≥ 1000 µg/plate the substance showed cytotoxicity in preincubation test. Slight increase in revertant colonies using TA 100 with S9 mix was not confirmed in further studies with this strain; therefore, it was considered as accidental.