[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]diethylammonium acetate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Experimental study according to guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]diethylammonium acetate

EC Number:

278-585-9

EC Name:

[4-[[4-(diethylamino)phenyl]phenylmethylene]-2,5-cyclohexadien-1-ylidene]diethylammonium acetate

Cas Number:

76994-37-1

Molecular formula:

C27H33N2.C2H3O2

IUPAC Name:

N-(4-{[4-(diethylamino)phenyl](phenyl)methylene}cyclohexa-2,5-dien-1-ylidene)-N-ethylethanaminium acetate

Details on test material:

- Physical state: Liquid, high viscous, green
- Storage: Room temperature (avoid temperatures > 60 °C)
- Stability: The stability of the test substance under storage conditions is guaranteed until 01 July 2020
- Expiry Date: 2020-07-01

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 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 (Phenobarbital/β-naphthoflavone induced rat liver)

Test concentrations with justification for top dose:

Experiment 1 (standard plate test): 0, 33, 100, 333, 1000, 2750 and 5500 μg/plate
Experiment 2 (standard plate test): 0, 0.1, 0.3, 1.0, 3.3, 10 and 33 μg/plate (TA strains) and 0, 0.3, 1.0, 3.3, 10, 33 and 100 μg/plate (E.coli)
Experiment 3 (preincubation test): 0, 0.03, 0.1, 0.3, 1.0, 3.3 and 10 μg/plate (TA strains) and 0, 0.1, 0.3, 1.0, 3.3, 10 and 33 μg/plate (E.coli)

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

(MNNG) TA 1535, TA 100 (without activation)

Controls 2

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

(NOPD) TA 98 (without activation)

Controls 3

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

Remarks:

(4-NQO) WP2 uvrA (without activation)

Controls 4

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(AAC) TA 1537 (without activation)

Controls 5

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2-AA) TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA (with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation, in agar (plate incorporation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth). It was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

The test substance was considered positive in this assay if 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 data 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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: starting at 5500 μg/plate with and without S9 mix


ADDITIONAL INFORMATION ON CYTOTOXICITY: It was dependent on test strain used but independent of metabolic activation. The lowest concentrations inducing a decreased revertant number were 10 µg/plate (standard plate test) and 3.3 µg/plate (preincubation test).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1 (standard plate)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
DMSO	7.7 ± 2.1	95.7 ± 1.5	7.0 ± 2.0	17.0 ± 3.6	18.7 ± 0.6
33	0 ± 0	0 ± 0	0 ± 0	0 ± 0	7.7 ± 0.6
100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
333	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
1000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2750	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
5500	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
MNNG (5)	4633 ± 203	3648 ± 86.5
AAC (100)			1686.7 ± 190
NOPD (10)				407.3 ± 25.3
4-NQO (5)					1337 ± 88.7
	Results with S9
DMSO	9.3 ± 2.5	92.3 ± 5.5	9.3 ± 1.2	26 ± 3.5	26.3 ± 2.1
33	7.0 ± 4.0	56.3 ± 3.5	0 ± 0	15 ± 1	21 ± 1
100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	11.7 ± 3.1
333	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
1000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2750	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
5500	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2-AA (2.5)	240.3 ± 22.8	2082.3 ± 105	145 ± 8.2	1519.7 ± 182.8
2-AA (60.0)					71.3 ± 10

Experiment 2 (standard plate)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
DMSO	9 ± 2	83.3 ± 7.4	6 ± 1	20 ± 4.4	21.7 ± 2.5
0.1	9.7 ± 3.8	84 ± 17.1	7.3 ± 2.1	16.3 ± 3.2
0.3	7 ± 2.6	90 ± 5.3	5.7 ± 2.1	18 ± 2.6	21.3 ± 4.9
1	7.7 ± 0.6	73.7 ± 14	6.7 ± 3.2	15.3 ± 2.1	17.3 ± 2.5
3.3	9.7 ± 5.1	73.7 ± 7.2	5.3 ± 3.5	17.3 ± 1.5	19 ± 2.6
10	6 ± 1	34 ± 8	2.7 ± 0.6	9 ± 3.6	12.7 ± 2.5
33	0 ± 0	0 ± 0	0 ± 0	0 ± 0	11 ± 3.6
100					0 ± 0
MNNG (5)	4562.7 ± 89.6	3249 ± 145.7
AAC (100)			1444.3 ± 205.7
NOPD (10)				377.3 ± 24.8
4-NQO (5)					1109.7 ± 17.9
	Results with S9
DMSO	11 ± 1.7	105.3 ± 10	8 ± 2	23.7 ± 1.2	24 ± 4.4
0.1	8 ± 1.7	84.7 ± 3.2	7.7 ± 1.2	28.3 ± 7.6
0.3	8 ± 3	99 ± 7	5.7 ± 0.6	20.7 ± 2.1	18.7 ± 3.8
1	10.3 ± 1.5	95.3 ± 12.1	5.7 ± 1.2	20.3 ± 2.3	18 ± 6.6
3.3	7 ± 1	88 ± 11.3	6 ± 1	19 ± 3.6	17.3 ± 4.7
10	8.3 ± 4	86.7 ± 11.6	5.7 ± 0.6	8.7 ± 3.8	21.7 ± 3.1
33	4 ± 3.5	55 ± 7.5	0 ± 0	6 ± 2.6	15.7 ± 4
100					4.3 ± 0,6
2-AA (2.5)	241.3 ± 15.8	2033.3 ± 236.1	124.7 ± 15.5	1529.7 ± 326
2-AA (60.0)					90 ± 8.9

Experiment 3 (preincubation)

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA100	TA1537	TA98	WP2 uvrA
	Results without S9
DMSO	11 ± 1.7	81.7 ± 8.5	6.7 ± 3.1	13.7 ± 4.2	22 ± 7.2
0.03	12.7 ± 3.5	85 ± 4	5.7 ± 2.9	12 ± 4.4
0.1	13.7 ± 3.5	84.3 ± 8.1	5.7 ± 0.6	16.3 ± 2.3	22 ± 3.6
0.3	9 ± 3.5	75.3 ± 10.4	5.7 ± 1.2	22 ± 6.6	16 ± 2.6
1	10.3 ± 1.5	84.3 ± 5	8 ± 3	14.7 ± 2.1	19.3 ± 2.9
3.3	9.7 ± 4	52 ± 3.5	3.3 ± 1.5	11 ± 3.6	20.7 ± 3.8
10	7.3 ± 1.5	20.3 ± 5.5	0 ± 0	10.7 ± 1.2	19.7 ± 1.2
33					12 ± 5.3
MNNG (5)	2228 ± 67.8	2206.7 ± 195.8
AAC (100)			688.3 ± 232.8
NOPD (10)				416.7 ± 13.5
4-NQO (5)					447 ± 3
	Results with S9
DMSO	5.7 ± 2.1	89.3 ± 6.5	11 ± 4.6	20 ± 2	25 ± 8.7
0.03	10 ± 4	80.7 ± 7.6	9.3 ± 3.2	21.3 ± 11
0.1	11.7 ± 4.5	80.7 ± 12.1	6 ± 2	25 ± 3.6	20 ± 5.3
0.3	8.7 ± 2.9	19.7 ± 10.5	9.3 ± 0.6	22 ± 7.9	23.7 ± 7.2
1	13.3 ± 4.2	76.3 ± 9.2	10.3 ± 6.5	28.7 ± 1.2	20 ± 0
3.3	10.3 ± 2.5	13.7 ± 13	6 ± 1	23 ± 5	14.3 ± 2.1
10	5.7 ± 2.3	82.3 ± 10.1	6.3 ± 2.3	16.7 ± 3.1	18.3 ± 4
33					14.3 ± 3.2
2-AA (2.5)	168.3 ± 28.2	1467.3 ± 26.3	107.7 ± 15.5	1242.3 ± 11.6
2-AA (60.0)					60 ± 9.5