Bis (3‐(diethylamino)‐7‐hydroxy‐5‐phenylphenazinium) sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: Key study. Test method according to OECD 471, GLP study. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 500 µg/plate. Based on the available data, the test item is not mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2019 - 26 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his D (S. typhimurium TA 98); his C (S. typhimurium TA 1537); his G (S. typhimurium TA 100 and TA1535); tryp E (E. coli WP2 uvrA pKM101)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: ΔuvrB and rfa mutated

Remarks:

(TA 98 and TA 100: pKM 101)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA, pKM 101 mutated

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 fraction prepared from Sprague Dawley rat liver homogenate and provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA).
- method of preparation of S9 mix : 10% S9 fraction, 8 mM MgCL2-6H2O, 33 mM KCl, 5 mM Glucose-6-Phosphate Na2, 4 mM NADP Na2 and 0.1 M Phosphate buffer pH 7.4.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL of S9-mix.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test: 500 μL of S9-mix were added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates were incubated for 48 - 72 hours at 37°C and then examined.

Test concentrations with justification for top dose:

Initial mutation test (plate incorporation) / Confirmatory mutation test (pre-incubation +S9): 5, 15, 50, 150, 250 and 500 µg/plate.
In the preliminary cytotoxicity test (strain TA100) a very high toxicity was found for doses from 700 to 5000 μg/plate. Therefore, the test item was tested at the highest dose of 500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: the test item was found soluble in water at 5 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO), acetone, NaCl 0.15M

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (1, 2 μg/plate; S. typhimurium strains, + S9), cis-Platinum (II) Diamine Dichloride (1 μg/plate; E.coli, - S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
1. Plate incorporation (initial mutation test): A stock solution of the test item was prepared at 5 mg/mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9 E09 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled at 45ºC containing 10% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains, or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain. In the assay with metabolic activation, the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.
2. Pre-incubation (confirmatory mutation test +S9): The test item solution with the test strain, and 500 μL of S9-mix fraction are preincubated with shaking for 30 min., at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.. After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes (confirmatory mutation test)
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 E03 bacteria /mL) and 0.1 mL of the stock solution and dilutions were successively added to 2 mL of top agar at 45ºC, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone was run in parallel. In case of bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
In the bacterial reverse mutation test, mutations are detected which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
After a 48-72-hour incubation period at 37ºC, revertant colonies were manually counted in each plate. The following ratio was calculated per plate: R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item.

- OTHER:
- Sterility test: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.

Rationale for test conditions:

Results of sterility controls show the absence of any bacterial growth in the presence of test item S9-mix. Results of the bacteriostatic activity control show no toxicity. Values and frequency are within the laboratory's historical control ranges.

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight to high thinning of the bacterial lawn for doses of 150 to 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight to high thinning of the bacterial lawn for doses of 150 to 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight thinning or thinning of the bacterial lawn for doses of 250 and 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS : None observed.

RANGE-FINDING/SCREENING STUDIES: In the preliminary cytotoxicity test (strain TA100) a very high toxicity was found for doses from 700 to 5000 μg/plate. Therefore, the test item was tested at the highest dose of 500 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: see tables below.

Ames test:
- Signs of toxicity: see table 5 below.
- Individual plate counts: see table 5 below.
- Mean number of revertant colonies per plate and standard deviation: see table 5 below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 6 below
- Negative (solvent/vehicle) historical control data: see table 6 below
- There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory.

Table 3. Sterility control.

Series	Doses	Colony number/plate
Control n° 1		1	2	3
	500 µg /plate	0	0	0
Solution of	250 µg /plate	0	0	0
VIOLET LCC6D dried BATCH: 3/19	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
(LEMI code : 19/0180-160919-S1)	15 µg /plate	0	0	0
	5 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 2	Doses	1	2	3
	500 µg /plate	0	0	0
Solution of	250 µg /plate	0	0	0
VIOLET LCC6D dried BATCH: 3/19	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
(LEMI code : 19/0180-230919-S1)	15 µg /plate	0	0	0
	5 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

Table 4. Bacteriostatic activity controls.

		0 (negative control)	Water	150µg	250µg	350µg	500µg	700µg	1000µg	2500µg	5000µg
Solution of VIOLET LCC6D dried BATCH: 3/19   LEMI code : 19/0180-110919-S1	N1	776	759	432	370	184	186	2	0	0	0
	N2	674	725	382	329	181	156	9	0	0	0
	N3	758	711	438	368	194	177	8	0	0	0
	N	736±54	732±25	417±31	356±23	186±7	173±15	6±4	0±0	0±0	0±0
	%	-	99%	57%	48%	25%	24%	1%	0%	0%	0%

		0 (negative control)	Water	50 µg	150 µg	250 µg	500 µg	700 µg	1000 µg
Solution of VIOLET LCC6D dried BATCH: 3/19   LEMI code : 19/0180-130919-S1	N1	623	633	631	396	240	180	0	0
	N2	667	641	645	198	256	138	0	0
	N3	616	652	634	139	314	191	0	0
	N	635±28	642±10	637±7	244± 135	270±39	170± 28	0±0	0±0
	%	-	101%	100%	38%	42%	27%	0%	0%

		0 (negative control)	Water	5 µg	15 µg	50 µg	150 µg	250 µg	500 µg
Solution of VIOLET LCC6D dried BATCH: 3/19   LEMI code : 19/0180-160919-S1	N1	669	657	751	678	583	314	280	181
	N2	718	671	683	712	588	293	258	133
	N3	704	709	711	659	592	339	256	201
	N	697±25	679±27	715 ± 34	683±27	588±5	315 ± 23	265±13	172±35
	%	-	97%	103%	98%	84%	45%	38%	25%

		0 (negative control)	Water	5 µg	15 µg	50 µg	150 µg	250 µg	500 µg
Solution of VIOLET LCC6D dried BATCH: 3/19   LEMI code : 19/0180-230919-S1	N1	671	636	629	613	526	402	301	195
	N2	687	644	675	671	584	356	259	123
	N3	699	686	681	643	601	333	236	215
	N	686±14	655±27	662±28	642±29	570±39	364 ± 35	265±33	178±48
	%	-	96%	96%	94%	83%	53%	39%	26%

Table 5. Result tables.

TA1535 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	13	11	13	12.33	1.15	_
Positive control solvent	5 µL	12	12	12	12.00	0.00	_
Positive control : Sodium azide	5 µg in 5 µL	1023	987	1058	1022.67	35.50	85.22
Vehicle	100 µL	9	10	10	9.67	0.58	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg***	2	4	1	2.33	1.53	0.24
	250 µg**	8	7	15	10.00	4.36	1.03
	150 µg*	16	10	14	13.33	3.06	1.38
	50 µg	18	12	15	15.00	3.00	1.55
	15 µg	12	14	18	14.67	3.06	1.52
	5 µg	17	12	18	15.67	3.21	1.62

TA1535 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	11	16	13.00	2.65	_
Positive control solvent	20 µL	17	15	17	16.33	1.15	_
Positive control : 2-Anthramine	2 µg in 20 µL	87	85	95	89.00	5.29	5.45
Vehicle	100 µL	13	10	15	12.67	2.52	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg***	4	5	3	4.00	1.00	0.32
	250 µg*	7	23	3	11.00	10.58	0.87
	150µg	10	17	13	13.33	3.51	1.05
	50 µg	18	14	7	13.00	5.57	1.03
	15 µg	18	10	5	11.00	6.56	0.87
	5 µg	10	18	17	15.00	4.36	1.18

TA1535 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	23	11	14	16.00	6.24	_
Positive control solvent	5 µL	15	12	18	15.00	3.00	_
Positive control : Sodium azide	5 µg in 5 µL	1175	1147	1245	1189.00	50.48	79.27
Vehicle	100 µL	15	12	11	12.67	2.08	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg***	1	8	3	4.00	3.61	0.32
	250 µg**	8	3	12	7.67	4.51	0.61
	150 µg*	11	8	17	12.00	4.58	0.95
	50 µg	8	13	8	9.67	2.89	0.76
	15 µg	4	16	13	11.00	6.24	0.87
	5 µg	8	15	14	12.33	3.79	0.97

TA1535 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	21	18	11	16.67	5.13	_
Positive control solvent	10 µL	19	13	8	13.33	5.51	_
Positive control : 2-Anthramine	1 µg in 10 µL	77	80	99	85.33	11.93	6.40
Vehicle	100 µL	16	13	19	16.00	3.00	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg***	6	9	3	6.00	3.00	0.38
	250 µg*	15	14	12	13.67	1.53	0.85
	150 µg	13	17	13	14.33	2.31	0.90
	50 µg	10	9	19	12.67	5.51	0.79
	15 µg	17	16	10	14.33	3.79	0.90
	5 µg	10	19	10	13.00	5.20	0.81

TA1537 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	16	4	8.67	6.43	_
Positive control solvent	20 µL	8	5	5	6.00	1.73	_
Positivecontrol: 9-Aminoacridine	50 µg in 20 µL	1856	1958	1482	1765.33	250.62	294.22
Vehicle	100 µL	12	2	8	7.33	5.03	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	3	4	5	4.00	1.00	0.55
	250 µg*	12	8	10	10.00	2.00	1.36
	150 µg	12	15	8	11.67	3.51	1.59
	50 µg	9	10	11	10.00	1.00	1.36
	15 µg	7	3	8	6.00	2.65	0.82
	5 µg	8	6	7	7.00	1.00	0.95

TA1537 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	16	6	8	10.00	5.29	_
Positive control solvent	20 µL	10	11	9	10.00	1.00	_
Positive control : 2-Anthramine	2 µg in 20 µL	26	38	39	34.33	7.23	3.43
Vehicle	100 µL	12	7	11	10.00	2.65	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	4	4	5	4.33	0.58	0.43
	250 µg*	6	9	9	8.00	1.73	0.80
	150µg	12	9	11	10.67	1.53	1.07
	50 µg	15	9	19	14.33	5.03	1.43
	15 µg	9	15	19	14.33	5.03	1.43
	5 µg	6	10	12	9.33	3.06	0.93

TA1537 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	11	15	15.00	4.00	_
Positive control solvent	20 µL	8	8	14	10.00	3.46	_
Positivecontrol: 9-Aminoacridine	50 µg in 20 µL	1058	1042	1312	1137.33	151.48	113.73
Vehicle	100 µL	18	16	12	15.33	3.06	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	4	1	6	3.67	2.52	0.24
	250 µg*	7	10	7	8.00	1.73	0.52
	150 µg	13	8	15	12.00	3.61	0.78
	50 µg	7	11	11	9.67	2.31	0.63
	15 µg	15	7	15	12.33	4.62	0.80
	5 µg	10	15	10	11.67	2.89	0.76

TA1537 Assay n°2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	20	15	18.00	2.65	_
Positive control solvent	10 µL	14	8	13	11.67	3.21	_
Positive control : 2-Anthramine	1 µg in 10 µL	45	32	49	42.00	8.89	3.60
Vehicle	100 µL	14	21	18	17.67	3.51	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	6	6	3	5.00	1.73	0.28
	250 µg*	17	15	19	17.00	2.00	0.96
	150 µg	13	12	11	12.00	1.00	0.68
	50 µg	15	12	20	15.67	4.04	0.89
	15 µg	20	16	18	18.00	2.00	1.02
	5 µg	17	13	11	13.67	3.06	0.77

TA98 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	12	17	18	15.67	3.21	_
Positive control solvent	20 µL	17	15	14	15.33	1.53	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	383	402	379	388.00	12.29	25.30
Vehicle	100 µL	17	21	15	17.67	3.06	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	10	9	16	11.67	3.79	0.66
	250 µg*	21	25	38	28.00	8.89	1.58
	150 µg	38	34	30	34.00	4.00	1.92
	50 µg	26	26	29	27.00	1.73	1.53
	15 µg	23	23	15	20.33	4.62	1.15
	5 µg	16	18	20	18.00	2.00	1.02

TA98 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	17	28	19	21.33	5.86	_
Positive control solvent	20 µL	28	15	19	20.67	6.66	_
Positive control : 2-Anthramine	2 µg in 20 µL	342	436	416	398.00	49.52	19.26
Vehicle	100 µL	15	24	19	19.33	4.51	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	24	28	16	22.67	6.11	1.17
	250 µg*	27	37	27	30.33	5.77	1.57
	150 µg	12	24	33	23.00	10.54	1.19
	50 µg	28	28	22	26.00	3.46	1.34
	15 µg	30	29	24	27.67	3.21	1.43
	5 µg	30	32	23	28.33	4.73	1.47

TA98 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	24	16	11	17.00	6.56	_
Positive control solvent	20 µL	18	18	11	15.67	4.04	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	388	338	410	378.67	36.90	24.17
Vehicle	100 µL	18	18	26	20.67	4.62	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	15	13	17	15.00	2.00	0.73
	250 µg*	38	31	26	31.67	6.03	1.53
	150 µg	31	27	22	26.67	4.51	1.29
	50 µg	29	23	18	23.33	5.51	1.13
	15 µg	21	14	13	16.00	4.36	0.77
	5 µg	15	10	14	13.00	2.65	0.63

TA98 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	23	33	28	28.00	5.00	_
Positive control solvent	10 µL	28	17	18	21.00	6.08	_
Positive control : 2-Anthramine	1 µg in 10 µL	237	264	272	257.67	18.34	12.27
Vehicle	100 µL	31	31	29	30.33	1.15	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	32	26	35	31.00	4.58	1.02
	250 µg*	24	17	33	24.67	8.02	0.81
	150 µg	26	27	24	25.67	1.53	0.85
	50 µg	20	25	23	22.67	2.52	0.75
	15 µg	21	25	26	24.00	2.65	0.79
	5 µg	34	19	20	24.33	8.39	0.80

TA100 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	82	93	70	81.67	11.50	_
Positive control solvent	20 µL	91	82	85	86.00	4.58	_
Positive control : Sodium azide	20 µg in 20 µL	1590	1460	1149	1399.67	226.61	16.28
Vehicle	100 µL	80	79	90	83.00	6.08	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg***	5	5	3	4.33	1.15	0.05
	250 µg**	99	94	89	94.00	5.00	1.13
	150 µg*	100	106	97	101.00	4.58	1.22
	50 µg	90	95	98	94.33	4.04	1.14
	15 µg	84	101	106	97.00	11.53	1.17
	5 µg	60	75	77	70.67	9.29	0.85

TA100 Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	108	113	125	115.33	8.74	_
Positive control solvent	20 µL	70	114	94	92.67	22.03	_
Positive control : 2-Anthramine	2 µg in 20 µL	1047	696	792	845.00	181.40	9.12
Vehicle	100 µL	79	118	95	97.33	19.60	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg***	6	3	2	3.67	2.08	0.04
	250 µg*	132	150	123	135.00	13.75	1.39
	150 µg	124	118	95	112.33	15.31	1.15
	50 µg	110	107	94	103.67	8.50	1.07
	15 µg	98	103	99	100.00	2.65	1.03
	5 µg	79	94	104	92.33	12.58	0.95

TA100 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	105	53	63	73.67	27.59	_
Positive control solvent	20 µL	98	84	76	86.00	11.14	_
Positive control : Sodium azide	20 µg in 20 µL	1510	1413	1577	1500.00	82.46	17.44
Vehicle	100 µL	61	78	62	67.00	9.54	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg***	9	7	6	7.33	1.53	0.11
	250 µg**	95	95	74	88.00	12.12	1.31
	150 µg*	107	83	94	94.67	12.01	1.41
	50 µg	91	80	97	89.33	8.62	1.33
	15 µg	95	85	79	86.33	8.08	1.29
	5 µg	98	90	84	90.67	7.02	1.35

TA100 Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	104	93	79	92.00	12.53	_
Positive control solvent	10 µL	109	98	111	106.00	7.00	_
Positive control : 2-Anthramine	1 µg in 10 µL	383	420	552	451.67	88.84	4.26
Vehicle	100 µL	100	102	108	103.33	4.16	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg***	9	10	2	7.00	4.36	0.07
	250 µg*	69	99	58	75.33	21.22	0.73
	150 µg	76	84	90	83.33	7.02	0.81
	50 µg	92	93	85	90.00	4.36	0.87
	15 µg	105	95	111	103.67	8.08	1.00
	5 µg	112	103	90	101.67	11.06	0.98

E. COLI Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	87	111	82	93.33	15.50	_
Positive control solvent	10 µL	120	89	115	108.00	16.64	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	244	328	356	309.33	58.29	2.86
Vehicle	100 µL	97	127	121	115.00	15.87	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	115	101	128	114.67	13.50	1.00
	250 µg*	121	162	153	145.33	21.55	1.26
	150 µg	190	183	182	185.00	4.36	1.61
	50 µg	173	164	175	170.67	5.86	1.48
	15 µg	179	123	159	153.67	28.38	1.34
	5 µg	147	161	153	153.67	7.02	1.34

E. COLI Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	262	241	216	239.67	23.03	_
Positive control solvent	5 µL	229	231	214	224.67	9.29	_
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	471	510	499	493.33	20.11	2.20
Vehicle	100 µL	253	227	242	240.67	13.05	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-160919-S1	500 µg**	235	242	252	243.00	8.54	1.01
	250 µg*	218	239	263	240.00	22.52	1.00
	150 µg	227	200	214	213.67	13.50	0.89
	50 µg	225	211	225	220.33	8.08	0.92
	15 µg	236	194	236	222.00	24.25	0.92
	5 µg	258	236	253	249.00	11.53	1.03

E. COLI Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	132	133	130	131.67	1.53	_
Positive control solvent	10 µL	140	132	129	133.67	5.69	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	410	350	423	394.33	38.94	2.95
Vehicle	100 µL	121	115	126	120.67	5.51	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	88	105	131	108.00	21.66	0.90
	250 µg*	161	159	163	161.00	2.00	1.33
	150 µg	142	138	170	150.00	17.44	1.24
	50 µg	153	171	135	153.00	18.00	1.27
	15 µg	174	140	181	165.00	21.93	1.37
	5 µg	135	162	158	151.67	14.57	1.26

E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	183	188	198	189.67	7.64	_
Positive control solvent	5 µL	210	190	175	191.67	17.56	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	485	516	537	512.67	26.16	2.67
Vehicle	100 µL	207	236	221	221.33	14.50	_
Solution of VIOLET LCC6D dried BATCH: 3/19 LEMI code : 19/0180-230919-S1	500 µg**	211	192	195	199.33	10.21	0.90
	250 µg*	180	194	224	199.33	22.48	0.90
	150 µg	206	193	209	202.67	8.50	0.92
	50 µg	212	196	197	201.67	8.96	0.91
	15 µg	208	207	218	211.00	6.08	0.95
	5 µg	216	213	204	211.00	6.24	0.95

* slight thinning of the bacterial lawn

** thinning of the bacterial lawn

*** high thinning of the bacterial lawn

Conclusions:

The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 500 µg/plate. Based on the available data, the test item is not mutagenic.

Executive summary:

A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471 under GLP conditions. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA were exposed to concentrations of the test substance ranging from 5 to 500 µg/plate in water, with and without metabolic activation, according to preliminary assays. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate (provided by MOLTOX). Two independent assays were performed: an initial mutation test (plate incorporation method) and a confirmatory mutation test (plate incorporation method without S9 and pre-incubation method with S9) were carried out. Untreated, solvent controls and strain specific positive controls were included in the assays and were within the historical control range in all strains. All validity criteria were fulfilled. The test item did not induce any significant increase in the number of revertants in any of the strains tested, with and without metabolic activation, up to 500 µg/plate. Based on the available data, the test item is not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data (negative Ames test), the test substance is not classified for mutagenecity in accordance with CLP Regulation (EC) No. 1272/2008.