3-Quinolinecarboxylic acid, 7-chloro-1-cyclopropyl-1,4-dihydro-8-methyl-4-oxo-, ethyl ester

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 mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation.

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:

03 August 2020 - 15 Octubre 2020

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:

Without metabolic activation: 25, 7.5, 2.5, 0.75 and 0.25 μg/plate.
With metabolic activation: 1, 0.3, 0.1, 0.03 and 0.01 μg/plate.
With metabolic activation and with pre-incubation: 0.3, 0.1, 0.03, 0.01, and 0.003 μg/plate.

Top doses based on several preliminary cytotoxicity assays (see "Addtional information on results")

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was found soluble in DMSO at the highest tested concentration.

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 100 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: 48-72 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 and S9-mix. Concentrations were selected based on the results of the bacteriostatic activity controls. 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

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

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

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

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

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 high toxicity was found for doses from 250 to 5000 μg/plate. The doses in the mutagenic assay were selected according to these results. However, in this first mutagenic assay (data not shown) no revertant colonies were counted for all the doses studied in presence of metabolic activation. Evidence of toxicity was demonstrated by an absence or a high thinning of the background lawn of non -revertant bacteria, in all strains. In absence of metabolic activation, the toxicity was also observed by an absence or thinning of the background lawn of non -revertant bacteria, in all strains for the highest doses tested from 25 to 250 μg/plate. These results were not consistent with the first cytotoxicity study on TA100. In order to justify acceptable range of doses for the test additional assays were carried out under the same conditions than a mutagenicity test without metabolic activation. Reference substances were not used in this assay. Results of these assays are presented in the tables 4.1 and 4.2 below. According to these results, doses were selected for the definitive mutagenic assay (assays 2 and 3 in the table 5 below).

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.

Serie	Doses	Colony number/plate
Control n° 2		1	2	3
Solution of   T-A07 BATCH: M20165C (Solution code :20/0287-051020-S2)without metabolic activation	25 µg /plate	0	0	0
	7.5 µg /plate	0	0	0
	2.5 µg /plate	0	0	0
	0.75 µg /plate	0	0	0
	0.25 µg /plate	0	0	0
Solution of   T-A07 BATCH: M20165C (Solution code :20/0287-051020-S3)with metabolic activation	1 µg /plate	0	0	0
	0.3 µg /plate	0	0	0
	0.1 µg /plate	0	0	0
	0.03 µg /plate	0	0	0
	0.01 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 3		1	2	3
Solution of   T-A07 BATCH: M20165C (Solution code :20/0287-121020-S2)without metabolic activation	25 µg /plate	0	0	0
	7.5 µg /plate	0	0	0
	2.5 µg /plate	0	0	0
	0.75 µg /plate	0	0	0
	0.25 µg /plate	0	0	0
Solution of   T-A07 BATCH: M20165C (Solution code :20/0287-121020-S3)with metabolic activation	0.3 µg /plate	0	0	0
	0.1 µg /plate	0	0	0
	0.03 µg /plate	0	0	0
	0.01 µg /plate	0	0	0
	0.003 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

Table 4.1. Bacteriostatic activity controls. TA100

		Doses (/plate)
		0 (negativecontrol)	DMSO			50 µg	150 µg	500 µg		1 500 µg			2500 µg			5 000 µg
	N1	701	705			708	15	0		0			0			0
Solution of	N2	677	577			741	51	0		0			0			0
T-A07 BATCH: M20165C	N3	754	718			695	48	0		0			0			0
	N	711 ± 39	667±78			715±24	38±20	0±0		0±0			0±0			0±0
Solution code: 20/0287-030820-S1	%	-		94%		101%	5%	0%			0%			0%			0%

		Doses (/plate)
		0 (negative control)	DMSO	1000 µg			500 µg			250 µg			100 µg	50 µg
	N1	679	735	0			0			196			388	412
Solution of	N2	698	726	0			0			193			251	412
T-A07 BATCH: M20165C	N3	714	687	0			0			291			224	321
	N	697±18	716±26	0±0			0±0			227±56			288±88	382±53
Solution code: 20/0287-170820-S1	%	-	103%		0%			0%			33%		41%		55%

		Doses (/plate)
		0 (negative control)	DMSO		250 µg			70 µg			25 µg			7 µg	2.5 µg
	N1	730	740		114			254			552			745	741
Solution of	N2	724	736		128			239			547			712	689
T-A07 BATCH: M20165C	N3	711	738		137			245			536			736	758
	N	722±10	738±2		126±12			246±8			545±8			731±17	729±36
Solution code: 20/0287-240820-S1	%	-	102%			18%			34%			76%		101%	101%

		Doses (/plate)
		0 (negative control)	DMSO	25 µg			7.5 µg	2.5 µg			0.75 µg	0.25 µg
	N1	791	745	410			789	765			785	756
Solution of	N2	766	741	325			796	742			774	759
T-A07 BATCH: M20165C	N3	779	863	289			777	758			746	755
	N	779±13	783±69	341±62			787±10	755±12			768±20	757±2
Solution code: 20/0287-051020-S1	%	-	101%		44%		101%		97%		99%		97%

		Doses (/plate)
		0 (negativecontrol)	DMSO	25 µg			7.5 µg	2.5 µg	0.75 µg		0.25 µg
	N1	699	723	305			671	722	698		711
Solution of	N2	705	711	382			725	723	702		721
T-A07 BATCH: M20165C	N3	717	695	310			753	701	714		712
	N	707±9	710±14	332±43			716±42	715±12	705±8		715±6
Solution code: 20/0287-121020-S1	%	-	100%		47%		101%	101%	100%		101%

N1 Number of colonies in plate 1

N2 Number of colonies in plate 2

N3 Number of colonies in plate 3

N Mean per plate

% Percent of survival compared to negative control

Table 4.2. Bacteriostatic activity controls. Additional assays. Revertant colonies number

	STRAINS without metabolic activation
Doses	TA100	TA98	TA1537	TA1535	E. Coli
Spontaneous revertant colonies	68-60	14-23	2-7	18-8	43-46
250 µg/plate**	0	0	0	0	2
70 µg/plate*	0	3	0	2	38
25 µg/plate*	48	15	4	8	42
7 µg/plate	58	25	9	11	60
2.5 µg/plate	75	20	10	10	68

	STRAINS with metabolic activation
Doses	TA100	TA98	TA1537	TA1535	E. Coli
Spontaneous revertant colonies	73	30	11	17	69
10 µg/plate	0	0	0	0	0
5 µg/plate	0	0	0	0	0
2.5 µg/plate	0	0	0	0	0
1.25 µg/plate	0	0	0	0	0
0.625 µg/plate	0	0	0	0	39-34
0.3125 µg/plate	0**	9-13	0	3-0**	41-45*

	STRAINS with metabolic activation
Doses	TA100	TA98	TA1537	TA1535	E. Coli
Spontaneous revertant colonies	85-70-72	38-35-30	11-8-20	18-14-17	68-96-95
1 µg/plate***	0	0	0	0	11-20-18
0.3 µg/plate	2-5-4**	13-20-16**	5-4-2**	4-3-3**	46-39-32*
0.1 µg/plate	69-76-60*	36-44-33	11-6-11	15-19-11	97-117-117
0.03 µg/plate	105-104-77	34-31-38	11-20-11	14-21-12	94-97-93
0.01 µg/plate	87-92-107	30-33-28	16-9-12	19-13-17	80-104-93

*** high thinning of the bacterial lawn

** thinning of the bacterial lawn

*light thinning of the bacterial lawn

Table 5. Result tables.

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	14	10	14	12.67	2.31	_
Positive control solvent	5 µL	6	10	9	8.33	2.08	_
Positive control : Sodium azide	5 µg in 5 µL	723	696	789	736.00	47.84	88.32
Vehicle	50 µL	19	12	8	13.00	5.57	_
	25 µg*	7	5	6	6.00	1.00	0.46
Solution of	7.5 µg	16	7	11	11.33	4.51	0.87
T-A07 BATCH: M20165C	2.5 µg	17	2	13	10.67	7.77	0.82
	0.75 µg	15	16	8	13.00	4.36	1.00
Solution code:20/0287-051020-S2	0.25 µg	12	12	9	11.00	1.73	0.85

TA1535 Assay n°2 – 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	19	8	14.33	5.69	_
Positive control solvent	20 µL	18	14	17	16.33	2.08	_
Positive control : 2-Anthramine	2 µg in 20 µL	167	178	182	175.67	7.77	10.76
Vehicle	50 µL	3	14	16	11.00	7.00	_
	1 µg**	4	1	0	1.67	2.08	0.15
Solution of	0.3 µg*	1	5	4	3.33	2.08	0.30
T-A07 BATCH: M20165C	0.1 µg	14	8	10	10.67	3.06	0.97
	0.03 µg	10	12	13	11.67	1.53	1.06
Solution code:20/0287-051020-S3	0.01 µg	12	17	18	15.67	3.21	1.42

TA1535 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	13	10	9.67	3.51	_
Positive control solvent	5 µL	14	7	15	12.00	4.36	_
Positive control : Sodium azide	5 µg in 5 µL	767	569	685	673.67	99.49	56.14
Vehicle	50 µL	9	15	10	11.33	3.21	_
	25 µg*	4	5	5	4.67	0.58	0.41
Solution of	7.5µg	7	8	5	6.67	1.53	0.59
T-A07 BATCH: M20165C	2.5 µg	8	7	7	7.33	0.58	0.65
	0.75 µg	13	12	13	12.67	0.58	1.12
Solution code:20/0287-121020-S2	0.25 µg	10	12	15	12.33	2.52	1.09

TA1535 Assay n°3 – 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	11	22	12	15.00	6.08	_
Positive control solvent	10 µL	11	16	13	13.33	2.52	_
Positive control : 2-Anthramine	1 µg in 10 µL	57	54	68	59.67	7.37	4.48
Vehicle	50 µL	9	9	13	10.33	2.31	_
	0.3 µg**	1	1	3	1.67	1.15	0.16
Solution of	0.1µg*	5	8	6	6.33	1.53	0.61
T-A07 BATCH: M20165C	0.03µg	12	13	12	12.33	0.58	1.19
	0.01 µg	9	12	16	12.33	3.51	1.19
Solution code:20/0287-121020-S3	0.003 µg	11	7	8	8.67	2.08	0.84

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	6	4	10	6.67	3.06	_
Positive control solvent	20 µL	7	9	4	6.67	2.52	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	902	977	1143	1007.33	123.33	151.10
Vehicle	50 µL	2	8	4	4.67	3.06	_
	25 µg*	8	1	2	3.67	3.79	0.79
Solution of	7.5 µg	7	5	7	6.33	1.15	1.36
T-A07 BATCH: M20165C	2.5 µg	11	10	9	10.00	1.00	2.14
	0.75 µg	7	6	6	6.33	0.58	1.36
Solution code:20/0287-051020-S2	0.25 µg	4	5	3	4.00	1.00	0.86

TA1537 Assay n°2 – 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	5	6	7	6.00	1.00	_
Positive control solvent	20 µL	12	6	11	9.67	3.21	_
Positive control : 2-Anthramine	2 µg in 20 µL	49	51	46	48.67	2.52	5.03
Vehicle	50 µL	4	11	6	7.00	3.61	_
	1 µg***	1	0	1	0.67	0.58	0.10
Solution of	0.3 µg**	3	3	8	4.67	2.89	0.67
T-A07 BATCH: M20165C	0.1 µg	16	17	6	13.00	6.08	1.86
	0.03 µg	9	12	13	11.33	2.08	1.62
Solution code:20/0287-051020-S3	0.01 µg	20	10	12	14.00	5.29	2.00

TA1537 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	5	2	3	3.33	1.53	_
Positive control solvent	20 µL	2	6	8	5.33	3.06	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	625	794	529	649.33	134.17	121.75
Vehicle	50 µL	6	4	3	4.33	1.53	_
	25 µg*	2	4	3	3.00	1.00	0.69
Solution of	7.5µg	3	7	3	4.33	2.31	1.00
T-A07 BATCH: M20165C	2.5 µg	5	2	3	3.33	1.53	0.77
	0.75 µg	6	4	7	5.67	1.53	1.31
Solution code:20/0287-121020-S2	0.25 µg	5	6	9	6.67	2.08	1.54

TA1537 Assay n°3 – 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	4	7	5	5.33	1.53	_
Positive control solvent	10 µL	7	4	4	5.00	1.73	_
Positive control : 2-Anthramine	1 µg in 10 µL	41	50	54	48.33	6.66	9.67
Vehicle	50 µL	8	7	8	7.67	0.58	_
	0.3 µg***	3	2	2	2.33	0.58	0.30
Solution of	0.1 µg**	5	6	4	5.00	1.00	0.65
T-A07 BATCH: M20165C	0.03 µg	6	8	7	7.00	1.00	0.91
	0.01 µg	6	10	7	7.67	2.08	1.00
Solution code:20/0287-121020-S3	0.003 µg	5	5	11	7.00	3.46	0.91

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	19	10	23	17.33	6.66	_
Positive control solvent	20 µL	17	18	18	17.67	0.58	_
Positivecontrol: 2-Nitrofluorene	2 µg in 20 µL	286	338	226	283.33	56.05	16.04
Vehicle	50 µL	18	23	16	19.00	3.61	_
	25 µg*	17	24	26	22.33	4.73	1.18
Solution of	7.5 µg	22	29	17	22.67	6.03	1.19
T-A07 BATCH: M20165C	2.5 µg	16	21	18	18.33	2.52	0.96
	0.75 µg	16	27	20	21.00	5.57	1.11
Solution code:20/0287-051020-S2	0.25 µg	26	18	23	22.33	4.04	1.18

TA98 Assay n°2 – 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	31	27	32	30.00	2.65	_
Positive control solvent	20 µL	38	30	30	32.67	4.62	_
Positive control : 2-Anthramine	2 µg in 20 µL	678	703	594	658.33	57.10	20.15
Vehicle	50 µL	27	29	14	23.33	8.14	_
	1 µg**	3	4	3	3.33	0.58	0.14
Solution of	0.3 µg*	4	13	19	12.00	7.55	0.51
T-A07 BATCH: M20165C	0.1 µg	29	22	22	24.33	4.04	1.04
	0.03 µg	34	27	19	26.67	7.51	1.14
Solution code:20/0287-051020-S3	0.01 µg	22	31	17	23.33	7.09	1.00

TA98 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	19	20	20	19.67	0.58	_
Positive control solvent	20 µL	26	23	27	25.33	2.08	_
Positivecontrol: 2-Nitrofluorene	2 µg in 20 µL	486	401	373	420.00	58.85	16.58
Vehicle	50 µL	27	21	24	24.00	3.00	_
	25 µg*	9	12	9	10.00	1.73	0.42
Solution of	7.5µg	20	13	19	17.33	3.79	0.72
T-A07 BATCH: M20165C	2.5 µg	23	22	11	18.67	6.66	0.78
	0.75 µg	19	14	19	17.33	2.89	0.72
LEMI code :20/0287-121020-S2	0.25 µg	10	13	14	12.33	2.08	0.51

TA98 Assay n°3 – 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	31	30	33	31.33	1.53	_
Positive control solvent	10 µL	32	34	33	33.00	1.00	_
Positive control : 2-Anthramine	1 µg in 10 µL	302	257	289	282.67	23.16	8.57
Vehicle	50 µL	34	31	36	33.67	2.52	_
	0.3 µg**	11	17	13	13.67	3.06	0.41
Solution of	0.1 µg*	33	32	26	30.33	3.79	0.90
T-A07 BATCH: M20165C	0.03 µg	22	31	35	29.33	6.66	0.87
	0.01 µg	28	28	25	27.00	1.73	0.80
Solution code:20/0287-121020-S3	0.003 µg	30	26	24	26.67	3.06	0.79

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	90	84	84	86.00	3.46	_
Positive control solvent	20 µL	79	87	91	85.67	6.11	_
Positive control : Sodium azide	20 µg in 20 µL	1121	1322	1277	1240.00	105.48	14.47
Vehicle	50 µL	66	83	66	71.67	9.81	_
	25 µg*	34	27	24	28.33	5.13	0.40
Solution of	7.5 µg	59	56	82	65.67	14.22	0.92
T-A07 BATCH: M20165C	2.5 µg	73	60	77	70.00	8.89	0.98
	0.75 µg	71	85	61	72.33	12.06	1.01
Solution code:20/0287-051020-S2	0.25 µg	69	83	62	71.33	10.69	1.00

TA100 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	71	61	72	68.00	6.08	_
Positive control solvent	20 µL	67	78	65	70.00	7.00	_
Positive control : Sodium azide	20 µg in 20 µL	1120	1023	1084	1075.67	49.03	15.37
Vehicle	50 µL	71	71	62	68.00	5.20	_
	25 µg*	23	31	17	23.67	7.02	0.35
Solution of	7.5µg	72	62	74	69.33	6.43	1.02
T-A07 BATCH: M20165C	2.5 µg	73	81	66	73.33	7.51	1.08
	0.75 µg	57	66	58	60.33	4.93	0.89
Solution code:20/0287-121020-S2	0.25 µg	69	59	65	64.33	5.03	0.95

TA100 Assay n°3 – 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	86	69	84	79.67	9.29	_
Positive control solvent	10 µL	82	79	84	81.67	2.52	_
Positive control : 2-Anthramine	1 µg in 10 µL	512	502	525	513.00	11.53	6.28
Vehicle	50 µL	73	83	88	81.33	7.64	_
	0.3 µg***	15	21	18	18.00	3.00	0.22
Solution of	0.1 µg**	50	64	58	57.33	7.02	0.70
T-A07 BATCH: M20165C	0.03 µg	85	75	73	77.67	6.43	0.95
	0.01 µg	80	80	69	76.33	6.35	0.94
Solution code:20/0287-121020-S3	0.003 µg	79	77	58	71.33	11.59	0.88

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	61	67	59	62.33	4.16	_
Positive control solvent	10 µL	58	62	60	60.00	2.00	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	297	309	312	306.00	7.94	5.10
Vehicle	50 µL	65	60	59	61.33	3.21	_
	25 µg*	45	40	46	43.67	3.21	0.71
Solution of	7.5 µg	52	45	43	46.67	4.73	0.76
T-A07 BATCH: M20165C	2.5 µg	64	84	34	60.67	25.17	0.99
	0.75 µg	49	51	47	49.00	2.00	0.80
Solution code:20/0287-051020-S2	0.25 µg	61	52	68	60.33	8.02	0.98

E Coli Assay n°2 – 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	93	106	101	100.00	6.56	_
Positive control solvent	5 µL	98	101	91	96.67	5.13	_
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	401	372	421	398.00	24.64	4.12
Vehicle	50 µL	84	107	102	97.67	12.10	_
	1 µg*	13	9	15	12.33	3.06	0.13
Solution of	0.3 µg*	34	40	32	35.33	4.16	0.36
T-A07 BATCH: M20165C	0.1 µg	104	110	106	106.67	3.06	1.09
	0.03 µg	80	88	96	88.00	8.00	0.90
Solution code:20/0287-051020-S3	0.01 µg	81	70	87	79.33	8.62	0.81

E. Coli Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standarddeviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	67	59	63	63.00	4.00	_
Positive control solvent	10 µL	80	81	81	80.67	0.58	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	310	327	341	326.00	15.52	4.04
Vehicle	50 µL	68	57	81	68.67	12.01	_
	25 µg*	35	39	43	39.00	4.00	0.57
Solution of	7.5µg	36	47	48	43.67	6.66	0.64
T-A07 BATCH: M20165C	2.5 µg	65	80	77	74.00	7.94	1.08
	0.75 µg	51	48	60	53.00	6.24	0.77
Solution code:20/0287-121020-S2	0.25 µg	62	55	61	59.33	3.79	0.86

E Coli Assay n°3 – 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	99	99	81	93.00	10.39	_
Positive control solvent	5 µL	106	93	81	93.33	12.50	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	295	301	326	307.33	16.44	3.29
Vehicle	50 µL	81	77	85	81.00	4.00	_
	0.3 µg*	17	23	25	21.67	4.16	0.27
Solution of	0.1 µg	97	102	90	96.33	6.03	1.19
T-A07 BATCH: M20165C	0.03 µg	89	91	100	93.33	5.86	1.15
	0.01 µg	78	89	87	84.67	5.86	1.05
Solution code:20/0287-121020-S3	0.003 µg	101	90	85	92.00	8.19	1.14

Conclusions:

The test item did not induce any mutagenic change in the bacterial reverse mutation test in any of the strains tested with and without metabolic activation.

Executive summary:

A bacterial reverse mutation test was conducted on the test substance according to OECD guideline 471 under GLP conditions. The study was performed in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the absence and presence of metabolic activation. The test substance was found soluble in DMSO which was used as vehicle. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate. In the preliminary cytotoxicity test (strain TA100) a high toxicity was found for doses from 250 to 5000 μg/plate. The doses in the mutagenic assay were selected according to these results. However, in this first mutagenic assay (assay nº1) high toxicity was found which was not consistent with the results of the first cytotoxicity study on TA100. Therefore, additional cytotoxicity assays in all strains were carried out in order to justify acceptable range of doses. According to the results obtained, the second (plate incorporation method with and without S9) and third (plate incorporation method without S9 and pre-incubation method with S9) mutagenic assays were performed with the following doses: 0.25 to 25 µg/plate without S9, 0.01 to 1 µg/plate (plate incorporation method) and 0.003 to 0.3 µg/plate (pre-incubation) with S9. Untreated, solvent controls and strain specific positive controls were included in the assays and the values obtained were within ranges of the historical control values of the laboratory 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. Based on these results, the test item can be considered as not mutagenic according to the OECD TG 471.

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.