3-Quinolinecarboxylic acid, 7-[6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-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 up to 5000 µg/plate.

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 - 01 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: 1500, 500, 150, 50 and 15 μg/plate
With metabolic activation: 5, 1.5, 0.5, 0.15 and 0.05 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 1500 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 the absence or a thinning of the background lawn of non -revertant bacteria, in all strains. In absence of metabolic activation, the toxicity was also observed by the absence or a thinning of the background lawn of non -revertant bacteria, in all strains for the highest doses tested from 3000 to 5000 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, 4.2 and 4.3 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	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
T-A12 BATCH: I20196A	150 µg /plate	0	0	0
(LEMI code :20/0285-210920-S1)
	50 µg /plate	0	0	0
without metabolic activation
	15 µg /plate	0	0	0
Solution of	5 µg /plate	0	0	0
	1.5 µg /plate	0	0	0
T-A12 BATCH: I20196A	0.5 µg /plate	0	0	0
(LEMI code :20/0285-290920-S2)
	0.15 µg /plate	0	0	0
with metabolic activation
	0.05 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 3		1	2	3
Solution of	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
T-A12 BATCH: I20196A	150 µg /plate	0	0	0
(LEMI code :20/0285-280920-S1)
	50 µg /plate	0	0	0
without metabolic activation
	15 µg /plate	0	0	0
Solution of	5 µg /plate	0	0	0
	1.5 µg /plate	0	0	0
T-A12 BATCH: I20196A	0.5 µg /plate	0	0	0
(LEMI code :20/0285-280920-S2)
	0.15 µg /plate	0	0	0
with metabolic activation
	0.05 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

Table 4.1. Bacteriostatic activity controls. TA100. Preliminary assay

		Doses (/plate)
		0 (negative control)	DMSO			50 µg	150 µg	500 µg			1 500 µg	2500 µg			5 000 µg (p)
	N1	701	765			710	690	520			130	135			53
Solution of	N2	677	577			752	723	510			175	189			70
T-A12 BATCH: I20196A	N3	754	718			742	718	474			192	201			60
	N	711±39	687±98			735±22	710±18	501±24			166±32	175±35			61±9
LEMI code : 20/0285-030820-S1	%	-		97%		103%	100%		71%		23%		25%			9%

		Doses (/plate)
		0 (negative control)	DMSO	500 µg			1000 µg			2000 µg			3000 µg	5 000 µg (p)
	N1	679	735	510			250			190			185	67
Solution of	N2	698	726	520			221			222			102	71
T-A12 BATCH: I20196A	N3	714	687	444			265			175			163	81
	N	697±18	716±26	491±41			245±22			196±24			150±43	73±7
LEMI code : 20/0285-170820-S1	%	-	103%		70%			35%			28%		22%		10%

(p) presence of precipitates

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
5000µg/plate(p)	0	0	0	5	2-8
3000 µg/plate	3	3	1	0	2-4
1500 µg/plate	1*	6*	0	2*	32*
500 µg/plate	20	14	2	4	41
Spontaneous revertant colonies	68-60	14 -23	2-7	18-8	43-46

	STRAINS with metabolic activation
Doses	TA100	TA98	TA1537	TA1535	E. Coli
500 µg/plate	0	0	0	0	0
250 µg/plate	0	0	0	0	0
150 µg/plate	0	0	0	0	0
50 µg/plate	0	0	0	0	0
25 µg/plate	0	7*** - 9***	1***-0***	0	11***-6***
10 µg/plate	0	6**-3**	x	x	6**-7**
5 µg/plate	3*-0*	8*-1*	1*-0*	1*-0*	15*-16*
Spontaneous revertant colonies	73	30	11	17	65

(p) presence of precipitates

*** high thinning of the bacterial lawn

** thinning of the bacterial lawn

*light thinning of the bacterial lawn

Table 4.3. Bacteriostatic activity controls. Additional assays. TA100

		Doses (/plate)
		0 (negative control)	DMSO			15 µg			50 µg	150 µg			500 µg	1500 µg
	N1	760	717			705			734	761			526	198
Solution of	N2	699	701			711			731	697			544	200
T-A12 BATCH: I20196A	N3	687	703			689			682	599			510	166
	N	715±39	707±9			702±11			716±29	686±82			527±17	188±19
LEMI code : 20/0285-210920-S1	%	-		99%			98%		100%		96%		74%		26%

		Doses (/plate)
		0 (negative control)	DMSO			15 µg			50 µg			150 µg			500 µg	1500 µg
	N1	761	687			638			644			588			475	162
Solution of	N2	774	716			574			655			584			423	174
T-A12 BATCH: I20196A	N3	714	728			686			618			593			463	178
	N	750±32	710±21			633±56			639±19			588±5			454±27	171±8
LEMI code : 20/0285-280920-S1	%	-		95%			84%			85%			78%		61%		23%

Table 5. Result tables.

TA1535 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	5	16	9	10.00	5.57	_
Positive control solvent	5 µL	13	11	11	11.67	1.15	_
Positive control: Sodium azide	5 µg in 5 µL	653	699	670	674.00	23.26	57.77
Vehicle	50 µL	8	5	5	6.00	1.73	_
	1500 µg*	1	5	1	2.33	2.31	0.39
Solution of	500 µg*	7	5	3	5.00	2.00	0.83
T-A12 BATCH: I20196A	150 µg	10	15	9	11.33	3.21	1.89
	50 µg	8	5	12	8.33	3.51	1.39
LEMI code :20/0285-210920-S1	15 µg	9	9	4	7.33	2.89	1.22

TA1535 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	13	11	13	12.33	1.15	_
Positive control solvent	20 µL	8	12	13	11.00	2.65	_
Positive control: 2-Anthramine	2 µg in 20 µL	197	194	188	193.00	4.58	17.55
Vehicle	50 µL	7	4	15	8.67	5.69	_
	5 µg**	4	3	4	3.67	0.58	0.42
Solution of	1.5 µg*	5	6	8	6.33	1.53	0.73
T-A12 BATCH: I20196A	0.5 µg	9	7	12	9.33	2.52	1.08
	0.15µg	13	9	5	9.00	4.00	1.04
LEMI code :20/0285-210920-S2	0.05 µg	11	10	13	11.33	1.53	1.31

TA1535 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	5	10	7.00	2.65	_
Positive control solvent	5 µL	5	11	11	9.00	3.46	_
Positive control: Sodium azide	5 µg in 5 µL	834	793	810	812.33	20.60	90.26
Vehicle	50 µL	8	9	5	7.33	2.08	_
	1500 µg*	3	2	1	2.00	1.00	0.27
Solution of	500 µg*	8	6	3	5.67	2.52	0.77
T-A12 BATCH: I20196A	150 µg	4	14	7	8.33	5.13	1.14
	50 µg	11	9	8	9.33	1.53	1.27
LEMI code :20/0285-280920-S1	15 µg	8	9	7	8.00	1.00	1.09

TA1535 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	14	7	7	9.33	4.04	_
Positive control solvent	10 µL	12	7	8	9.00	2.65	_
Positive control: 2-Anthramine	1 µg in 10 µL	64	48	40	50.67	12.22	5.63
Vehicle	50 µL	9	7	9	8.33	1.15	_
	5 µg***	1	2	1	1.33	0.58	0.16
Solution of	1.5 µg**	4	5	1	3.33	2.08	0.40
T-A12 BATCH: I20196A	0.5 µg*	10	9	8	9.00	1.00	1.08
	0.15µg	9	4	5	6.00	2.65	0.72
LEMI code :20/0285-280920-S2	0.05 µg	12	7	9	9.33	2.52	1.12

TA1537 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	11	12	7	10.00	2.65	_
Positive control solvent	20 µL	14	12	13	13.00	1.00	_
Positivecontrol: 9-Aminoacridine	50 µg in 20 µL	1569	904	1495	1322.67	364.46	101.74
Vehicle	50 µL	11	7	8	8.67	2.08	_
	1500 µg*	2	1	2	1.67	0.58	0.19
Solution of	500 µg	5	8	5	6.00	1.73	0.69
T-A12 BATCH: I20196A	150 µg	9	7	6	7.33	1.53	0.85
	50 µg	8	7	12	9.00	2.65	1.04
LEMI code :20/0285-210920-S1	15 µg	10	9	12	10.33	1.53	1.19

TA1537 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	8	6	18	10.67	6.43	_
Positive control solvent	20 µL	15	8	7	10.00	4.36	_
Positive control: 2-Anthramine	2 µg in 20 µL	73	71	60	68.00	7.00	6.80
Vehicle	50 µL	13	14	15	14.00	1.00	_
	5 µg**	3	4	4	3.67	0.58	0.26
Solution of	1.5 µg*	11	3	4	6.00	4.36	0.43
T-A12 BATCH: I20196A	0.5 µg	6	9	8	7.67	1.53	0.55
	0.15µg	8	13	11	10.67	2.52	0.76
LEMI code :20/0285-210920-S2	0.05 µg	7	9	11	9.00	2.00	0.64

TA1537 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	5	9	6	6.67	2.08	_
Positive control solvent	20 µL	2	9	6	5.67	3.51	_
Positivecontrol: 9-Aminoacridine	50 µg in 20 µL	918	1461	834	1071.00	340.35	189.00
Vehicle	50 µL	9	2	4	5.00	3.61	_
	1500 µg*	2	1	1	1.33	0.58	0.27
Solution of	500 µg	5	2	3	3.33	1.53	0.67
T-A12 BATCH: I20196A	150 µg	3	2	2	2.33	0.58	0.47
	50 µg	5	4	8	5.67	2.08	1.13
LEMI code :20/0285-280920-S1	15 µg	7	5	7	6.33	1.15	1.27

TA1537 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	12	9	9.33	2.52	_
Positive control solvent	10 µL	4	4	3	3.67	0.58	_
Positive control : 2-Anthramine	1 µg in 10 µL	62	59	51	57.33	5.69	15.64
Vehicle	50 µL	4	3	7	4.67	2.08	_
	5 µg***	4	2	4	3.33	1.15	0.71
Solution of	1.5 µg**	6	2	2	3.33	2.31	0.71
T-A12 BATCH: I20196A	0.5 µg*	7	4	9	6.67	2.52	1.43
	0.15 µg	4	5	7	5.33	1.53	1.14
LEMI code :20/0285-280920-S2	0.05 µg	5	4	3	4.00	1.00	0.86

TA98 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	17	20	20	19.00	1.73	_
Positive control solvent	20 µL	24	22	26	24.00	2.00	_
Positivecontrol: 2-Nitrofluorene	2 µg in 20 µL	258	381	281	306.67	65.39	12.78
Vehicle	50 µL	19	20	23	20.67	2.08	_
	1500 µg*	9	13	16	12.67	3.51	0.61
Solution of	500 µg	14	16	22	17.33	4.16	0.84
T-A12 BATCH: I20196A	150 µg	18	15	18	17.00	1.73	0.82
	50 µg	21	24	16	20.33	4.04	0.98
LEMI code :20/0285-210920-S1	15 µg	22	20	20	20.67	1.15	1.00

TA98 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	27	35	38	33.33	5.69	_
Positive control solvent	20 µL	35	31	37	34.33	3.06	_
Positive control : 2-Anthramine	2 µg in 20 µL	566	526	704	598.67	93.39	17.44
Vehicle	50 µL	37	31	36	34.67	3.21	_
	5 µg*	7	8	3	6.00	2.65	0.17
Solution of	1,5 µg	10	18	13	13.67	4.04	0.39
T-A12 BATCH: I20196A	0.5 µg	30	28	40	32.67	6.43	0.94
	0,15 µg	33	41	34	36.00	4.36	1.04
LEMI code :20/0285-210920-S2	0.05 µg	47	28	29	34.67	10.69	1.00

TA98 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	6	8	7	7.00	1.00	_
Positive control solvent	20 µL	9	6	9	8.00	1.73	_
Positivecontrol: 2-Nitrofluorene	2 µg in 20 µL	254	252	315	273.67	35.81	34.21
Vehicle	50 µL	9	9	6	8.00	1.73	_
	1500 µg*	2	14	7	7.67	6.03	0.96
Solution of	500 µg	5	8	11	8.00	3.00	1.00
T-A12 BATCH: I20196A	150 µg	10	11	9	10.00	1.00	1.25
	50 µg	8	10	10	9.33	1.15	1.17
LEMI code :20/0285-280920-S1	15 µg	10	11	8	9.67	1.53	1.21

TA98 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	14	10	15	13.00	2.65	_
Positive control solvent	10 µL	10	12	10	10.67	1.15	_
Positive control: 2-Anthramine	1 µg in 10 µL	255	257	219	243.67	21.39	22.84
Vehicle	50 µL	14	14	11	13.00	1.73	_
	5 µg**	14	18	6	12.67	6.11	0.97
Solution of	1,5 µg*	24	16	4	14.67	10.07	1.13
T-A12 BATCH: I20196A	0,5 µg	19	14	11	14.67	4.04	1.13
	0,15 µg	14	19	13	15.33	3.21	1.18
LEMI code :20/0285-280920-S2	0.05 µg	12	12	12	12.00	0.00	0.92

TA100 Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	66	71	65	67.33	3.21	_
Positive control solvent	20 µL	70	59	60	63.00	6.08	_
Positive control: Sodium azide	20 µg in 20 µL	1246	1125	1233	1201.33	66.43	19.07
Vehicle	50 µL	65	61	62	62.67	2.08	_
	1500 µg**	6	2	4	4.00	2.00	0.06
Solution of	500 µg*	18	27	18	21.00	5.20	0.34
T-A12 BATCH: I20196A	150 µg*	67	52	70	63.00	9.64	1.01
	50 µg	58	71	67	65.33	6.66	1.04
LEMI code :20/0285-210920-S1	15 µg	67	59	56	60.67	5.69	0.97

TA100 Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	85	70	72	75.67	8.14	_
Positive control solvent	20 µL	70	75	69	71.33	3.21	_
Positive control: 2-Anthramine	2 µg in 20 µL	629	1051	954	878.00	221.03	12.31
Vehicle	50 µL	80	77	73	76.67	3.51	_
	5 µg**	4	6	7	5.67	1.53	0.07
Solution of	1.5 µg**	17	12	12	13.67	2.89	0.18
T-A12 BATCH: I20196A	0.5 µg*	70	79	90	79.67	10.02	1.04
	0.15 µg	83	83	66	77.33	9.81	1.01
LEMI code :20/0285-210920-S2	0.05 µg	70	64	62	65.33	4.16	0.85

TA100 Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	52	70	59	60.33	9.07	_
Positive control solvent	20 µL	56	62	62	60.00	3.46	_
Positive control: Sodium azide	20 µg in 20 µL	1029	1284	1024	1112.33	148.69	18.54
Vehicle	50 µL	46	51	48	48.33	2.52	_
	1500 µg**	2	5	6	4.33	2.08	0.09
Solution of	500 µg*	8	12	15	11.67	3.51	0.24
T-A12 BATCH: I20196A	150 µg*	79	56	36	57.00	21.52	1.18
	50 µg	56	75	65	65.33	9.50	1.35
LEMI code :20/0285-280920-S1	15 µg	80	69	72	73.67	5.69	1.52

TA100 Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	72	71	63	68.67	4.93	_
Positive control solvent	10 µL	86	88	73	82.33	8.14	_
Positive control: 2-Anthramine	1 µg in 10 µL	297	370	301	322.67	41.04	3.92
Vehicle	50 µL	72	63	84	73.00	10.54	_
	5 µg***	4	6	5	5.00	1.00	0.07
Solution of	1.5 µg**	10	18	13	13.67	4.04	0.19
T-A12 BATCH: I20196A	0.5 µg*	55	53	49	52.33	3.06	0.72
	0.15 µg	79	79	74	77.33	2.89	1.06
LEMI code :20/0285-280920-S2	0.05 µg	66	86	69	73.67	10.79	1.01

E. COLI Assay n°2 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	68	115	95	92.67	23.59	_
Positive control solvent	10 µL	84	100	104	96.00	10.58	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	409	511	399	439.67	61.98	4.58
Vehicle	50 µL	112	87	87	95.33	14.43	_
	1500 µg*	26	25	17	22.67	4.93	0.24
Solution of	500 µg	70	75	51	65.33	12.66	0.69
T-A12 BATCH: I20196A	150 µg	76	81	71	76.00	5.00	0.80
	50 µg	93	85	84	87.33	4.93	0.92
LEMI code :20/0285-210920-S1	15 µg	62	70	75	69.00	6.56	0.72

E. COLI Assay n°2 – with metabolic activation (10 % S9-mix) – without pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	68	96	95	86.33	15.89	_
Positive control solvent	5 µL	94	98	98	96.67	2.31	_
Positive control: Dimethylbenzanthracene	5 µg in 5 µL	608	599	566	591.00	22.11	6.11
Vehicle	50 µL	93	115	84	97.33	15.95	_
	5 µg*	29	46	27	34.00	10.44	0.35
Solution of	1,5 µg	75	91	53	73.00	19.08	0.75
T-A12 BATCH: I20196A	0.5 µg	100	102	110	104.00	5.29	1.07
	0.15 µg	97	98	105	100.00	4.36	1.03
LEMI code :20/0285-210920-S2	0.05 µg	97	105	73	91.67	16.65	0.94

E. COLI Assay n°3 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	60	56	53	56.33	3.51	_
Positive control solvent	10 µL	59	58	54	57.00	2.65	_
Positivecontrol: cis-Platinum(II)	1 µg in 10 µL	424	389	412	408.33	17.79	7.16
Vehicle	50 µL	44	59	68	57.00	12.12	_
	1500 µg*	30	23	29	27.33	3.79	0.48
Solution of	500 µg	57	63	51	57.00	6.00	1.00
T-A12 BATCH: I20196A	150 µg	58	61	71	63.33	6.81	1.11
	50 µg	47	55	57	53.00	5.29	0.93
LEMI code :20/0285-280920-S1	15 µg	45	41	42	42.67	2.08	0.75

E. COLI Assay n°3 – with metabolic activation (10 % S9-mix) – with pre-incubation
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	69	74	91	78.00	11.53	_
Positive control solvent	5 µL	77	77	70	74.67	4.04	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	389	415	437	413.67	24.03	5.54
Vehicle	50 µL	75	79	69	74.33	5.03	_
	5 µg**	21	23	20	21.33	1.53	0.29
Solution of	1,5 µg*	73	79	89	80.33	8.08	1.08
T-A12 BATCH: I20196A	0,5 µg	88	77	114	93.00	19.00	1.25
	0.15 µg	81	70	74	75.00	5.57	1.01
LEMI code :20/0285-280920-S2	0.05 µg	75	69	88	77.33	9.71	1.04

*** high thinning of the bacterial lawn

** thinning of the bacterial lawn

*light thinning of the bacterial lawn

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 1500 to 5000 μg/plate. The doses in the mutagenic assay were selected according to these results. However, in this first mutagenic assay (assay nº1) no revertant colonies were counted for all the doses studied in presence of metabolic activation and toxicity 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: 1500, 500, 150, 50 and 15 μg/plate without S9 and 5, 1.5, 0.5, 0.15 and 0.05 μg/plate 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.