Boronic acid, [6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2020 - 28 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Boronic acid, [6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-

Cas Number:

446299-81-6

Molecular formula:

C14H15BN2O3

IUPAC Name:

Boronic acid, [6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-

Test material form:

solid: particulate/powder

Method

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

Species / strain 1

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 2

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): 50, 150, 500, 1500 and 5000 µg/plate.
In the preliminary cytotoxicity test (strain TA100) no toxicity was found for doses up to 5000 μg/plate. Therefore, the test item was tested at the recommended maximum test concentration of 5000 μg/plate.

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.

Controls

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. 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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) no toxicity was found for doses up to 5000 μg/plate. Therefore, the test item was tested at the recommended maximum test concentration of 5000 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.

Any other information on results incl. tables

Table 3. Sterility control.

Serie	Doses	Colony number/plate
Control n° 1		1	2	3
Solution of   T-A11 BATCH: l19319E   (LEMI code :20/0151-190520-S1)	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0
Control n° 2		1	2	3
Solution of   T-A11 BATCH: l19319E   (LEMI code :20/0151-250520-S1)	5000 µg /plate	0	0	0
	1500 µg /plate	0	0	0
	500 µg /plate	0	0	0
	150 µg /plate	0	0	0
	50 µg /plate	0	0	0
S9-mix	500 µL/plate	0	0	0

Table 4. Bacteriostatic activity controls.

		Doses (/plate)
		0 (negative control)			DMSO			50 µg			150 µg			500 µg			1 500 µg	2 500 µg			5 000 µg
	N1		738			819			754			784			783		797		800			802
Solution of	N2		871			766			798			751			811		769		767			788
T-A11 BATCH: l19319E	N3		798			699			737			796			780		768		779			753
	N	802	±	67	761	±	60	763	±	31	777	±	23	791	±	17	778±16	782	±	17	781	±	25
LEMI code : 20/0151-270420-S1	%		-			95%			95%			97%			99%		97%		97%			97%

		Doses (/plate)
		0 (negative control)			DMSO			50 µg			150 µg			500 µg			1 500 µg	5 000 µg
	N1		794			812			757			742			801		730		815
Solution of	N2		779			769			778			803			818		766		803
T-A11 BATCH: l19319E	N3		801			795			809			761			794		805		746
	N	791	±	11	792	±	22	781	±	26	769	±	31	804	±	12	767±38	788	±	37
LEMI code : 20/0151-190520-S1	%		-			100%			99%			97%			102%		97%		100%

Table 5. Result tables.

TA1535 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	9	9	10	9.33	0.58	_
Positive control solvent	5 µL	16	14	5	11.67	5.86	_
Positive control : Sodium azide	5 µg in 5 µL	1005	929	1065	999.67	68.16	85.69
Vehicle	50µL	18	10	9	12.33	4.93	_
	5000 µg	11	9	7	9.00	2.00	0.73
Solution of	1500 µg	10	10	11	10.33	0.58	0.84
T-A11 BATCH: l19319E	500 µg	9	20	8	12.33	6.66	1.00
	150 µg	11	22	10	14.33	6.66	1.16
LEMI code : 20/0151-190520-S1	50 µg	18	11	16	15.00	3.61	1.22

TA1535 Assay n°1 – 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	16	12	14	14.00	2.00	_
Positive control solvent	20 µL	18	23	9	16.67	7.09	_
Positive control : 2-Anthramine	2 µg in 20 µL	206	140	133	159.67	40.28	9.58
Vehicle	50µL	17	18	8	14.33	5.51	_
	5000 µg	7	7	14	9.33	4.04	0.65
Solution of	1500 µg	13	13	12	12.67	0.58	0.88
T-A11 BATCH: l19319E	500 µg	16	10	15	13.67	3.21	0.95
	150 µg	14	16	16	15.33	1.15	1.07
LEMI code : 20/0151-190520-S1	50 µg	13	13	13	13.00	0.00	0.91

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	12	16	9	12,33	3,51	_
Positive control solvent	5 µL	14	11	10	11,67	2,08	_
Positive control : Sodium azide	5 µg in 5 µL	1009	980	1020	1003,00	20,66	85,97
Vehicle	50µL	9	14	11	11,33	2,52	_
	5000 µg	12	9	11	10,67	1,53	0,94
Solution of	1500 µg	10	14	10	11,33	2,31	1,00
T-A11 BATCH: l19319E	500 µg	13	12	10	11,67	1,53	1,03
	150 µg	10	18	16	14,67	4,16	1,29
LEMI code : 20/0151-250520-S1	50 µg	7	16	13	12,00	4,58	1,06

TA1535 Assay n°2 – 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	15	19	22	18,67	3,51	_
Positive control solvent	10 µL	16	13	15	14,67	1,53	_
Positive control : 2-Anthramine	1 µg in 10 µL	98	80	68	82,00	15,10	5,59
Vehicle	50µL	13	12	16	13,67	2,08	_
	5000 µg	11	9	10	10,00	1,00	0,73
Solution of	1500 µg	16	11	20	15,67	4,51	1,15
T-A11 BATCH: l19319E	500 µg	15	13	14	14,00	1,00	1,02
	150 µg	17	12	18	15,67	3,21	1,15
LEMI code : 20/0151-250520-S1	50 µg	9	20	19	16,00	6,08	1,17

TA1537 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	7	6	6	6.33	0.58	_
Positive control solvent	20 µL	5	7	5	5.67	1.15	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	858	1586	1250	1231.33	364.36	217.29
Vehicle	50µL	6	7	11	8.00	2.65	_
	5000 µg	3	2	2	2.33	0.58	0.29
Solution of	1500 µg	7	5	4	5.33	1.53	0.67
T-A11 BATCH: l19319E	500 µg	8	5	5	6.00	1.73	0.75
	150 µg	6	4	6	5.33	1.15	0.67
LEMI code : 20/0151-190520-S1	50 µg	4	4	10	6.00	3.46	0.75

TA1537 Assay n°1 – 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	10	8	8.67	1.15	_
Positive control solvent	20 µL	8	6	8	7.33	1.15	_
Positive control : 2-Anthramine	2 µg in 20 µL	56	48	73	59.00	12.77	8.05
Vehicle	50µL	8	7	7	7.33	0.58	_
	5000 µg	5	3	8	5.33	2.52	0.73
Solution of	1500 µg	10	4	5	6.33	3.21	0.86
T-A11 BATCH: l19319E	500 µg	3	6	6	5.00	1.73	0.68
	150 µg	5	5	6	5.33	0.58	0.73
LEMI code : 20/0151-190520-S1	50 µg	13	13	8	11.33	2.89	1.55

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	6	7	5	6.00	1.00	_
Positive control solvent	20 µL	8	6	6	6.67	1.15	_
Positive control : 9-Aminoacridine	50 µg in 20 µL	862	1280	1480	1207.33	315.34	181.10
Vehicle	50µL	7	5	9	7.00	2.00	_
	5000 µg	3	2	2	2.33	0.58	0.33
Solution of	1500 µg	4	6	5	5.00	1.00	0.71
T-A11 BATCH: l19319E	500 µg	5	4	7	5.33	1.53	0.76
	150 µg	6	7	5	6.00	1.00	0.86
LEMI code : 20/0151-250520-S1	50 µg	5	3	6	4.67	1.53	0.67

TA1537 Assay n°2 – 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	8	7	9	8.00	1.00	_
Positive control solvent	10 µL	8	8	7	7.67	0.58	_
Positive control : 2-Anthramine	1 µg in 10 µL	26	32	27	28.33	3.21	3.70
Vehicle	50µL	7	6	9	7.33	1.53	_
	5000 µg	3	5	4	4.00	1.00	0.55
Solution of	1500 µg	4	9	4	5.67	2.89	0.77
T-A11 BATCH: l19319E	500 µg	12	9	9	10.00	1.73	1.36
	150 µg	11	8	7	8.67	2.08	1.18
LEMI code : 20/0151-250520-S1	50 µg	12	7	8	9.00	2.65	1.23

TA98 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	20	16	14	16.67	3.06	_
Positive control solvent	20 µL	19	12	16	15.67	3.51	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	210	218	225	217.67	7.51	13.89
Vehicle	50µL	19	16	18	17.67	1.53	_
	5000 µg	17	16	21	18.00	2.65	1.02
Solution of	1500 µg	17	18	16	17.00	1.00	0.96
T-A11 BATCH: l19319E	500 µg	16	21	19	18.67	2.52	1.06
	150 µg	16	13	21	16.67	4.04	0.94
LEMI code : 20/0151-190520-S1	50 µg	21	18	19	19.33	1.53	1.09

TA98 Assay n°1 – 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	23	25	25	24.33	1.15	_
Positive control solvent	20 µL	26	22	21	23.00	2.65	_
Positive control : 2-Anthramine	2 µg in 20 µL	437	400	505	447.33	53.26	19.45
Vehicle	50µL	22	24	27	24.33	2.52	_
	5000 µg	21	25	26	24.00	2.65	0.99
Solution of	1500 µg	24	30	19	24.33	5.51	1.00
T-A11 BATCH: l19319E	500 µg	19	34	25	26.00	7.55	1.07
	150 µg	25	26	24	25.00	1.00	1.03
LEMI code : 20/0151-190520-S1	50 µg	23	30	27	26.67	3.51	1.10

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	16	16	13	15.00	1.73	_
Positive control solvent	20 µL	19	15	16	16.67	2.08	_
Positive control : 2-Nitrofluorene	2 µg in 20 µL	329	279	244	284.00	42.72	17.04
Vehicle	50µL	19	17	13	16.33	3.06	_
	5000 µg	15	22	17	18.00	3.61	1.10
Solution of	1500 µg	12	17	21	16.67	4.51	1.02
T-A11 BATCH: l19319E	500 µg	22	20	16	19.33	3.06	1.18
	150 µg	24	21	17	20.67	3.51	1.27
LEMI code : 20/0151-250520-S1	50 µg	22	19	29	23.33	5.13	1.43

TA98 Assay n°2 – 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	19	22	25	22.00	3.00	_
Positive control solvent	10 µL	25	21	20	22.00	2.65	_
Positive control : 2-Anthramine	1 µg in 10 µL	306	339	292	312.33	24.13	14.20
Vehicle	50µL	24	21	20	21.67	2.08	_
	5000 µg	26	30	25	27.00	2.65	1.25
Solution of	1500 µg	27	24	31	27.33	3.51	1.26
T-A11 BATCH: l19319E	500 µg	29	26	38	31.00	6.24	1.43
	150 µg	29	33	22	28.00	5.57	1.29
LEMI code : 20/0151-250520-S1	50 µg	33	21	26	26.67	6.03	1.23

TA100 Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	84	100	98	94.00	8.72	_
Positive control solvent	20 µL	89	98	91	92.67	4.73	_
Positive control : Sodium azide	20 µg in 20 µL	1299	1338	1170	1269.00	87.93	13.69
Vehicle	50µL	83	82	105	90.00	13.00	_
	5000 µg	72	76	70	72.67	3.06	0.81
Solution of	1500 µg	93	75	89	85.67	9.45	0.95
T-A11 BATCH: l19319E	500 µg	73	79	80	77.33	3.79	0.86
	150 µg	71	85	70	75.33	8.39	0.84
LEMI code : 20/0151-190520-S1	50 µg	82	80	76	79.33	3.06	0.88

TA100 Assay n°1 – 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	87	89	111	95.67	13.32	_
Positive control solvent	20 µL	91	109	89	96.33	11.02	_
Positive control : 2-Anthramine	2 µg in 20 µL	832	750	681	754.33	75.59	7.83
Vehicle	50µL	101	86	121	102.67	17.56	_
	5000 µg	108	105	115	109.33	5.13	1.06
Solution of	1500 µg	117	123	120	120.00	3.00	1.17
T-A11 BATCH: l19319E	500 µg	74	99	105	92.67	16.44	0.90
	150 µg	94	100	97	97.00	3.00	0.94
LEMI code : 20/0151-190520-S1	50 µg	92	90	99	93.67	4.73	0.91

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	81	77	70	76.00	5.57	_
Positive control solvent	20 µL	80	74	74	76.00	3.46	_
Positive control : Sodium azide	20 µg in 20 µL	1233	1226	1168	1209.00	35.68	15.91
Vehicle	50µL	72	68	76	72.00	4.00	_
	5000 µg	79	84	83	82.00	2.65	1.14
Solution of	1500 µg	72	71	81	74.67	5.51	1.04
T-A11 BATCH: l19319E	500 µg	95	85	88	89.33	5.13	1.24
	150 µg	81	63	82	75.33	10.69	1.05
LEMI code : 20/0151-250520-S1	50 µg	85	90	78	84.33	6.03	1.17

TA100 Assay n°2 – 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	93	90	80	87.67	6.81	_
Positive control solvent	10 µL	91	87	89	89.00	2.00	_
Positive control : 2-Anthramine	1 µg in 10 µL	571	557	473	533.67	53.00	6.00
Vehicle	50µL	73	74	87	78.00	7.81	_
	5000 µg	89	88	72	83.00	9.54	1.06
Solution of	1500 µg	82	97	105	94.67	11.68	1.21
T-A11 BATCH: l19319E	500 µg	96	110	89	98.33	10.69	1.26
	150 µg	93	82	86	87.00	5.57	1.12
LEMI code : 20/0151-250520-S1	50 µg	100	75	97	90.67	13.65	1.16

E. COLI Assay n°1 – without metabolic activation (-S9-mix)
Serie	Dose/Plate	Plate			Mean	Standard deviation	R
		n° 1	n° 2	n° 3
Negative control	100 µL	54	62	76	64.00	11.14	_
Positive control solvent	10 µL	48	65	49	54.00	9.54	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	258	310	440	336.00	93.74	6.22
Vehicle	50µL	60	44	57	53.67	8.50	_
	5000 µg	75	46	48	56.33	16.20	1.05
Solution of	1500 µg	51	53	44	49.33	4.73	0.92
T-A11 BATCH: l19319E	500 µg	52	43	55	50.00	6.24	0.93
	150 µg	58	67	56	60.33	5.86	1.12
LEMI code : 20/0151-190520-S1	50 µg	49	50	51	50.00	1.00	0.93

E. COLI Assay n°1 – 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	79	69	72	73.33	5.13	_
Positive control solvent	5 µL	80	68	79	75.67	6.66	_
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	483	575	524	527.33	46.09	6.97
Vehicle	50µL	76	76	72	74.67	2.31	_
	5000 µg	73	79	79	77.00	3.46	1.03
Solution of	1500 µg	72	63	74	69.67	5.86	0.93
T-A11 BATCH: l19319E	500 µg	77	76	85	79.33	4.93	1.06
	150 µg	78	69	68	71.67	5.51	0.96
LEMI code : 20/0151-190520-S1	50 µg	69	79	67	71.67	6.43	0.96

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	72	73	70	71,67	1,53	_
Positive control solvent	10 µL	74	67	62	67,67	6,03	_
Positive control : cis-Platinum (II)	1 µg in 10 µL	310	343	413	355,33	52,60	5,25
Vehicle	50µL	66	66	70	67,33	2,31	_
	5000 µg	70	83	61	71,33	11,06	1,06
Solution of	1500 µg	75	84	67	75,33	8,50	1,12
T-A11 BATCH: l19319E	500 µg	66	63	70	66,33	3,51	0,99
	150 µg	72	76	68	72,00	4,00	1,07
LEMI code : 20/0151-250520-S1	50 µg	71	78	69	72,67	4,73	1,08

E. COLI Assay n°2 – 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	106	109	97	104,00	6,24	_
Positive control solvent	5 µL	89	65	79	77,67	12,06	_
Positive control : Dimethylbenzanthracene	2.5 µg in 5 µL	450	481	505	478,67	27,57	6,16
Vehicle	50µL	81	67	75	74,33	7,02	_
	5000 µg	85	79	83	82,33	3,06	1,11
Solution of	1500 µg	81	74	86	80,33	6,03	1,08
T-A11 BATCH: l19319E	500 µg	92	85	87	88,00	3,61	1,18
	150 µg	73	88	91	84,00	9,64	1,13
LEMI code : 20/0151-250520-S1	50 µg	90	99	72	87,00	13,75	1,17

Applicant's summary and conclusion

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 up to 5000 µg/plate.

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 50 to 5000 µg/plate in DMSO, with and without metabolic activation, based on preliminary solubility and cytotoxicity tests. The metabolic activation system (S9 fraction) was prepared from Sprague Dawley rat liver homogenate. Two independent assays were performed in all strains: an initial mutation test (plate incorporation method) and a confirmatory mutation test (plate incorporation method without S9 and pre-incubation method 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, up to 5000 µg/plate. Based on these results, the test item can be considered as not mutagenic according to the OECD TG 471.