Benzamide, N-(5-bromo-3-methyl-2-pyridinyl)-N-methyl-

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:

From 08-08-2022 to 26-08-2022

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Preparation os the metabolic activation system:
- Obtention of S9: S9 fraction prepared from Sprague Dawley rat liver homogenate, provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 mOLTOX-11-101.5-447 VALIDATED ON 11.10.2021 - EXPIRY DATE: 13.07.2023).
- Preparation of S9 mix 10%: 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.
- preliminary cytotoxicity testing: Sterility test: test item and the corresponding dilutions were added to 2 mL of top agar at 45ºC, homogenized and poured on the agar (20 ml) onto a Petri plate in three plates. Plates were incubated for 48 - 72 hours at 37°C.

Test concentrations with justification for top dose:

Initial test concentrations: 50, 150, 500, 1500 and 5000 µg/plate.
No toxicity was found for doses up to 5000 μg/plate. That's why the test item was tested at concentration of 5000 μg/plate (maximum one).

Vehicle / solvent:

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

Details on test system and experimental conditions:

PRELIMINARY CYTOTOXICITY TEST:
In a test tube 0.1 mL of the bacterial suspension (1-9 X 10^3 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.

METHOD:
1. Without metabolic activation: Salmonella Typhimurium strains: 0.1 mL of the bacterial suspension containing 1-9 x10^9 bacteria/mL and 50µl 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 Escherichia coli strain containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.
With metabolic activation, 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: 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.

DATA PRESENTATION
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.

Evaluation criteria:

The following validity criteria were checked to validate each experiment:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75%,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- in presence of the solvent the spontaneous reversion rate shall comply with the historial values of the absolute negative control of the laboratory
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
- negative and positive values should not show significant difference with the historical values of the laboratory (± standard deviations).

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

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

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

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

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

Table 3. Sterility control.

 

Serie	Doses	Colony number/plate
Control n° 1		1	2	3
Solution of   T-A10 BATCH: M22154C   (Identification code :PH-22/0484)	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-A10 BATCH: M22154C   (Identification code :PH-22/0484)	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 control			50 µg			150 µg			500 µg			1 500 µg	5 000 µg
	N1		900			864			869			831			867		860		184
Solution of	N2		885			900			896			854			890		894		264
T-A10 BATCH: M22154C	N3		880			844			854			876			899		781		254
	N	888	±	10	869	±	28	873	±	21	854	±	23	885	±	17	845±58	234	±	44
	%		-			98%			98%			96%			100%		95%		26%

 

		Doses (/plate)
		0 (negative control)			DMSO			50 µg			150 µg			500 µg			1 500 µg	5 000 µg
	N1		684			701			702			698			701		645		210
Solution of	N2		676			689			698			685			626		621		168
T-A10 BATCH: M22154C	N3		699			677			652			674			691		632		174
	N	686	±	12	689	±	12	684	±	28	686	±	12	676	±	44	633±12	788	±	37
	%		-			100%			100%			100%			98%		92%		27%

 

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	7	13	9	9.67	3.06	_
Positive control solvent	5 µL	11	19	12	14.00	4.36	_
Positive control : Sodium azide	5 µg in 5 µL	462	513	577	517.33	57.62	36.95
Vehicle	50µL	11	12	15	12.67	2.08	_
	5000 µg	12	13	11	9.00	1.00	0.95
Solution of	1500 µg	13	15	16	10.33	1.53	1.16
T-A10 BATCH: M22154C	500 µg	17	12	12	12.33	2.89	1.08
	150 µg	12	20	15	14.33	4.04	1.24
	50 µg	17	9	8	11.33	4.93	0.89

 

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	9	12	14	11.67	2.52	_
Positive control solvent	20 µL	11	13	8	10.67	2.52	_
Positive control : 2-Anthramine	2 µg in 20 µL	164	125	112	133.67	27.06	12.53
Vehicle	50µL	7	10	10	9.00	1.73	_
	5000 µg	14	8	11	11.00	3.00	1.22
Solution of	1500 µg	11	18	12	13.67	3.79	1.52
T-A10 BATCH: M22154C	500 µg	13	10	16	13.00	3.00	1.44
	150 µg	12	15	20	15.67	4.04	1.74
	50 µg	9	20	14	14.33	5.51	1.59

 

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	15	11	12	12.67	2.08	_
Positive control solvent	5 µL	7	12	8	9.00	2.65	_
Positive control : Sodium azide	5 µg in 5 µL	807	960	855	874.00	78.25	97.11
Vehicle	50µL	6	9	10	8.33	2.08	_
	5000 µg	7	17	8	10.67	5,51	1.28
Solution of	1500 µg	17	15	11	14.33	3.06	1.72
T-A10 BATCH: M22154C	500 µg	9	6	10	8.33	1,53	1.00
	150 µg	7	10	8	8.33	4,16	1.00
	50 µg	9	9	13	10.33	4,58	1.24

 

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	9	11	8	9.33	1.53	_
Positive control solvent	10 µL	11	10	7	9.33	2.08	_
Positive control : 2-Anthramine	1 µg in 10 µL	108	83	73	88.00	18.03	9.43
Vehicle	50µL	11	6	12	9.67	3.21	_
	5000 µg	9	13	11	11.00	2.00	1.14
Solution of	1500 µg	14	16	12	14.00	2.00	1.45
T-A10 BATCH: M22154C	500 µg	17	14	19	16.67	2.52	1.72
	150 µg	10	17	18	15.00	4.36	1.55
	50 µg	17	12	13	14.00	2.65	1.45

 

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	6	7	6	6.33	0.58	_
Positive control solvent	20 µL	10	4	7	7.00	3.00	_
Positive control : 9-Aminoacridine	50 µg	645	793	845	761.00	103.77	108.71
Vehicle	50µL	11	5	5	7.00	3.46	_
	5000 µg	6	3	6	5.00	1.73	0.71
Solution of	1500 µg	6	8	7	7.00	1.00	1.00
T-A10 BATCH: M22154C	500 µg	5	6	4	5.00	1.00	0.71
	150 µg	8	7	3	6.00	2.65	0.86
	50 µg	4	10	7	7.00	3.00	1.700

 

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	12	7	16	11.67	4.51	_
Positive control solvent	20 µL	10	10	11	10.33	0.58	_
Positive control : 2-Anthramine	50 µL	38	40	39	39.00	1.00	3.77
Vehicle	50µL	10	8	16	11.33	4.16	_
	5000 µg	7	5	8	6.67	1.53	0.59
Solution of	1500 µg	18	16	11	15.00	3.61	1.32
T-A10 BATCH: M22154C	500 µg	9	10	12	10.33	1.53	0.91
	150 µg	6	12	18	12.00	6.00	1.06
	50 µg	18	13	10	13.67	4.04	1.21

 

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	3	13	5	7.00	5.29	_
Positive control solvent	20 µL	4	10	7	7.00	3.00	_
Positive control : 9-Aminoacridine	50 µg	878	1293	666	945.67	318.93	135.10
Vehicle	50µL	6	5	8	6.33	1.53	_
	5000 µg	1	1	2	1.33	0.58	0.21
Solution of	1500 µg	2	8	5	5.00	3.00	0.79
T-A10 BATCH: M22154C	500 µg	8	6	4	6.00	2.00	0.95
	150 µg	10	9	8	9.00	1.00	1.42
	50 µg	3	7	7	5.67	2.31	0.89

 

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	5	8	8	7.00	1.73	_
Positive control solvent	10 µL	12	8	10	10.00	2.500	_
Positive control : 2-Anthramine	50 µL	26	28	25	26.33	1.53	2.63
Vehicle	50µL	6	4	8	6.00	2.00	_
	5000 µg	1	6	2	3.00	2.65	0.50
Solution of	1500 µg	4	8	10	7.33	3.06	1.22
T-A10 BATCH: M22154C	500 µg	8	12	10	10.00	2.00	1.67
	150 µg	4	10	11	8.33	3.79	1.39
	50 µg	8	12	9	9.67	2.08	1.61

 

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	23	23	18	21.33	2.89	_
Positive control solvent	20 µL	15	19	20	18.00	2.65	_
Positive control : 2-Nitrofluorene	50 µL	427	336	426	396.33	52.25	22.02
Vehicle	50µL	19	18	23	20.00	2.65	_
	5000 µg	28	24	14	22.00	7.21	1.10
Solution of	1500 µg	18	21	18	19.00	1.73	0.95
T-A10 BATCH: M22154C	500 µg	26	27	26	26.33	0.58	1.32
	150 µg	18	15	29	20.67	7.37	1.03
	50 µg	12	18	20	16.67	4.16	0.83

 

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	26	25	25	25.33	0.58	_
Positive control solvent	20 µL	31	25	23	26.33	4.16	_
Positive control : 2-Anthramine	50 µL	407	321	293	340.33	59.41	12.92
Vehicle	50µL	21	36	24	27.00	7.94	_
	5000 µg	24	27	14	21.67	6.81	0.80
Solution of	1500 µg	33	31	34	32.67	1.53	1.21
T-A10 BATCH: M22154C	500 µg	34	26	27	29.00	4.36	1.07
	150 µg	31	34	38	34.33	3.51	1.27
	50 µg	30	36	26	30.67	5.03	1.14

 

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	15	23	14	17.33	4.93	_
Positive control solvent	20 µL	19	16	16	17.00	1.73	_
Positive control : 2-Nitrofluorene	2 µL	423	423	441	429.00	10.39	17.04
Vehicle	50µL	14	17	12	14.33	2.52	_
	5000 µg	22	6	13	13.67	8.02	0.95
Solution of	1500 µg	16	24	17	19.00	4.36	1.33
T-A10 BATCH: M22154C	500 µg	15	18	16	16.33	1.53	1.14
	150 µg	17	18	18	17.67	0.58	1.23
	50 µg	16	24	21	20.33	4.04	1.42

 

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	31	29	130	30.00	1.00	_
Positive control solvent	10 µL	24	31	27	27.33	3.51	_
Positive control : 2-Anthramine	1 µg	215	218	301	244.67	48.81	8.95
Vehicle	50µL	29	23	22	24.67	3.79	_
	5000 µg	16	27	15	19.33	6.66	0.78
Solution of	1500 µg	33	26	28	29.00	3.61	1.18
T-A10 BATCH: M22154C	500 µg	35	28	21	28.00	7.00	1.14
	150 µg	31	34	32	32.33	1.53	1.31
	50 µg	127	27	32	28.67	2.89	1.16

 

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	68	60	70	66.00	5.29	_
Positive control solvent	20 µL	69	57	74	66.67	8.74	_
Positive control : Sodium azide	20 µg	1137	996	1137	1069.00	81.41	16.35
Vehicle	50µL	69	69	81	73.00	6.93	_
	5000 µg	43	39	33	38.33	5.03	0.53
Solution of	1500 µg	82	76	85	81.00	4.58	1.11
T-A10 BATCH: M22154C	500 µg	66	77	56	66.33	10.50	0.91
	150 µg	55	62	71	62.67	8.02	0.86
	50 µg	71	85	636	73.00	11.14	1.00

 

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	54	54	62	56.67	4.62	_
Positive control solvent	20 µL	68	69	65	67.33	2.08	_
Positive control : 2-Anthramine	2 µg	527	557	531	538.33	16.29	8.00
Vehicle	50µL	54	57	66	59.00	6.24	_
	5000 µg	35	45	33	37.67	6.43	0.64
Solution of	1500 µg	82	67	53	67.33	14.50	1.14
T-A10 BATCH: M22154C	500 µg	74	82	67	74.33	7.51	1.26
	150 µg	99	69	66	78.00	18.25	1.32
	50 µg	72	59	82	71.00	11.53	1.20

 

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	68	87	66	73.67	11.59	_
Positive control solvent	20 µL	68	84	57	69.67	13.58	_
Positive control : Sodium azide	20 µg	1188	1124	1115	1142.33	39.80	16.40
Vehicle	50µL	80	69	46	65.00	17.35	_
	5000 µg	49	32	29	36.67	10.79	0.56
Solution of	1500 µg	56	60	62	59.33	3.06	0.91
T-A10 BATCH: M22154C	500 µg	61	52	62	58.33	5.51	0.90
	150 µg	69	72	60	67.00	6.24	1.03
	50 µg	59	70	57	62.00	7.00	0.95

 

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	57	66	60	61.00	4.58	_
Positive control solvent	10 µL	67	71	65	67.67	3.06	_
Positive control : 2-Anthramine	1 µg	509	498	456	487.67	27.97	7.21
Vehicle	50µL	69	61	72	67.33	5.96	_
	5000 µg	32	47	49	42.67	9.29	0.63
Solution of	1500 µg	74	61	71	68.67	6.81	1.02
T-A10 BATCH: M22154C	500 µg	74	82	71	75.67	5.69	1.12
	150 µg	69	59	82	70.00	11.53	1.04
	50 µg	82	79	71	77.33	5.69	1.15

 

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	50	49	56	51.67	3.79	_
Positive control solvent	10 µL	61	49	55	55.00	6.00	_
Positive control : MMS	100 µg	524	531	601	552.00	42.58	10.04
Vehicle	50µL	47	66	69	60.67	11.93	_
	5000 µg	42	38	35	38.33	3.51	0.63
Solution of	1500 µg	55	62	63	60.00	4.36	0.99
T-A10 BATCH: M22154C	500 µg	51	49	48	49.33	1.53	0.81
	150 µg	49	56	55	53.33	3.79	0.88
	50 µg	51	49	52	50.67	1.53	0.84

 

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	70	80	82	77.33	6.43	_
Positive control solvent	50 µL	83	83	90	85.33	4.04	_
Positive control : 2-Anthramine	50 µg	1034	1088	1003	1041.67	43.02	12.21
Vehicle	50µL	85	79	91	85.00	6.00	_
	5000 µg	59	58	56	57.67	1.53	0.68
Solution of	1500 µg	79	84	82	81.67	2.52	0.96
T-A10 BATCH: M22154C	500 µg	81	80	90	83.67	5.51	0.98
	150 µg	89	87	79	85.00	5.29	1.00
	50 µg	83	85	81	83.00	2.00	0.98

 

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	47	45	43	45.00	2.00	_
Positive control solvent	10 µL	54	63	43	53.33	10.02	_
Positive control : MMS	100 µg	445	535	374	451.33	80.69	8.46
Vehicle	50µL	51	48	65	54.67	9.07	_
	5000 µg	38	35	32	35.00	3.00	0.64
Solution of	1500 µg	45	56	60	53.67	7.77	0.98
T-A10 BATCH: M22154C	500 µg	54	62	61	59.00	4.36	1.08
	150 µg	59	46	45	50.00	7.81	0.91
	50 µg	43	63	44	50.00	11.27	0.91

 

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	70	69	76	71.67	6,24	_
Positive control solvent	12,5 µL	80	100	63	81.00	12,06	_
Positive control : Dimethylbenzanthracene	12.5 µg	1118	894	772	4928.00	27,57	11.16
Vehicle	50µL	81	72	86	79.67	7,02	_
	5000 µg	45	44	39	42.67	3,06	0.54
Solution of	1500 µg	75	83	82	80.00	6,03	1.00
T-A10 BATCH: l19319E	500 µg	81	77	76	78.00	3,61	0.98
	150 µg	80	76	72	76.00	9,64	0.95
	50 µg	80	83	68	77.00	13,75	0.97

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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.