Methyl 5-mesyl-o-anisate

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: According to OECD 471 Guideline. GLP study. Test item do not induce any mutagenic change in tested bacteria (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) and Escherichia coli WP2) under the test conditions.

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:

October 26, 2016 - February 22, 2017

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25ºC)
- Stability under test conditions: Stable under normal storage condition.
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO

Target gene:

Histidine-requiring gene in Salmonella typhimurium, Tryptophan-requiring gene for Escherichia coli.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: ∆ uvr B, rfa mutated

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: ∆ uvr A mutated

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from Sprague Dawley rat liver homogenate

Test concentrations with justification for top dose:

Tested doses of test item diluted in DMSO: 50,150,500,1500,5000 ug/plate.
The preliminary study showed no cytotoxicity of the test item at the highest dose tested (5000 μg/plate); therefore this concentrations range was used for the mutagenicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: It does not react with test item and is compatible with the survival of the bacteria and S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

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: cis-Platinum (II) Diammine Dichloride and 2-Anthramine (see remarks)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
ASSAY 1: Plate incorporation method. A first experiment of genotoxicity was conducted, with and without metabolic activation, on the range of concentrations defined by the preliminary study: 5000µg, 1500µg, 500µg, 150µg, and 50µg dose/plate.
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x109 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 supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL.
For the assay with metabolic activation 500 μL of S9-mix fraction were quickly added, before pouring the mixture onto the plates.
Afterwards, plates were incubated at 37° C over a 48-72 hour period. The number of revertant colonies per plate was counted.

ASSAY 2: Preincubation method: a second experiment was performed, also with and without metabolic activation, with dose levels defined after analysis of results obtained on the first experiment. This second experiment was performed in order to confirm or for complement results of the first one. The protocol is similar to the Assay 1, except for that the solution of the test item solution with the test strain are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate. When metabolic activation is used, 500 μL of S9-mix fraction are added before the pre-incubation.

- Cell density at seeding: 1.9E09 bacteria/mL

DURATION
- Preincubation period: 30min at 37ºC, only for the Test 2.
- Exposure duration: 48-72h

SELECTION AGENT: absence of Histidin and Tryptophan, the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Cytotoxicity may be detected by a reduction in the number of revertant colonie.s.
A preliminary cytotoxicity test was performed with the strain TA100 to determine any sign of cytotoxicity. The doses used were 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg dose/plate.
In a test tube 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 0.1 mL of the stock solution and dilutions, are successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 hours at 37°C, and the colonies counted. Petri plates were observed and any cytotoxicity sign was reported (bottom bacterial layer reduction). A negative control containing the blank alone is run in parallel.In case 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.

OTHER EXAMINATIONS:
Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45°C, 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 37°C 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:

Test conditions according to the OECD nº471. Dose selection for main assays were perfromed based on results of the preliminary cytotoxicity test.

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test item was observed.

RANGE-FINDING/SCREENING STUDIES:
Preliminary study was carried out with S. thyphymurium TA 100 with doses up to 5000µg/plate. Neither original solution nor dilutions have bacteriostatic effect. For this reason, on the Assay 1, the following doses were used: 5 000, 1 500, 500, 150 and 50 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

- Positive control data

Without metabolic activation:
TA 1535 N=912,Mean=668.2±202.4 (min/max=45.0/1481.0),
TA 1537 N=912,Mean=787,5±385.0 (min/max=219.0/1967.0)
TA 98 N=912,Mean=532.2±214.4 (min/max=187.0/1667.0)
TA 100 N=912,Mean=862.4±270.3 (min/max=381.0-1690.0)
Escherichia coli WP2(pKM101) (uvrA-) N=675, Mean=531.1±160.5 (min/max=280.0/1089.0)

With metabolic activation - without pre-incubation
TA 1535 N=492,Mean=95.2±46.9 (min/max=26.0/269.0)
TA 1537 N=492,Mean=53.6±21.7 (min/max=24.0/170.0)
TA 98 N=492,Mean=566.3±195.1 (min/max=219.0/1280.0)
TA 100 N=489,Mean=898.1±374.0 (min/max=361.0/2163.0)
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=739.1±234.7 (min/max=384.0/1680.0)

With metabolic activation - with pre-incubation
TA 1535 N=489,Mean=69.9±29.2 (min/max=25.0/185.0),
TA 1537 N=489,Mean=69.9±29.2 (min/max=21.0/182.0),
TA 98 N=489,Mean=479.4±183.9(min/max=174.0/1370.0),
TA 100 N=492,Mean=708.0±295.9(min/max=309.0/1889.0)
Escherichia coli WP2(pKM101) (uvrA-)N=351,Mean=731.7±217.3 (min/max=405.0/1680.0)

- Negative (solvent/vehicle) historical control data:

Without metabolic activation:
TA 1535 N=912,Mean=10.7±3.4 (min/max=4.0/23.0)
TA 1537 N=912,Mean=5.8±2.4 (min/max=1.0/14.0)
TA 98 N=912,Mean=15.7±3.7 (min/max=6.0/29.0)
TA 100 N=912,Mean=65.2±17.1(min/max=41.0/158.0)
Escherichia coli WP2(pKM101) (uvrA-) N=675,Mean=67.8±20.0(min/max=40.0-/65.0)

With metabolic activation - without pre-incubation
TA 1535 N=492,Mean=12.2±4.0 (min/max=3.0/23.0)
TA 1537 N=492,Mean 7.9±3.0, (min/max=1.0/24.0)
TA 98 N=492,Mean=22.9±4.6 (min/max=12.0/35.0)
TA 100 N=489,Mean=108.3±25.1 (min/max=58.0/220.0),
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=153.1±28.9 (min/max=80.0/264.0)

With metabolic activation, with pre-incubation
TA 1535 N=489,Mean=12.6±4.1 (min/max=5.0/25.0),
TA 1537 N=489,Mean=8.2±3.0(min/max=2.0/19.0)
TA 98 N=489,Mean=23.0±4.9(min/max=11.0/35.0)
TA 100 N=489,Mean=108.4±27.1(min/max=51.0/217.0)
Escherichia coli WP2(pKM101) (uvrA-) N=351,Mean=158.5±31.4 (min/max=69.0/250.0)

Table No.6: Sterility control:

Serie	Doses	Colony number/plate
Control nº1		1	2	3
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	0	0	0
	1500ug/plate	0	0	0
	500ug/plate	0	0	0
	150ug/plate	0	0	0
	50ug/plate	0	0	0
S9-mix	500ul/plate	0	0	0
Control nº2		1	2	3
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	0	0	0
	1500ug/plate	0	0	0
	500ug/plate	0	0	0
	150ug/plate	0	0	0
	50ug/plate	0	0	0
S9-mix	500ul/plate	0	0	0

Table No.7: Bacteriostatic activity control (nº1 and nº2)

		0 Negative control	DMSO	50ug	150ug	500ug	1500ug	2500ug	5000ug
Solution of test item Lemi code :GFF211116-S2	N1	249	236	249	255	264	249	238	261
	N2	265	248	258	265	258	243	251	246
	N3	259	249	245	257	246	249	263	240
	N	258+-8	244+-7	251+-7	259+-5	256+-9	247+-3	251+-13	249+-11
	%	-	95	97	101	99	96	97	97
		0 Negative control	DMSO	50ug	150ug	500ug	1500ug	5000ug
Solution of test item Lemi code :GFF281116-S2	N1	231	265	237	276	223	231	223
	N2	281	285	228	260	242	264	261
	N3	272	225	252	249	261	275	267
	N	261+-27	258+-31	239+-12	262+-14	242+-19	257+-23	250+-24
	%	-	99	91	100	93	98	96

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 No.8: TA 1535 Assay nº1- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	N3	mean	Standart deviation	R
Negative control	100uL	14	14	14	14.00	0.00	-
Positive control solvent	5uL	15	7	15	12.33	4.62	-
Positive control Sodium azide	5ug In 5uL	740	561	696	665.67	93.28	53.97
Vehicle	50uL	17	7	12	12.00	5.00	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	10	4	12	8.67	4.16	0.72
	1500ug/plate	11	10	13	11.33	1.53	0.94
	500ug/plate	8	18	9	11.67	5.51	0.97
	150ug/plate	8	11	10	9.67	1.53	0.81
	50ug/plate	7	15	13	11.67	4.16	0.97

Table No.9: TA 1535 Assay nº1- with metabolic activation (10% S9 -mix) - without preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	21	5	18	14.67	8.50	-
Positive control solvent	20uL	9	11	9	9.67	1.15	-
Positive control 2-Anthramide	2ug In 20uL	152	161	128	147.00	17.06	15.21
Vehicle	50uL	18	9	15	14.00	4.58	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	9	12	8	9.67	2.08	0.69
	1500ug/plate	14	12	13	13.00	1.00	0.93
	500ug/plate	14	15	13	14.00	1.00	1.00
	150ug/plate	7	15	8	10.00	4.36	0.71
	50ug/plate	14	9	12	11.67	2.52	0.83

Table No.10: TA 1535Assay nº2- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	10	13	15	12.67	2.52	-
Positive control solvent	5uL	8	9	7	8.00	1.00	-
Positive control Sodium Azide	5ug In 5uL	743	759	772	758	14.53	94.75
Vehicle	50uL	16	12	11	13.00	2.65	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	11	8	13	10.67	2.52	0.82
	1500ug/plate	12	14	7	11.00	3.61	0.85
	500ug/plate	11	16	14	13.67	2.52	1.05
	150ug/plate	6	11	14	10.33	4.04	0.79
	50ug/plate	15	8	18	13.67	5.13	1.05

Table No.11: TA 1535 Assay nº2- with metabolic activation (10% S9 -mix) - with preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	12	12	17	13.67	2.89	-
Positive control solvent	10uL	10	11	9	10.00	1.00	-
Positive control 2-Anthramide	1ug In 10uL	52	65	45	54.00	10.15	5.40
Vehicle	50uL	13	12	16	13.67	2.08	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	12	10	10	10.67	1.15	0.78
	1500ug/plate	8	16	8	10.67	4.62	0.78
	500ug/plate	16	10	13	13.00	3.00	0.95
	150ug/plate	17	15	8	13.33	4.73	0.98
	50ug/plate	15	12	13	13.33	1.53	0.98

Table No.12 TA1537 Assay nº1 -without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	2	8	6	5.33	3.06	-
Positive control solvent	20uL	3	5	3	3.67	1.15	-
Positive control 9-Aminoacridine	50ug In 20uL	1563	1673	1645	1627.00	57.17	443.73
Vehicle	50uL	6	6	5	5.33	0.58	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	5	5	9	6.67	2.08	1.25
	1500ug/plate	8	12	4	8.00	4.00	1.50
	500ug/plate	6	9	5	6.67	2.08	1.25
	150ug/plate	5	5	3	4.33	1.15	0.81
	50ug/plate	3	7	5	5.00	2.00	0.94

Table No.13 TA1537 Assay nº1 -with metabolic activation (10% -S9 -mix) - without preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	16	12	19	15.67	3.51	-
Positive control solvent	20uL	12	11	12	11.67	0.58	-
Positive control 2-Anthramine	2ug In 20uL	43	72	63	59.33	14.84	5.09
Vehicle	50uL	11	10	13	11.33	1.53	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	8	10	5	7.67	2.52	0.68
	1500ug/plate	14	10	9	11.00	2.65	0.97
	500ug/plate	7	8	14	9.67	3.79	0.85
	150ug/plate	6	14	7	9.00	4.36	0.79
	50ug/plate	4	13	14	10.33	5.51	0.91

Table No.14 TA1537 Assay nº2 -without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	4	2	5	3.67	1.53	-
Positive control solvent	20uL	3	7	11	7.00	4.00	-
Positive control 9-Aminoacridine	50ug In 20uL	1581	727	1591	1299.67	495.97	185.67
Vehicle	50uL	11	11	13	11.67	1.15	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	6	9	4	6.33	2.52	0.54
	1500ug/plate	5	5	6	5.33	0.58	0.46
	500ug/plate	11	8	3	7.33	4.04	0.63
	150ug/plate	6	6	6	6.00	0.00	0.51
	50ug/plate	10	4	8	7.33	3.06	0.63

Table No.15 TA1537 Assay nº2 -with metabolic activation (10%-S9 -mix) - with preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	10	14	11	11.67	2.08	-
Positive control solvent	10uL	8	9	10	9.00	1.00	-
Positive control 2-Anthramine	1ug In 10uL	27	31	40	32.67	6.66	3.63
Vehicle	50uL	8	12	16	12.00	4.00	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	5	9	13	9.00	4.00	0.75
	1500ug/plate	13	6	19	12.67	6.51	1.06
	500ug/plate	10	13	10	11.00	1.73	0.92
	150ug/plate	11	10	10	10.33	0.58	0.86
	50ug/plate	12	9	7	9.33	2.52	0.78

Table No.16 TA98 Assay nº1- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	18	11	18	15.67	4.04	-
Positive control solvent	20uL	18	10	17	15.00	4.36	-
Positive control 2-Nitrofluorene	2ug In 20uL	286	268	298	284.00	15.10	18.93
Vehicle	50uL	12	15	16	14.33	2.08	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	15	11	16	14.00	2.65	0.98
	1500ug/plate	12	11	15	12.67	2.08	0.88
	500ug/plate	11	14	22	15.67	5.69	1.09
	150ug/plate	15	17	11	14.33	3.06	1.00
	50ug/plate	16	24	19	19.67	4.04	1.37

Table No.17 TA98 Assay nº1- with metabolic activation (10% -S9 -mix)- without preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	18	21	25	21.33	3.51	-
Positive control solvent	20uL	23	16	19	19.33	3.51	-
Positive control 2-Anthramine	2ug In 20uL	442	429	293	388.00	82.53	20.07
Vehicle	50uL	19	18	12	16.33	3.79	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	16	15	22	17.67	3.79	1.08
	1500ug/plate	31	19	19	23.00	6.93	1.41
	500ug/plate	21	24	17	20.67	3.51	1.27
	150ug/plate	12	12	23	15.67	6.35	0.96
	50ug/plate	23	22	24	23.00	1.00	1.41

Table No.18 TA98 Assay nº2- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	15	11	18	14.67	3.51	-
Positive control solvent	20uL	12	18	17	15.67	3.21	-
Positive control 2-Nitrofluorene	2ug In 20uL	302	198	195	231.67	60.93	14.79
Vehicle	50uL	22	18	19	19.67	2.08	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	9	14	11	11.33	2.52	0.58
	1500ug/plate	15	15	15	15.00	0.00	0.76
	500ug/plate	11	17	15	14.33	3.06	0.73
	150ug/plate	14	25	17	18.67	5.69	0.95
	50ug/plate	16	16	21	17.67	2.89	0.90

Table No.19 TA98 Assay nº2- with metabolic activation (10%-S9 -mix) - with preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	28	30	20	26.00	5.29	-
Positive control solvent	10uL	17	11	13	17.00	8.73	-
Positive control 2-Anthramine	1ug In 10uL	178	199	203	193.33	13.43	11.37
Vehicle	50uL	27	22	21	23.33	3.21	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	29	28	22	26.33	3.79	1.13
	1500ug/plate	18	28	20	22.00	5.29	0.94
	500ug/plate	22	20	23	21.67	1.53	0.93
	150ug/plate	27	21	30	26.00	4.58	1.11
	50ug/plate	23	32	29	28.00	4.58	1.20

Table No.20 TA 100 Assay nº1- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	49	41	64	51.33	11.68	-
Positive control solvent	20uL	50	60	60	56.67	5.77	-
Positive control Sodium Azide	20ug In 20uL	1646	1510	1589	1581.67	68.30	27.91
Vehicle	50uL	72	76	74	74.00	2.00	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	81	50	46	59.00	19.16	0.80
	1500ug/plate	63	81	65	69.67	9.87	0.94
	500ug/plate	52	71	54	59.00	10.44	0.80
	150ug/plate	66	68	73	69.00	3.61	0.93
	50ug/plate	75	73	70	72.67	2.52	0.98

Table No.21 TA 100 Assay nº1- with metabolic activation (10% -S9 -mix) without preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	72	91	69	76.67	12.42	-
Positive control solvent	20uL	72	77	76	75.00	2.65	-
Positive control 2-Anthramine	2ug In 20uL	746	563	555	621.33	108.04	8.28
Vehicle	50uL	72	73	74	73.00	1.00	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	91	82	72	81.67	9.50	1.12
	1500ug/plate	71	72	71	71.33	0.58	0.98
	500ug/plate	78	84	72	78.00	6.00	1.07
	150ug/plate	101	82	98	93.67	10.21	1.28
	50ug/plate	79	77	75	77.00	2.00	1.05

Table No.22 TA 100 Assay nº2- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	59	76	112	82.33	27.06	-
Positive control solvent	20uL	59	71	60	63.33	6.66	-
Positive control Sodium Azide	20ug In 20uL	1622	1274	1561	1485.67	185.83	23.46
Vehicle	50uL	73	66	71	70.00	3.61	-
Solution of test itemLemi code :GFF281116-S2	5000ug/plate	73	62	64	66.33	5.86	0.95
	1500ug/plate	57	57	68	60.67	6.35	0.87
	500ug/plate	78	71	62	70.33	8.02	1.00
	150ug/plate	72	61	77	70.00	8.19	1.00
	50ug/plate	61	68	68	65.67	4.04	0.94

Table No.23 TA 100 Assay nº2- without metabolic activation (10% -S9 -mix) with preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	84	79	62	75.00	11.53	-
Positive control solvent	10uL	79	86	70	78.33	8.02	-
Positive control 2-Anthramine	1ug In 10uL	544	426	561	510.33	73.53	6.51
Vehicle	50uL	54	68	59	60.33	7.09	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	65	85	72	74.00	10.15	1.23
	1500ug/plate	80	84	65	76.33	10.02	1.27
	500ug/plate	79	107	97	94.33	14.19	1.56
	150ug/plate	93	108	90	97.00	9.64	1.61
	50ug/plate	87	75	92	84.67	8.74	1.40

Table No.24 E.coli Assay nº1- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	47	478	59	51.33	6.66	-
Positive control solvent	10uL	48	61	49	52.67	7.23	-
Positive control Cis-Platinum (II)	1ug In 10uL	280	311	297	296.00	15.52	5.62
Vehicle	50uL	129	164	138	143.67	18.18	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	102	24	43	56.33	40.67	0.39
	1500ug/plate	78	78	102	86.00	13.86	0.6
	500ug/plate	77	84	69	76.67	7.51	0.53
	150ug/plate	71	68	74	71.00	3.00	0.49
	50ug/plate	73	124	91	96.00	25.87	0.67

Table No.25 E.coli Assay nº1- with metabolic activation (10% -S9 -mix) without preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	206	196	216	206.00	10.00	-
Positive control solvent	5uL	217	179	184	193.33	20.65	-
Positive control DimethylbenzathracEne	5ug In 5uL	428	435	415	426.00	10.15	2.20
Vehicle	50uL	215	208	210	211.00	3.61	-
Solution of test item Lemi code :GFF211116-S2	5000ug/plate	59	131	154	114.67	49.56	0.54
	1500ug/plate	162	149	191	167.33	21.50	0.79
	500ug/plate	154	145	176	158.33	15.95	0.75
	150ug/plate	170	187	183	180.00	8.89	0.85
	50ug/plate	173	184	191	182.67	9.07	0.87

Table No.26 E.coli Assay nº2- without metabolic activation (-S9 -mix)

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	91	108	107	102.00	9.54	-
Positive control solvent	10uL	126	133	119	126.00	7.00	-
Positive control Cis-Platinum (II)	1ug In 10uL	348	327	301	325.33	23.54	2.58
Vehicle	50uL	118	133	134	128.33	8.96	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	50	24	79	51.00	27.51	0.40
	1500ug/plate	82	78	53	71.00	15.72	0.55
	500ug/plate	114	97	100	103.67	9.07	0.81
	150ug/plate	132	123	126	127.00	4.58	0.99
	50ug/plate	114	138	115	122.33	13.58	0.95

Table No.27 E.coli Assay nº2- with metabolic activation (10% -S9 -mix) with preincubation

Serie	Dose/plate	Nº1	Nº2	Nº3	mean	Standart deviation	R
Negative control	100uL	161	155	187	167.67	17.01	-
Positive control solvent	5uL	186	179	199	188.00	10.15	-
Positive control DimethylbenzathracEne	2.5ug In 5uL	512	537	428	492.33	57.10	2.62
Vehicle	50uL	169	197	188	184.67	14.29	-
Solution of test item Lemi code :GFF281116-S2	5000ug/plate	124	184	82	130.00	51.26	0.70
	1500ug/plate	136	125	117	126.00	9.54	0.68
	500ug/plate	197	163	186	182.00	17.35	0.99
	150ug/plate	191	184	209	194.67	12.90	1.05
	50ug/plate	184	159	170	171.00	12.53	0.93

Conclusions:

Test item do not induce any mutagenic change in tested bacterias (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) and Escherichia coli WP2 (uvrA-) (pKM101) with and without metabolic activation up to 5000 μg/plate.

Executive summary:

The aim of this test was to determine the ability of the test item to induce mutation, it was assessed using the bacterial reverse mutation test (Ames test). The test was performed following the OECD Guideline 471 with GLP on four Salmonella typhimurium strains and on Escherichia coli WP2. The test item dilutions were prepared in DMSO for analysis. Firstly, a preliminary cytotoxicity test was performed on S. typhimurium TA100 strain with the following concentrations: 5 000, 2500, 1 500, 500, 150 and 50μg/plate, for triplicate. Plates were incubated at 37ºC for 48-72h and after this period colonies were counted. As the preliminary study showed no cytotoxicity of the test item, the concentrations 5 000, 1 500, 500, 150 and 50 μg/plate were used for the genotoxicity Test 1 (direct method) and Test 2 (pre-incubation method), and positive and negative/solvent controls were included. The same test conditions as the preliminary test were used, with and without metabolic activation system S9 mix. The revertant analysis showed no cytotoxic effect. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. Based on the result of this study, the test item was not found to be mutagenic under the test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key Study - In vitro gene mutation study in bacteria: According to OECD 471 Guideline. GLP study. The test was performed on four Salmonella typhimurium strains and on Escherichia coli WP2. After a preliminary test which showed no cytotoxicity of the test item, a direct method test and a pre-incubation method test were performed with the doses of 5 000, 1 500, 500, 150 and 50 μg/plate of test item. There was no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions without and with metabolic activation. Based on the result of this study, the test item was not found to be mutagenic under the test conditions.

Justification for classification or non-classification

Based on the available information, the test item is not classified as mutagen according to CLP Regulation (EC) no.1272/2008.