4-amino-5-(ethylsulphonyl)-o-anisic acid

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:

3 September 2014 - 2 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

4-amino-5-(ethylsulphonyl)-o-anisic acid

EC Number:

275-833-8

EC Name:

4-amino-5-(ethylsulphonyl)-o-anisic acid

Cas Number:

71675-87-1

Molecular formula:

C10H13NO5S

IUPAC Name:

4-amino-5-(ethanesulfonyl)-2-methoxybenzoic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

- Name of test material (as cited in study report): 4-amino-5-ethylsulfonyl-2-methoxybenzoic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31
- Expiration date of the lot/batch: 16 June 2017

Method

Target gene:

Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.

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: rfa, uvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solubility of the test item was examined in Distilled water, Dimethyl sulfoxide (DMSO) and Acetone as vehicles. The test item was insoluble at 100 mg/mL concentration in Distilled water. However, it was soluble at the same concentration using DMSO or Acetone as vehicles (after 5 minutes of vortex). Due to the better biocompatibility to the test system, DMSO was selected as vehicle (solvent) for the study. The behaviour of the obtained test item formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test). The selected vehicle was compatible with the survival of the cells and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

untreated control - used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO and distilled water)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level).The
equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test
strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of
minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into
appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were
gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content
was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were
incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.

Evaluation criteria:

CRITERIA FOR A POSITIVE RESPONSE
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

CRITERIA FOR A NEGATIVE RESPONSE
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(Confirmatory Mutation Test in TA1537 strain at 5000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

Test results 5

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

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

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

RANGE-FINDING/SCREENING STUDIES: The numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate or signs of inhibitory, cytotoxic effect were detected on the plates in the preliminary experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. see below

Positive contrrol data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 331.5 1369.2 1230.9 524.3 1107.4 2458.9 2472.1 239.3 229.8 266.5
St. dev. 81.9 230.3 238.4 244.4 154.3 437.0 427.8 198.7 79.7 52.2
Range 152-1224 912-2120 208-2440 592-1708 592-1708 496-4918 1192-5240 125-2216 117-838 125-496
n 549 546 549 543 546 546 540 543 543 546

Untreated control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 23.4 105.8 9.5 6.9 31.1 30.8 116.6 11.2 8.8 39.1
St. dev. 6.2 28.2 4.5 3.5 10.0 7.2 24.8 3.8 3.7 10.2
Range 9-46 54-210 1-46 1-24 11-76 10-56 65-204 3-35 1-28 16-81
n 549 546 549 543 546 546 540 543 543 546

DMSO control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 22.1 102.4 9.8 7.1 29.8 29.9 1118.0 11.4 9.0 38.5
St. dev. 6.5 28.1 4.3 3.7 9.5 7.4 25.3 3.7 4.0 10.1
Range 9-55 40-217 1-43 1-25 7-81 11-67 62-229 3-24 2-29 18-84
n 558 555 558 552 555 554 549 551 552 555

Distilled water control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 23.8 106.9 9.7 7.8 31.9 31.5 120.3 11.1 9.4 40.5
St. dev. 6.2 30.4 4.4 3.4 9.9 7.5 27.2 3.7 3.5 9.7
Range 13-43 45-215 2-47 2-24 12-77 10-50 70-222 3-39 1-20 18-91
n 129 546 549 129 549 129 546 549 129 552

Acetone control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 331.5 1369.2 1230.9 524.3 1107.4 2458.9 2472.1 239.3 229.8 266.5
St. dev. 81.9 230.3 238.4 244.4 154.3 437.0 427.8 198.7 79.7 52.2
Range 152-1224 912-2120 208-2440 592-1708 592-1708 496-4918 1192-5240 125-2216 117-838 125-496
n 549 546 549 543 546 546 540 543 543 546

n: number of cases

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effect of the test item (reduced background lawn and reduced revertant counts) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain at 5000 μg/plate concentration without metabolic activation.

Any other information on results incl. tables

 Table No.4 Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA98

Cell count (overnight culture): 2.71 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	19	33
	20	38
	29	40
	22.7	37.0
	5.51	3.61
	1.01	1.16
1581   MEAN SD MF	23	28
	28	36
	28	41
	26.3	35.0
	2.89	6.56
	1.18	1.09
500   MEAN SD MF	25	32
	25	36
	27	45
	25.7	37.7
	1.15	6.66
	1.15	1.18
158.1   MEAN SD MF	18	30
	19	36
	20	38
	19.0	34.7
	1.00	4.16
	0.85	1.08
50   MEAN SD MF	21	35
	24	37
	25	42
	23.3	38.0
	2.08	3.61
	1.04	1.19
15.81   MEAN SD MF	22	37
	23	39
	29	39
	24.7	38.3
	3.79	1.15
	1.10	1.20
5   MEAN SD MF	16	27
	23	30
	25	34
	21.3	30.3
	4.73	3.51
	0.96	0.95
Untreated control     MEAN SD MF	27	36
	30	27
	20	33
	25.7	32.0
	5.13	4.58
	1.15	1.00
DMSO control     MEAN SD MF	25	33
	21	30
	21	33
	22.3	32.0
	2.31	1.73
	1.00	1.00
Positive control NPD (4ug)       MEAN SD MF	372	2488
	320	2524
	384	2480
	358.7	2497.3
	34.02	23.44
	16.06	78.04

Table No.5: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA100

Cell count (overnight culture): 3.19 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	83	98
	117	95
	107	115
	102.3	102.7
	17.47	10.79
	0.97	0.95
1581   MEAN SD MF	109	115
	92	101
	109	111
	103.3	109.0
	9.81	7.21
	0.98	1.01
500   MEAN SD MF	108	106
	100	93
	112	92
	106.7	97.0
	6.11	7.81
	1.02	0.9
158.1   MEAN SD MF	99	99
	97	89
	116	100
	104.0	96.0
	10.44	6.08
	0.99	0.89
50   MEAN SD MF	105	98
	85	86
	109	86
	99.7	90.0
	12.86	6.93
	0.95	0.83
15.81   MEAN SD MF	105	101
	118	104
	85	97
	102.7	100.7
	16.62	3.51
	0.98	0.93
5   MEAN SD MF	101	104
	94	87
	114	98
	103.0	96.3
	10.15	8.62
	0.98	0.89
Untreated control     MEAN SD MF	111	102
	121	102
	131	121
	121.0	108.3
	10.00	10.97
	1.15	1.00
DMSO control     MEAN SD MF	86	104
	108	106
	121	114
	105.0	108.0
	17.69	5.29
	1.00	1.00
Distilled water control*       MEAN SD MF	106	103
	116	113
	117	115
	105.0	110.3
	17.69	6.43
	1.00	1.02
Positive control SAZ (2ug)   MEAN SD MF	1188                       2AA	2412
	1204                       (2ug)	2424
	1212	2488
	1201.3	2441.3
	12.22	40.86
	10.63	22.60

*Distilled water control was used because of SAZ

Table No.6: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA1535

Cell count (overnight culture): 2.03 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	14	11
	18	15
	19	13
	17.0	13.0
	2.65	2.00
	0.85	1.18
1581   MEAN SD MF	16	14
	14	7
	17	13
	15.7	11.3
	1.53	3.79
	0.78	1.03
500   MEAN SD MF	11	16
	15	9
	19	11
	15.00	12.0
	4.00	3.61
	0.75	1.09
158.1   MEAN SD MF	18	10
	11	11
	15	10
	14.7	10.3
	3.51	0.58
	0.73	0.94
50   MEAN SD MF	10	11
	13	8
	21	10
	14.7	9.7
	5.69	1.53
	0.73	0.88
15.81   MEAN SD MF	17	14
	15	15
	14	7
	15.3	12.0
	1.53	4.36
	0.77	1.09
5   MEAN SD MF	17	6
	16	14
	13	11
	15.3	10.3
	2.08	4.04
	0.77	0.94
Untreated control     MEAN SD MF	16	8
	16	13
	11	11
	14.3	10.7
	2.89	2.52
	0.72	0.97
DMSO control     MEAN SD MF	20	11
	19	13
	21	9
	20.0	11.0
	1.00	2.00
	1.00	1.00
Distilled water control*       MEAN SD MF	18	9
	16	13
	17	17
	17.00	13.0
	1.00	4.00
	0.85	1.18
Positive control SAZ (2ug)   MEAN SD MF	1200                   2AA	208
	1100                    2ug	214
	1050	101
	1116.7	174.3
	76.38	63.58
	65.69	15.85

Table No.7: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA1537

Cell count (overnight culture): 2.47 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	5	4
	3	9
	10	16
	6.0	9.7
	3.61	6.03
	0.64	1.04
1581   MEAN SD MF	7	6
	7	7
	7	6
	7.0	6.3
	0.00	0.58
	0.75	0.95
500   MEAN SD MF	6	10
	6	8
	5	9
	5.7	9.0
	0.58	1.00
	0.61	1.35
158.1   MEAN SD MF	6	6
	4	7
	7	8
	5.7	7.0
	1.53	1.00
	0.61	1.05
50   MEAN SD MF	9	7
	8	6
	8	5
	8.3	6.00
	0.58	1.00
	0.89	0.90
15.81   MEAN SD MF	6	5
	7	6
	6	7
	6.3	6.0
	0.58	1.00
	0.68	0.9
5   MEAN SD MF	7	10
	3	11
	3	6
	4.3	9.0
	2.31	2.65
	0.46	1.35
Untreated control     MEAN SD MF	6	8
	8	8
	9	6
	7.7	7.3
	1.53	1.15
	0.82	1.10
DMSO control     MEAN SD MF	8	5
	6	8
	14	7
	9.3	6.7
	4.16	1.53
	1.00	1.00
Positive control 9AA (50ug)     MEAN SD MF	404                     2AA	201
	396                      (2ug)	197
	352	204
	384.0	200.7
	28.00	3.51
	41.14	30.10

Table No.8: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Escherichia coli WP2 uvrA

Cell count (overnight culture): 3.03 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	35	35
	48	43
	31	42
	38.0	40.0
	8.89	4.36
	0.95	1.09
1581   MEAN SD MF	28	49
	42	45
	39	37
	36.3	43.7
	7.37	6.11
	0.91	1.19
500   MEAN SD MF	37	45
	30	32
	39	34
	35.3	37.0
	4.73	7.00
	0.88	1.01
158.1   MEAN SD MF	34	32
	34	41
	52	53
	40.0	42.0
	10.39	10.54
	1.00	1.15
50   MEAN SD MF	29	45
	36	46
	38	39
	34.3	43.3
	4.73	3.79
	0.86	1.18
15.81   MEAN SD MF	43	38
	39	51
	36	43
	39.3	44.0
	3.51	6.56
	0.98	1.20
5   MEAN SD MF	26	35
	30	46
	42	36
	32.7	39.0
	8.33	6.08
	0.82	1.06
Untreated control     MEAN SD MF	35	31
	32	39
	35	35
	34.0	35.0
	1.73	4.00
	0.85	0.95
DMSO control     MEAN SD MF	43	43
	32	33
	45	34
	40.0	36.7
	7.00	5.51
	1.00	1.00
Distilled water control*     MEAN SD MF	38	36
	36	37
	35	41
	36.3	38.0
	1.53	2.65
	0.91	1.04
Positive control MMS (2uL)   MEAN SD MF	1140             2AA	321
	1124             (50ug)	298
	1240	314
	1168.0	311.0
	62.86	11.79
	32.15	8.48

*Distilled water control was used because of MMS

Table No.9: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA98

Cell count (overnight culture): 2.20 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	28	32
	20	31
	23	32
	23.7	31.7
	4.04	0.58
	1.08	1.04
1581   MEAN SD MF	26	38
	22	27
	14	33
	20.7	32.7
	6.11	5.51
	0.94	1.08
500   MEAN SD MF	19	32
	17	31
	23	38
	19.7	33.7
	3.06	3.79
	0.89	1.11
158.1   MEAN SD MF	19	30
	35	38
	22	36
	25.3	34.7
	8.50	4.16
	1.15	1.14
50   MEAN SD MF	27	33
	22	35
	22	36
	23.7	34.7
	2.89	1.53
	1.08	1.14
15.81   MEAN SD MF	27	34
	28	38
	24	39
	26.3	37.0
	2.08	2.65
	1.20	1.22
5   MEAN SD MF	26	26
	22	36
	22	34
	23.3	32.0
	2.31	5.29
	1.06	1.05
Untreated control     MEAN SD MF	23	27
	23	30
	21	34
	22.3	30.3
	1.15	3.51
	1.02	1.00
DMSO control     MEAN SD MF	24	25
	20	35
	22	31
	22.0	30.3
	2.00	5.03
	1.00	1.00
Positive control NPD (4ug)   MEAN SD MF	392                  2AA	2416
	384                  (2ug)	2380
	420	2424
	398.7	2406.7
	18.90	23.44
	18.12	79.34

Table No.10: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA100

Cell count (overnight culture): 1.90 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	98	130
	98	98
	97	109
	97.7	112.3
	0.58	16.26
	1.09	1.14
1581   MEAN SD MF	129	91
	123	117
	106	108
	119.3	105.3
	11.93	13.20
	1.33	1.07
500   MEAN SD MF	121	103
	96	101
	101	102
	106.0	102.0
	13.23	1.00
	1.18	1.04
158.1   MEAN SD MF	103	108
	126	116
	111	126
	113.3	116.7
	11.68	9.02
	1.26	1.19
50   MEAN SD MF	122	100
	111	109
	112	110
	115.0	106.3
	6.08	5.51
	1.28	1.08
15.81   MEAN SD MF	96	101
	99	102
	111	104
	102.0	102.3
	7.94	1.53
	1.13	1.04
5   MEAN SD MF	127	122
	124	108
	110	94
	120.3	108.0
	9.07	14.00
	1.34	1.10
Untreated control     MEAN SD MF	121	92
	113	91
	113	112
	115.7	98.3
	4.62	11.85
	1.29	1.00
DMSO control     MEAN SD MF	91	105
	90	93
	89	97
	90.0	98.3
	1.00	6.11
	1.00	1.00
Distilled water control*     MEAN SD MF	83	92
	129	104
	91	130
	101.0	108.7
	24.58	19.43
	1.12	1.11
Positive control SAZ (2ug)   MEAN SD MF	1120          2AA	2520
	1084           (2ug)	2480
	1100	2452
	1101.3	2484.0
	18.04	34.18
	10.90	25.26

*Distilled water control was used because of SAZ

Table No.11: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA1535

Cell count (overnight culture): 3.15 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	11	9
	18	10
	7	11
	12.0	10.00
	5.57	1.00
	0.77	1.25
1581   MEAN SD MF	17	10
	19	11
	12	10
	16.0	10.3
	3.61	0.58
	1.02	1.29
500   MEAN SD MF	12	11
	14	7
	11	7
	12.3	8.3
	1.53	2.31
	0.79	1.04
158.1   MEAN SD MF	13	15
	16	13
	9	10
	12.7	12.7
	3.51	2.52
	0.81	1.58
50   MEAN SD MF	12	9
	17	10
	19	11
	16.0	10.0
	3.61	1.00
	1.02	1.25
15.81   MEAN SD MF	15	6
	13	11
	19	8
	15.7	8.3
	3.06	2.52
	1.00	1.04
5   MEAN SD MF	11	9
	11	8
	6	16
	9.3	11.0
	2.89	4.36
	0.60	1.38
Untreated control     MEAN SD MF	16	9
	14	7
	17	8
	15.7	8.0
	1.53	1.00
	1.00	1.00
DMSO control     MEAN SD MF	19	10
	14	6
	14	8
	15.7	8.0
	2.89	2.00
	1.00	1.00
Distilled water control*     MEAN SD MF	13	8
	18	12
	14	6
	15.0	8.7
	2.65	3.06
	0.96	1.08
Positive control SAZ (2ug)   MEAN SD MF	1012              2AA	201
	1140              (2ug)	204
	1084	211
	1078.7	205.3
	64.17	5.13
	71.91	25.67

*Distilled water control was used because of SAZ

Table NO.12 Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimuriu

Cell count (overnight culture): 2.49 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	n.d	14
	1R	15
	2R	15
	1.5	14.7
	0.71	0.58
	0.17	1.26
1581   MEAN SD MF	5	6
	9	8
	14	16
	9.3	10.0
	4.51	5.29
	1.08	0.86
500   MEAN SD MF	6	11
	9	14
	10	16
	8.3	13.7
	2.08	2.52
	0.96	1.17
158.1   MEAN SD MF	8	9
	9	13
	12	13
	9.7	11.7
	2.08	2.31
	1.12	1.00
50   MEAN SD MF	6	13
	11	14
	14	15
	10.3	14.0
	4.04	1.00
	1.19	1.20
15.81   MEAN SD MF	3	10
	8	11
	14	12
	8.3	11.0
	5.51	1.00
	0.96	0.94
5   MEAN SD MF	10	11
	10	13
	15	14
	11.7	12.7
	2.89	1.53
	1.35	1.09
Untreated control     MEAN SD MF	5	10
	7	12
	10	16
	7.3	12.7
	2.52	3.06
	0.85	1.09
DMSO control     MEAN SD MF	5	10
	9	12
	12	13
	8.7	11.7
	3.51	1.53
	1.00	1.00
Positive control 9AA (50ug)   MEAN SD MF	404               2AA	206
	412                (2ug)	208
	424	216
	13.3	210.0
	10.07	5.29
	47.69	18.00

n.d.: no data (plate was infected)

R: Reduced background

Table No.13 Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Escherichia coli WP2 uvrA

Cell count (overnight culture): 3.84 x 109 CFU/mL

	Revertant colony number
Concentration (μg/plate)	-S9	+S9
5000   MEAN SD MF	45	44
	46	51
	47	56
	46.0	50.3
	1.00	6.03
	1.02	0.93
1581   MEAN SD MF	43	47
	49	57
	51	61
	47.7	55.0
	4.16	7.21
	1.06	1.02
500   MEAN SD MF	41	52
	41	59
	50	71
	44.0	60.7
	5.20	9.61
	0.98	1.12
158.1   MEAN SD MF	31	52
	36	60
	47	61
	38.0	57.7
	8.19	4.93
	0.84	1.07
50   MEAN SD MF	39	56
	48	56
	57	56
	48.0	56.0
	9.00	0.00
	1.07	1.04
15.81   MEAN SD MF	41	49
	44	56
	45	57
	43.3	54.0
	2.08	4.36
	0.96	1.00
5   MEAN SD MF	36	54
	49	62
	68	64
	51.0	60.0
	16.09	5.29
	1.13	1.11
Untreated control     MEAN SD MF	42	59
	60	40
	36	60
	46.0	43.0
	12.49	11.27
	1.02	0.98
DMSO control     MEAN SD MF	33	52
	45	50
	57	60
	45.0	54.0
	12.00	5.29
	1.00	1.00
Distilled water control*     MEAN SD MF	59	65
	44	62
	46	58
	49.7	61.7
	8.14	3.51
	1.10	1.14
Positive control MMS (2uL)   MEAN SD MF	1140             2AA	324
	1082                (50 ug)	312
	1020	296
	1080.7	310.7
	60.01	14.05
	21.76	5.74

*Distilled water control was used because of MMS

Summary Table of the Initial Mutation Test (test item)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
5000	Mean	22.7	37.0	102.3	102.7	17.0	13.0	6.0	9.7	38.0	40.0
	MF	1.01	1.16	0.97	0.95	0.85	1.18	0.64	1.04	0.95	1.09
1581	Mean	26.3	35.0	103.3	109.0	15.7	11.3	7.0	6.3	36.3	43.7
	MF	1.18	1.09	0.98	1.01	0.78	1.03	0.75	0.95	0.91	1.19
500	Mean	25.7	37.7	106.7	97.0	15.0	12.0	5.7	9.0	35.3	37.0
	MF	1.15	1.18	1.02	0.90	0.75	1.09	0.61	1.35	0.88	1.01
158.1	Mean	19.0	34.7	104.0	96.0	14.7	10.3	5.7	7.0	40.0	42.0
	MF	0.85	1.08	0.99	0.89	0.73	0.94	0.61	1.05	1.00	1.15
50	Mean	23.3	38.0	99.7	90.0	14.7	9.7	8.3	6.0	34.3	43.3
	MF	1.04	1.19	0.95	0.83	0.73	0.88	0.89	0.90	0.86	1.18
15.81	Mean	24.7	38.3	102.7	100.7	15.3	12.0	6.3	6.0	39.3	44.0
	MF	1.10	1.20	0.98	0.93	0.77	1.09	0.68	0.90	0.98	1.20
5	Mean	21.3	30.3	103.0	96.3	15.3	10.3	4.3	9.0	32.7	39.0
	MF	0.96	0.95	0.98	0.89	0.77	0.94	0.46	1.35	0.82	1.06

Summary Table of the Confirmatory Mutation Test (test item):

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
5000	Mean	23.7	31.7	97.7	112.3	12.0	10.0	1.5	14.7	46.0	50.3
	MF	1.08	1.04	1.09	1.14	0.77	1.25	0.17	1.26	1.02	0.93
1581	Mean	20.7	32.7	119.3	105.3	16.0	10.3	9.3	10.0	47.7	55.0
	MF	0.94	1.08	1.33	1.07	1.02	1.29	1.08	0.86	1.06	1.02
500	Mean	19.7	33.7	106.0	102.0	12.3	8.3	8.3	13.7	44.0	60.7
	MF	0.89	1.11	1.18	1.04	0.79	1.04	0.96	1.17	0.98	1.12
158.1	Mean	25.3	34.7	113.3	116.7	12.7	12.7	9.7	11.7	38.0	57.7
	MF	1.15	1.14	1.26	1.19	0.81	1.58	1.12	1.00	0.84	1.07
50	Mean	23.7	34.7	115.0	106.3	16.0	10.0	10.3	14.0	48.0	56.0
	MF	1.08	1.14	1.28	1.08	1.02	1.25	1.19	1.20	1.07	1.04
15.81	Mean	26.3	37.0	102.0	102.3	15.7	8.3	8.3	11.0	43.3	54.0
	MF	1.20	1.22	1.13	1.04	1.00	1.04	0.96	0.94	0.96	1.00
5	Mean	23.3	32.0	120.3	108.0	9.3	11.0	11.7	12.7	51.0	60.0
	MF	1.06	1.05	1.34	1.10	0.60	1.38	1.35	1.09	1.13	1.11

Applicant's summary and conclusion

Conclusions:

The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471. The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test. Since the revertant colonies were in the normal range and no precipitate or cytotoxic effect were observed, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the solvent control and were within the normal biological variability of the test system. No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid. In conclusion, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.