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EC number: 275-833-8 | CAS number: 71675-87-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Key study: Test method OECD 471.GLP study. The test item had no mutagenic activity in the bacterium tester strains.
Link to relevant study records
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- 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
- 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 - Target gene:
- Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
- 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 / 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. - 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.
- 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
- 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
- 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 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
- 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. - 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.
Reference
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 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ggenetic toxicity in vitro:
Key study: 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) up to 5000 μg/plate. 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. Inhibitory, cytotoxic effect was observed in the confirmatory test in Salmonella typhimurium strain TA1537 at 5000 µg/plate without metabolic activation. The test was 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.
Justification for classification or non-classification
Based on available data, the substance is not classified according to CLP Regulation (EC) no. 1272/2008.
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