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EC number: 275-203-2 | CAS number: 71119-23-8
- 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
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:
- 2016-08-29 to 2016-09-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
- Version / remarks:
- 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-(4-morpholinyl)ethansulfonic acid hydrate
- Cas Number:
- 1266615-59-1
- Molecular formula:
- C6H13NO4S * xH2O
- IUPAC Name:
- 2-(4-morpholinyl)ethansulfonic acid hydrate
- Test material form:
- solid: particulate/powder
Constituent 1
Method
Species / strainopen allclose all
- 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:
- Type and composition of metabolic activation system
- source of S9 : The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2.; D-35394 Giessen, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium : 10% S9 mix in final culture medium
- quality controls of S9: enzymatic activity, sterility, metabolic capability - Test concentrations with justification for top dose:
- First and second experiment: 16, 50, 160, 500, 1600, 5000 µg/plate, where 5000 µg/plate is the recommended maximum test concentration for soluble non-cytotoxic substances
- Vehicle / solvent:
- - Vehicle/solvent used: ultrapure water
Controlsopen allclose all
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylenediamine
- Remarks:
- without S9 mix, TA98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with S9 mix, S. typhimurium strains and E. coli WP2 uvrA
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without S9 mix, TA100 and TA1535
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix, TA1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9 mix, E.coli WP2 uvrA
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation); preincubation
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: number of revertant colonies (with TA98 and TA100) - Evaluation criteria:
- A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces 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:
- According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Results and discussion
Test resultsopen allclose all
- 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
- True negative controls validity:
- not applicable
- 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
- True negative controls validity:
- not applicable
- 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
- True negative controls validity:
- not applicable
- 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
- True negative controls validity:
- not applicable
- 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
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
The pH of the test item stock solution (prepared for the highest concentration) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at the highest concentration of 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the different overlays without test item stock solution was in the range of 7.12 - 7.30, and the pH of overlays completed with test item stock solution was ~7 in all cases.
Extremes of pH could have influencing effect on the mutagenicity results; however in this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the result interpretation.
- Water solubility: 50 mg/mL
- Precipitation and time of the determination: no
RANGE-FINDING/SCREENING STUDIES:
Based on the solubility test, a stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 μg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
STUDY RESULTS
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
Sporadically increased revertant colony numbers were noticed in the performed experiments; these increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA1537 strain at 16 μg/plate, in presence of metabolic activation (+S9). This higher value however remained in the range of the corresponding vehicle historical control data. The mutation rate was 1.81, which was far below the genotoxicological threshold for being positive, was a unique value without any biological significance.
- Signs of toxicity : no cytotoxicity observed
- Individual plate counts : see attached results
- Mean number of revertant colonies per plate and standard deviation : see attached results
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see attached results
- Negative (solvent/vehicle) historical control data: see attached results
Any other information on results incl. tables
Table 1: Summary Table of the Results of the Concentration Range Finding Test
Range finding Test (Informatory Toxicity Test) |
||||||||
Concentration (µg/plate) |
Salmonella typhimurium tester strains |
|||||||
TA 98 |
TA 100 |
|||||||
-S9 |
+ S9 |
-S9 |
+S9 |
|||||
Mean values of revertants per plate and Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
16.7 |
0.88 |
21.3 |
1.02 |
95.7 |
1.03 |
115.0 |
0.93 |
DMSO Control |
19.0 |
1.00 |
23.0 |
1.00 |
- |
- |
108.7 |
1.00 |
Ultrapure Water Control |
19.0 |
1.00 |
21.0 |
1.00 |
93.3 |
1.00 |
123.3 |
1.00 |
5000 |
18.0 |
0.95 |
25.3 |
1.21 |
107.3 |
1.15 |
129.7 |
1.05 |
1600 |
19.3 |
1.02 |
30.7 |
1.46 |
100.0 |
1.07 |
112.7 |
0.91 |
500 |
20.7 |
1.09 |
26.7 |
1.27 |
131.3 |
1.41 |
122.0 |
0.99 |
160 |
19.3 |
1.02 |
22.3 |
1.06 |
94.0 |
1.01 |
120.0 |
0.97 |
50 |
20.3 |
1.07 |
29.7 |
1.41 |
102.7 |
1.10 |
126.7 |
1.03 |
16 |
19.0 |
1.00 |
21.7 |
1.03 |
96.0 |
1.03 |
120.7 |
0.98 |
5 |
15.7 |
0.82 |
29.7 |
1.41 |
104.0 |
1.11 |
114.7 |
0.93 |
NPD (4 µg) |
283.3 |
14.91 |
- |
- |
- |
- |
- |
- |
SAZ (2 µg) |
- |
- |
- |
- |
12240 |
13.11 |
- |
- |
2 AA (2 µg) |
- |
- |
1114.7 |
48.46 |
- |
- |
17387 |
6.00 |
MR: Mutation Rate
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.
Table 2: Summary Table of the Results of the Initial Mutation Test
Confirmatory Mutation Test (Pre-Incubation Test) |
||||||||||||||||||||
Concentration (µg/plate) |
Salmonella typhimurium tester strains |
Escherichia coli WP2uvrA |
||||||||||||||||||
TA 98 |
TA 100 |
TA 1535 |
TA 1537 |
|||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|||||||||||
Mean values of revertants per plate Mutation rate (MR) |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Mean |
MR |
Untreated Control |
17.0 |
0.98 |
20.3 |
0.94 |
88.7 |
0.81 |
144.7 |
1.17 |
9.0 |
0.96 |
11.7 |
1.09 |
8.7 |
1.63 |
5.3 |
1.00 |
22.3 |
1.24 |
24.3 |
1.00 |
DMSO Control |
17.3 |
1.00 |
22.3 |
1.00 |
- |
- |
109.7 |
1.00 |
- |
- |
9.0 |
1.00 |
7.7 |
1.00 |
7.7 |
1.00 |
- |
- |
25.3 |
1.00 |
Ultrapure Water Control |
17.3 |
1.00 |
21.7 |
1.00 |
110.0 |
1.00 |
123.7 |
1.00 |
9.3 |
1.00 |
10.7 |
1.00 |
5.3 |
1.00 |
5.3 |
1.00 |
18.0 |
1.00 |
24.3 |
1.00 |
5000 |
24.7 |
1.42 |
28.7 |
1.32 |
105.0 |
0.95 |
114.7 |
0.93 |
8.3 |
0.89 |
13.3 |
1.25 |
5.3 |
1.00 |
6.7 |
1.25 |
23.7 |
1.31 |
28.0 |
1.15 |
1600 |
25.7 |
1.48 |
29.7 |
1.37 |
99.7 |
0.91 |
111.0 |
0.90 |
10.3 |
1.11 |
10.3 |
0.97 |
5.7 |
1.06 |
7.3 |
1.38 |
25.0 |
1.39 |
31.0 |
1.27 |
500 |
19.3 |
1.12 |
21.0 |
0.97 |
105.3 |
0.96 |
100.3 |
0.81 |
10.7 |
1.14 |
10.3 |
0.97 |
4.7 |
0.88 |
6.0 |
1.13 |
21.7 |
1.20 |
27.3 |
1.12 |
160 |
20.0 |
1.15 |
27.7 |
1.28 |
96.0 |
0.87 |
91.0 |
0.74 |
10.3 |
1.11 |
13.3 |
1.25 |
6.0 |
1.13 |
7.0 |
1.31 |
20.3 |
1.13 |
24.0 |
0.99 |
50 |
18.0 |
1.04 |
17.7 |
0.82 |
88.7 |
0.81 |
104.3 |
0.84 |
10.3 |
1.11 |
10.3 |
0.97 |
9.0 |
1.69 |
5.7 |
1.06 |
22.0 |
1.22 |
25.0 |
1.03 |
16 |
22.3 |
1.29 |
32.7 |
1.51 |
91.0 |
0.83 |
96.3 |
0.78 |
8.7 |
0.93 |
11.7 |
1.09 |
7.3 |
1.38 |
9.7 |
1.81 |
16.0 |
0.89 |
25.0 |
1.03 |
NPD (4 µg) |
470.0 |
27.12 |
- |
|
|
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
SAZ (2 µg) |
- |
- |
- |
- |
1549.3 |
-14.08 |
- |
- |
979.3 |
104.93 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
9AA (50 µg) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
860.0 |
112.27 |
- |
- |
- |
- |
- |
- |
MMS (2 µL) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
2AA (2 µg) |
- |
- |
1176.0 |
52.66 |
- |
- |
1008.0 |
9.19 |
- |
- |
179.3 |
19.93 |
- |
- |
169.0 |
22.04 |
- |
- |
- |
- |
2AA (50 µg) |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
342.0 |
13.50 |
MR: Mutation Rate
Remarks: Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.
Table 3: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)
|
Bacterial strains |
||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|||
Historical control data of untreated control |
-S9 |
Average |
21.4 |
106.0 |
10.4 |
8.1 |
25.6 |
SD |
3.7 |
27.3 |
1.5 |
2.5 |
5.5 |
||
Minimum |
9 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
157 |
23 |
19 |
45 |
||
+S9 |
Average |
28.0 |
117.1 |
11.9 |
9.0 |
34.3 |
|
SD |
4.2 |
19.4 |
1.5 |
2.0 |
5.4 |
||
Minimum |
12 |
75 |
4 |
3 |
18 |
||
Maximum |
48 |
166 |
24 |
20 |
56 |
||
Historical control data of DMSO control |
-S9 |
Average |
20.9 |
101.4 |
10.3 |
7.9 |
24.9 |
SD |
3.5 |
26.2 |
1.4 |
2.5 |
4.9 |
||
Minimum |
10 |
65 |
3 |
2 |
11 |
||
Maximum |
39 |
150 |
23 |
20 |
44 |
||
+S9 |
Average |
27.1 |
114.7 |
12.0 |
8.8 |
34.2 |
|
SD |
4.0 |
19.3 |
1.5 |
2.1 |
5.2 |
||
Minimum |
15 |
71 |
4 |
3 |
16 |
||
Maximum |
48 |
161 |
24 |
20 |
56 |
||
Historical control data of Water control |
-S9 |
Average |
22.4 |
105.5 |
10.4 |
7.5 |
26.3 |
SD |
3.6 |
27.6 |
1.6 |
2.3 |
5.9 |
||
Minimum |
12 |
67 |
3 |
2 |
13 |
||
Maximum |
36 |
156 |
24 |
15 |
47 |
||
+S9 |
Average |
28.0 |
117.4 |
11.5 |
8.7 |
35.2 |
|
SD |
4.0 |
19.8 |
1.4 |
2.3 |
5.2 |
||
Minimum |
15 |
83 |
4 |
4 |
18 |
||
Maximum |
43 |
166 |
22 |
16 |
56 |
||
Historical control data of positive controls |
-S9 |
Average |
255.6 |
958.9 |
842.1 |
467.4 |
712.3 |
SD |
30.7 |
149.9 |
134.0 |
105.7 |
57.5 |
||
Minimum |
123 |
522 |
354 |
109 |
320 |
||
Maximum |
647 |
1927 |
1871 |
1498 |
1283 |
||
+S9 |
Average |
1224.8 |
1431.9 |
165.4 |
148.0 |
264.7 |
|
SD |
293.8 |
339.9 |
35.1 |
21.3 |
74.2 |
||
Minimum |
409 |
581 |
85 |
68 |
141 |
||
Maximum |
2587 |
2923 |
507 |
407 |
487 |
Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA
SD: Standard deviation;
DMSO: Dimethyl sulfoxide
Applicant's summary and conclusion
- Conclusions:
- The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
- Executive summary:
A study according OECD TG 471 was performed with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).
Based on the results of the Solubility and the Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity.
Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
5000; 1600; 500; 160; 50 and 16 μg/plate.
In the preliminary experiments the pH of the aqueous test item solution (50 mg/mL) was found as 3.47. Extremes of pH could have influencing effect on the mutagenicity results, therefore in the Initial and Confirmatory Mutation Tests the pH of the test item stock solution (prepared for the highest concentration of 50 mg/mL) and additionally the pH of the overlay (top agar-phosphate buffer-test item system (at 50 mg/mL)) was checked and recorded. The pH values of the test item stock solutions (50 mg/mL) were 3.50 and 3.52. The pH of the test item containing overlays was ~7 in both experiments. In this study the acidic test item solutions had only slight effect on the buffered overlay system. Unequivocal negative results were obtained and the additional information about the pH conditions was not involved into the mutagenicity result interpretation.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.
The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterium tester strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
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