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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in the Bacterial Reverse Mutation Assay according to OECD 471 (reference 7.6.1 -1).

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:
2016-08-29 to 2016-09-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
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; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix : The complete S9 Mix was freshly prepared with components: Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL + Rat liver homogenate (S9) 100 mL + Salt solution for S9 Mix 400 mL
- volume of S9 mix in the final culture medium: 0.5 mL of the S9 Mix (10 %) was added to each overlay tube.
Test concentrations with justification for top dose:
16, 50, 160, 500, 1600 and 5000 µg/plate
At the concentration choice the guideline criterion for soluble, non-toxic test compounds was taken into consideration where the recommended maximum test concentration is 5000 μg/plate.
Vehicle / solvent:
- Vehicle used: ultrapure water for test item, SAZ and MMS; DMSO for NPD, 9AA and 2AA

- Justification for choice of vehicle: The vehicle is compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine (NPD, TA98 without metabolic activation) and 2-aminoanthracene (2AA, all strains, with metabolic activation)
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 agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 ºC
- Exposure duration/duration of treatment: ca. 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; revertant colony numbers
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.
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:
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:
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
- 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 5.61 and 4.28. 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 in the range of 7.32 - 7.35 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: The test item was well soluble in water.
- Precipitation and time of the determination: No precipitation of the test item was observed in the Initial and Confirmatory Mutation Tests on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: Based on the solubility test, 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. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

STUDY RESULTS
Ames test:
- Signs of toxicity: No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.

HISTORICAL CONTROL DATA: Please refer to table 3.

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations
(µg/plate)
Salmonella typhimurium tester strains Escherichia coli 
TA 98 TA100 TA 1535 TA 1537 WP2 uvrA
-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 0.98 20.3 0.94 88.7 0.81 144.7 1.17 9 0.96 11.7 1.09 8.7 1.63 5.3 1 22.3 1.24 24.3 1
DMSO Control 17.3 1 22.3 1 - - 109.7 1 - - 9 1 7.7 1 7.7 1 - - 25.3 1
Ultrapure Water Control 17.3 1 21.7 1 110 1 123.7 1 9.3 1 10.7 1 5.3 1 5.3 1 18 1 24.3 1
5000 17 0.98 19 0.88 100.3 0.91 117.3 0.95 9.7 1.04 15.7 1.47 7 1.31 8 1.5 21 1.17 27 1.11
1600 15.3 0.88 19.3 0.89 101 0.92 114.7 0.93 7.3 0.79 8.7 0.81 5.7 1.06 5.7 1.06 19.7 1.09 20.7 0.85
500 20.3 1.17 19.7 0.91 96.3 0.88 101.3 0.82 10.7 1.14 11.7 1.09 6.7 1.25 5 0.4 20 1.11 25 1.03
160 17 0.98 21.3 0.98 95.3 0.87 136.7 1.11 13.7 1.46 9 0.84 4.3 0.81 7 1.31 21.7 1.2 30.3 1.25
50 14 0.81 24 1.11 102.7 0.93 109.7 0.89 9.3 1 13 1.22 4.7 0.88 6.7 1.25 19.7 1.09 29 1.19
16 19.3 1.12 26 1.2 95 0.86 132 1.07 10.3 1.11 12.3 1.16 3.7 0.69 6 1.13 22.3 1.24 24 0.99
NPD (4 µg/plate) 470 27.12 - - - - - - - - - - - - - - - - - -
SAZ (2 µg/plate) - - - - 1549.3 14.08 - - 979.3 104.93 - - - - - - - - - -
9AA (50 µg/plate) - - - - - - - - - - - - 860 112.17 - - - - - -
MMS (2 µL/plate) - - - - - - - - - - - - - - - - 617.3 34.3 - -
2AA (2 µg/plate) - - 1176 52.66 - - 1008 9.19 - - 179.3 19.93 - - 169 22.04 - - - -
2AA (50 µg/plate) - - - - - - - - - - - - - - - - - - 342 13.5

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

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 refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-incubation Test)

Concentrations
(µg/plate)
Salmonella typhimurium tester strains Escherichia coli
TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA
-S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean values of revertauts 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 15.7 1.09 20.3 1.02 74.7 0.86 115 0.97 12.3 1 9.7 1.04 7.3 1.47 6.7 1.17 19 1.04 29.7 0.75
DMSO control 13.3 1 14.7 1 - - 100.3 1 - - 10 1 5.3 1 6.3 1 - - 36 1
Ultrapure Water Control 14.3 1 20 1 86.3 1 118.3 1 12.3 1 9.3 1 5 1 5..7 1 18.3 1 39.7 1
5000 15.3 1.07 17.3 0.87 84.7 0.98 112.3 0.95 13 1.05 12.3 1.32 7 1.4 9.3 1.65 25.3 1.38 27.7 0.7
1600 12.7 0.88 23.3 1.17 81.3 0.94 130 1.1 10.7 0.86 12.3 1.32 5.7 1.13 7.7 1.35 23.3 1.27 30 0.76
500 21.3 1.49 15.7 0.78 79.7 0.92 111.7 0.94 11.3 0.92 12.7 1.36 4 0.8 6.7 1.18 20 1.09 25.3 0.64
160 15.3 1.07 18.3 0.92 84.3 0.98 119.3 1.01 10 0.81 10 1.07 6.3 1.27 11.7 2.06 32.3 1.76 31.7 0.8
50 18.7 1.3 17.7 0.88 80.7 0.93 114 0.96 10.7 0.86 9.3 1 3.3 0.67 9.7 1.71 27.3 1.49 30.7 0.77
16 13.3 0.93 17.3 0.87 86 1 122 1.03 12 0.97 13 1.39 4.3 0.87 6 1.06 30 1.64 31.7 0.8
NPD (4 µg/plate) 293.7 22.03 - - - - - - - - - - - - - - - - - -
SAZ (2 µg/plate) - - - - 852 9.87 - - 968 78.49 - - - - - - - - - -
9AA (50 µg/plate) - - - - - - - - - - - - 598.7 112.25 - - - - - -
MMS (2 µL/plate) - - - - - - - - - - - - - - - - 960 52.36 - -
2AA (2 µg/plate) - - 597.3 40.73 - - 1186.7 11.83 - - 120.7 12.07 - - 96 15.16 - - - -
2AA (50 µg/plate) - - - - - - - - - - - - - - - - - - 165.3 4.59

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoactidine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

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 refers to the ultrapure water; the mutation rate of NPD, 9AA and 2AA refers to DMSO.

Table 3: Historical Control Values for Revertants Plate (for the Period of 2008-2015)

  Bacterial strains
Historical control data of untreated control     TA98 TA100 TA1535 TA1537 E. coli
  Average 21.4 106 10.4 8.1 25.6
-S9 SD 3.7 27.3 1.5 2.5 2.5
  Minimum 9 65 3 2 11
  Maximum 39 157 23 19 45
    TA98 TA100 TA1535 TA1537 E. coli
  Average 28 117.1 11.9 9 34.3
+S9 SD 4.2 19.4 1.5 2 5.4
  Minimum 12 75 4 3 18
  Maximum 48 166 24 20 56
  Bacterial strains
Historical control data of DMSO control     TA98 TA100 TA1535 TA1537 E. coli
  Average 20.9 101.4 10.3 7.9 24.9
-S9 SD 3.5 26.2 1.4 2.5 4.9
  Minimum 10 65 3 2 11
  Maximum 39 150 23 20 44
    TA98 TA100 TA1535 TA1537 E. coli
  Average 27.1 114.7 12 8.8 34.2
+S9 SD 4 19.3 1.4 2.1 5.2
  Minimum 15 71 4 3 16
  Maximum 48 161 24 20 56
  Bacterial strains
Historical control data of Water control     TA98 TA100 TA1535 TA1537 E. coli
  Average 22.4 105.5 10.4 7.5 26.3
-S9 SD 3.6 27.6 1.6 2.3 5.9
  Minimum 12 67 3 2 13
  Maximum 36 156 24 15 47
    TA98 TA100 TA1535 TA1537 E. coli
  Average 28 117.4 11.5 8.7 35.2
+S9 SD 4 19.8 1.4 2.3 5.2
  Minimum 15 83 4 4 18
  Maximum 43 166 22 16 56
  Bacterial strains
Historical control data of positive controls     TA98 TA100 TA1535 TA1537 E. coli
  Average 255.6 958.9 842.1 467.4 712.3
-S9 SD 30.7 149.9 134 105.7 57.5
  Minimum 123 522 354 109 320
  Maximum 647 1927 1871 1498 1283
    TA98 TA100 TA1535 TA1537 E. coli
  Average 1224.8 1431.9 165.4 148 264.7
+S9 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

Conclusions:
The test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in the Bacterial Reverse Mutation Assay according to OECD 471.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471.

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 values of the aqueous test item solutions (50 mg/mL) were found as 3.77 and 3.82. 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 5.61 and 4.28. The pH of the test item containing overlays was in the range of 7.32-7.35 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 test item 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471.

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 values of the aqueous test item solutions (50 mg/mL) were found as 3.77 and 3.82. 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 5.61 and 4.28. The pH of the test item containing overlays was in the range of 7.32-7.35 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 test item 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.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.