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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria: Key study. Method according to OECD 471, GLP study. The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

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:
21 September 2020 to 15. October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his D, his C, his G, tryp E
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 Sprague Dawley rat liver homogenate by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox11101-5-4244 validated on 21.08.2020 – expiry date: 28.04.2022)
- method of preparation of S9 mix:
S9 fraction 10 %
MgCL2-6H2O 8 mM
KCl 33 mM
Glucose-6-Phosphate Na2 5 mM
NADP Na2 4 mM
Phosphate buffer pH 7.4 0.1 M
- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9-mix/plate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): sterility test
Test concentrations with justification for top dose:
Preliminary bacteriostatic activity controls with TA100: 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg.The highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. No bacterostatic activity could be detected.
Main Assay with 50µg, 150µg, 500µg, 1500µg, 2500µg and 5000µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
data concerning the solubility, the visual homogeneity and the stability of the test item
in the vehicle are available.

- Justification for percentage of solvent in the final culture medium:
No control of the concentration of the test item in the vehicle by means of an analytical method has been performed
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Anthramine & cis-Platinum (II) Diammine Dichloride
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2 assays, 5 bacteria , each with and without metabolic activation + sterility test and bacteriostatic activity control

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): (1-9 x 103 bacteria/mL
- Test substance added in suspension from stock solution 100mg/mL in EtOH

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Assay 2 with metabolic activation preincubation 30min @ 37C
- Exposure duration/duration of treatment: 48 to 72 hours
- Harvest time after the end of treatment (sampling/recovery times): after 48 to 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.:relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The determination of mutagenic activity of the test item is its capacity to induce revertant colonies in mutated Salmonella typhimurium and Escherichia coli strains. Revertant colonies grow in absence of the amino-acid of which they are originally dependant.
Evaluation criteria:
The following validity criteria were checked to validate each experiment:
• the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
• the spontaneous reversion rate of the absolute negative control shall comply with the historical
values of the laboratory,
• The spontaneous reversion rate of the blank control shall not be statistically different from
absolute negative control.
• the mean number of revertant colonies obtained for each strain and the corresponding positive
control, with and/or without metabolic activation shall comply with the historical values of the
laboratory.
• Negative and positive values should not show significant difference with the historical values of
the laboratory (± 2 standard deviations)
Statistics:
n/a
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
5000 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
5000 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
5000 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Remarks:
5000 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary cytotoxicity testing (strain TA100)


Ames test:
- Signs of toxicity Non observed
- Individual plate counts 3 plates per stain per dilution with and without s9
- Mean number of revertant colonies per plate and standard deviation See tables in 'any other information on results incl.tables'



HISTORICAL CONTROL DATA not speciefied. Historical data without S9, with S9 and without preincubation and with s9 and with preincubation mean and min max values available.

Table TA1538












































































































































































StrainTA 1535
Assay12
Metabolic activationWithoutWithWithoutWith
 MeanStandard deviationRMeanStandard deviationRMeanStandard deviationRMeanStandard deviationR
Negative Control12.672.31_14.335.69_9.673.51_156.08_
Positive Control solvent8.332.08_16.332.08_124.36_13.332.52_
Positive Control73647.8488.32175.677.7710.8673.6799.4956.1459.677.374.48
Vehicle13.331.53_112.65_72.65_112.65_
Test item:5000 µg*13.672.081.0318.332.081.67115.21.5712.332.521.12
1500 µg10.671.530.8181.731.649.332.521.3310.334.040.94
500 µg121.730.9124.361.097.672.081.16.672.080.61
150 µg1431.0516.671.151.526.672.080.9510.333.210.94
50 µg14.330.581.0814.335.861.37.331.531.0511.331.531.03

Table TA1537












































































































































































StrainTA 1537
Assay12
Metabolic activationWithoutWithWithoutWith
 MeanStandard deviationRMeanStandard deviationRMeanStandard deviationRMeanStandard deviationR
Negative Control6.673.06_61_3.331.53_5.331.53_
Positive Control solvent6.672.52_9.673.21_5.333.06_51.73_
Positive Control1007.33123.33151.148.672.525.03649.33134.17121.848.336.669.67
Vehicle5.331.15_74.36_5.331.15_52.65_
Test item:5000 µg*8.333.211.568.334.161.195.672.081.065.330.581.07
1500 µg711.318.333.211.194.331.150.813.670.580.73
500 µg8.331.531.5610.674.621.52310.564.673.210.93
150 µg6.332.081.197.332.081.055.670.581.064.670.580.93
50 µg5.331.5318.671.151.24520.945.672.081.13

Table TA98












































































































































































StrainTA 98
Assay12
Metabolic activationWithoutWithWithoutWith
 MeanStandard deviationRMeanStandard deviationRMeanStandard deviationRMeanStandard deviationR
Negative Control17.336.66_302.65_19.670.58_31.331.53_
Positive Control solvent17.670.58_32.674.62_25.332.08_331_
Positive Control283.3356.0516.04658.3357.120.242058.8516.58282.6723.168.57
Vehicle22.675.03_30.336.03_241.73_351_
Test item:5000 µg*19.332.080.8532.675.861.08205.570.8331.671.530.9
1500 µg16.334.040.7226.671.150.8818.333.510.7640.672.521.16
500 µg24.333.211.072920.96231.730.9632.672.520.93
150 µg27.672.521.2226.676.030.88231.730.9637.672.081.08
50 µg17.334.040.76294.360.9627.332.081.1436.334.041.04

Table TA100












































































































































































StrainTA 100
Assay12
Metabolic activationWithoutWithWithoutWith
 MeanStandard deviationRMeanStandard deviationRMeanStandard deviationRMeanStandard deviationR
Negative Control863.46_882_686.08_79.679.29_
Positive Control solvent85.676.11_90.337.57_707_81.672.52_
Positive Control1240105.4814.47962.6799.8910.71075.749.0315.3751311.536.28
Vehicle68.6717.01_72.3310.07_70.677.09_828.54_
Test item:5000 µg*70.332.521.0278.3310.691.0868.676.030.9779.675.510.97
1500 µg67.3314.640.9879.672.081.158.677.090.8381.677.231
500 µg68.677.64190.336.031.25536.080.757670.93
150 µg65.677.090.9676.338.741.06576.560.8171.676.510.87
50 µg5920.8675.3311.681.0456.673.510.867.672.520.83

Table Ecoli












































































































































































StrainE Coli
Assay12
Metabolic activationWithoutWithWithoutWith
 MeanStandard deviationRMeanStandard deviationRMeanStandard deviationRMeanStandard deviationR
Negative Control62.334.16_1006.56_634_9310.39_
Positive Control solvent602_96.675.13_80.670.58_93.3312.5_
Positive Control3067.945.139824.644.1232615.524.04307.3316.443.29
Vehicle57.334.51_89.338.02_625.2_877.94_
Test item:5000 µg*54.333.790.9568.677.090.77675.571.0890.679.871.04
1500 µg6031.05849.540.9474.677.371.288.3317.391.02
500 µg57.334.51168.333.060.76576.560.9283.6710.50.96
150 µg532.650.9270.677.020.7967.679.291.0991.6710.071.05
50 µg57.335.1316830.76702.651.1384.337.510.97
Conclusions:
The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.
Executive summary:

The mutagenic activity of the test item was determined according to OECD 471 under GLP conditions. Four different histidine dependent Salmonella typhimurium strains (TA1535, 1537, 98 & 100) and a tryptophan dependent Escherichia coli WP2 uvrA strain were exposed to the test subtance in EtOH solution ranging from 50 to 5000 μg/plate in triplicate, including negative, positive and positive-solvent controls with and without metabolic activation. The plates were screened for revertant colonies that gained the ability to be independent of the former dependent histidine or tryptophane.  No increase in revertant colonies could be observed. The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

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

Additional information

In-vitro gene mutation in bacteria:


The mutagenic activity of the test item was determined according to OECD 471 under GLP conditions. Four different histidine dependent Salmonella typhimurium strains (TA1535, 1537, 98 & 100) and a tryptophan dependent Escherichia coli WP2 uvrA strain were exposed to the test subtance in EtOH solution ranging from 50 to 5000 μg/plate in triplicate, including negative, positive and positive-solvent controls with and without metabolic activation. The plates were screened for revertant colonies that gained the ability to be independent of the former dependent histidine or tryptophane.  No increase in revertant colonies could be observed. The test item does not have mutagenic activity under the test conditions, with or without metabolic activation.

Justification for classification or non-classification

Based on the available information, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.

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