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Genetic toxicity in vitro

Description of key information

Genetic Toxicity in vitro: Ames (Gijsbrechts, 2018)

Under the conditions of this study, the test material was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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:
09 August 2018 to 29 August 2018
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine locus
E. coli: Tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames)
- The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- Stock cultures were stored in the freezer (-150 °C).
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK)
- The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was checked to confirm the tryptophanrequirement, UV-sensitivity and the number of spontaneous revertants at least every year.
- Stock cultures were stored in the freezer (-150 °C).
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Dose-range Finding Test: 1.7, 5.4, 17, 52, 164, 512, 1 600 and 5 000 μg/plate
First Experiment: Direct Plate Assay: 52, 164, 512, 1 600 and 5 000 µg/plate
Second Experiment: Pre-Incubation Assay: 17, 52, 164, 512, 1 600 and 5 000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a clear colourless solution in dimethyl sulfoxide.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191 (without S9, TA1537) and 2-aminoanthracene (with S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation

DOSE-RANGE FINDING TEST
- Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1 600 and 5 000 μg/plate were tested in triplicate.
-The highest concentration of the test material used in the subsequent mutation assays was 5 000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. To complete the data of the second experiment, an additional experiment was performed with the tester strains TA1535, TA1537, TA98 and TA100 in the absence and/or presence of S9-mix.
-The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

FIRST EXPERIMENT: DIRECT PLATE ASSAY
- The dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test material was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- The test material was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1 °C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in dimethyl sulfoxide. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: 3

COLONY COUNTING
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
- No formal hypothesis testing was done.
- Any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535 & TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
FIRST EXPERIMENT: DIRECT PLATE ASSAY
- Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1 600 and 5 000 μg/plate.
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at the concentrations of 1 600 and 5 000 μg/plate and no precipitate was observed at the end of the incubation period.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed tester strain TA1535 in the absence and presence of S9-mix and in TA1537, TA98 and TA100 in the absence of S9-mix.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- Based on the results of the first mutation assay, the test material was tested up to the dose level of 5 000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at the concentrations of 1 600 and 5 000 μg/plate and no precipitate was observed at the end of the incubation period.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix, in which no toxicity was observed.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

ADDITIONAL MUTATION EXPERIMENT
- In the second mutation experiment, due to cytotoxicity, only three analysable dose levels were left for the determination of the mutagenicity of the test material in the tester strains TA1535 and TA98 in the presence of S9-mix and TA1537 and TA100 in the absence and presence of S9-mix. Therefore an additional experiment was performed with these tester strains. Two additional dose levels, 1.7 and 5.4 μg/plate, were tested to complete the data of the second experiment.
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA1537 in the absence of S9-mix, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 5.4 μg/plate. However, since no dose-relationship was observed, no toxicity was observed at the higher dose level of 17 μg/plate, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies and is coupled to a low solvent control value.
- Mutagenicity: In the additional mutation experiment, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
- All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Conclusions:
Under the conditions of this study, the test material was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions.

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test material was initially tested up to concentrations of 5 000 μg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at dose levels of 512 μg/plate and above. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test material was tested up to concentrations of 5 000 μg/plate in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strain TA1535 in the absence and presence of S9-mix and in TA1537 and TA98 in the absence of S9-mix.

In the second mutation experiment, the test material was tested up to concentrations of 5 000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix, in which no toxicity was observed. In the second mutation experiment, due to cytotoxicity, only three analysable dose levels were left for the determination of the mutagenicity of the test material in the tester strains TA1535 and TA98 in the presence of S9-mix and TA1537 and TA100 in the absence and presence of S9-mix. Therefore an additional experiment was performed with these tester strains. Two additional dose levels, 1.7 and 5.4 μg/plate, were tested to complete the data of the second experiment. No toxicity or precipitate were observed at these dose levels.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

Under the conditions of this study, the test material was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic Toxicity in vitro: Ames (Gijsbrechts, 2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test material was initially tested up to concentrations of 5 000 μg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at dose levels of 512 μg/plate and above. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test material was tested up to concentrations of 5 000 μg/plate in the tester strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strain TA1535 in the absence and presence of S9-mix and in TA1537 and TA98 in the absence of S9-mix.

In the second mutation experiment, the test material was tested up to concentrations of 5 000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix, in which no toxicity was observed. In the second mutation experiment, due to cytotoxicity, only three analysable dose levels were left for the determination of the mutagenicity of the test material in the tester strains TA1535 and TA98 in the presence of S9-mix and TA1537 and TA100 in the absence and presence of S9-mix. Therefore an additional experiment was performed with these tester strains. Two additional dose levels, 1.7 and 5.4 μg/plate, were tested to complete the data of the second experiment. No toxicity or precipitate were observed at these dose levels.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

Under the conditions of this study, the test material was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.