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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
An Ames study (Verbaan I.A.J., PhD., 2016) is available which is key study. It is concluded that the test substance is equivocal in tester strain TA100 in the presence of S9-mix in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98) 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:
From 10 Mar to 07 Apr 2016
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
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: E010016692Purity: 99.3%
Target gene:
Histidine gene in Salmonella typhimurium, tryptophan gene in Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range finding test/first mutation experiment: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plateExperiment 2: 492, 878, 1568, 2800 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide- Justification for choice of solvent/vehicle: A solubility test was performed. The test item could not be dissolved in water. The test item was soluble in dimethyl sulfoxide. Therefore dimethyl sulfoxide was used as solvent in this project.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
In the absence of S-9 mix for TA1535 strains
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
In the absence of S-9 mix for TA1537 strains.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In the absence of S-9 mix for TA98 strains.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
In the absence of S-9 mix for TA100 strains.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
In the absence of S-9 mix for WP2uvrA strains.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
In the presence of S-9 mix for TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Dose range finding test, experiment 2 and experiment 3: in agar (plate incorporation)DURATION- Preincubation period:- Exposure duration: 48 ± 4 h both for dose range finding test, experiment 2 and experiment 3- Expression time (cells in growth medium):- Selection time (if incubation with a selection agent):- Fixation time (start of exposure up to fixation or harvest of cells):SELECTION AGENT (mutation assays):SPINDLE INHIBITOR (cytogenetic assays):STAIN (for cytogenetic assays):NUMBER OF REPLICATIONS: 3 replications both for dose range finding test, experiment 2 and experiment 3DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other:- Any supplementary information relevant to cytotoxicity:OTHER EXAMINATIONS:- Determination of polyploidy:- Determination of endoreplication:- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): - OTHER:
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Statistics:
A test item is considered negative (not mutagenic) in the test if:a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.b) The negative response should be reproducible in at least one follow-up experiment. A test item is considered positive (mutagenic) in the test if:a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.b) In case a follow up 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.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not specified
Vehicle 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:
not specified
Vehicle 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:
not specified
Vehicle 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:
not specified
Vehicle 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:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose range finding test/first mutation experiment:- Precipitate: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.- Toxicity: To determine the toxicity of test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. Toxicity, as evidenced by a biologically relevant decrease in the number of revertants, was observed in tester strains TA1535 and TA1537 in the absence of S9-mix at the highest tested concentration. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants was observed in all other tester strains.- Mutagenicity: No increase in the number of revertants was observed upon treatment with test substance under all conditions tested.Experiment 2:- Precipitate: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period.- Toxicity: In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. In strains TA1535, TA1537 and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no doserelationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.- Mutagenicity: In tester strain TA100, PF-06811569 induced up to 1.8-fold increases in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with test substance.Experiment 3:- Precipitate: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period.- Toxicity: In the third mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.- Mutagenicity: In tester strain TA100, test substance induced up to 2.1-fold increases in the number of revertant colonies compared to the solvent control in the presence of S9-mix. In the other tester strains, no increase in the number of revertants was observed upon treatment with test substance.
Remarks on result:
other: Since no dose relationship was observed the results are considered equivocal
Conclusions:
Based on the results of this study it is concluded that test substance is equivocal in tester strain TA100 in the presence of S9-mix in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98) and in the Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An Ames study was conducted according to OECD 471 using S.typhimurium and E. coli strains (Verbaan I.A.J., PhD., 2016). Key study.

It is concluded that the test substance is equivocal in tester strain TA100 in the presence of S9-mix in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98) and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Genetic toxicity in vitro: not positive result (equivocal in tester strain TA100 (with S9-mix), negative for tester strains TA1535, TA1537, TA98, and WP2uvrA).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1 the substance is not classified for the genetic toxicity endpoint. In the absence of other in-vitro/in-vivo data no definitive conclusion can be drawn on genetic toxicity endpoint.