Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-01-02 to 2018-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch: 17019FS4B4
Purity: 94.4%

Method

Target gene:
histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli (E. coli) strain
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver metabolic activation system (S9 homogenate)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver metabolic activation system (S9 homogenate)
Test concentrations with justification for top dose:
-Dose-range Finding Test:
TA100 and the WP2uvrA, both with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
-First Experiment:
TA1535, TA1537 and TA98, without S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
TA1535, TA1537 and TA98, with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
-Second Experiment:
TA1535, TA1537, TA98, TA100 and WP2uvrA, without S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
TA1535, TA1537, TA98, TA100 and WP2uvrA, with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
-Third Experiment:
TA1537, TA98, TA100 and WP2uvrA, without S9-mix: 164, 512, 1600, 2500 and 5000 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: Through solubility test based on visual assessment, the test item formed a clear ((dark-) brown) solution, the stock solution was treated with ultrasonic waves until the test item had completely dissolved.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA1535, without metabolic activation.
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
For TA1537, without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA1537, TA98, without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100, without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
For WP2uvrA, wthout metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA1535, TA1537, TA98, TA100 and WP2uvrA, with metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: Dose-Range Finding Test and Experiment 1: in agar (plate incorporation); Experiment 2 and 3: preincubation
- Cell density at seeding (if applicable): the optical density of 1.0 ± 0.1 at 700 nm (10+E9 cells/ml)

DURATION
- Preincubation period: 30 ± 2 minutes for Experiment 2.
- Exposure duration: 48 ± 4 h both for Experiment 1 and Experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
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 whencompared against relevant historical control data generated at the lab.
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 tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants intester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent 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 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 or TA98 is greater than three (3) 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Dose range finding test/first mutation experiment

-Precipitate: Precipitation of test item 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 the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

-Mutagenicity: In tester strain WP2uvrAin the absence of S9-mix, the test item induced an up to 2.2-fold increase in the number of revertant colonies compared to the solvent control. However, no dose-relationship was observed and the increases were within the historical control data range. Therefore, these increases are considered to be not biologically relevant. In all other tester strains no increase in the number of revertants was observed upon treatment with the test item.

 

Second Experiment

-Precipitate: Precipitation of test item on the plates was not observed at the start or at the end of the incubation period.

-Toxicity: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was only observed in tester strain TA1537 in the absence of S9-mix at the highest dose level tested. In all other tester strains no toxicity was observed.

-Mutagenicity: In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in four tester strains (TA1537, TA98, TA100 and WP2uvrA).

The increase observed in tester strain TA1537 was 3.5-fold the concurrent control at 1600 μg/plate. However, this increase was within the historical control data range. The increase observed in tester strain TA98 was within the laboratory historical control data range and was up to 2.9- fold the concurrent control. The increase observed in tester strain TA100 was above the laboratory historical control data range, and was up to 2.9-fold the concurrent vehicle control. The increase observed in tester strain WP2uvrAwas 4.8-fold the concurrent control. However, this increase was within the historical control data range.

In the presence of S9-mix no biologically relevant increases in the number of revertant colonies were observed.

In tester strain WP2uvrAin the presence of S9-mix, the test item induced a 3.6-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was within the historical control data range and coupled to a low solvent control value, and was therefore not considered to be biologically significant.

Third Experiment: Pre-Incubation Assay

-Precipitate: Precipitation of test item 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.

-Mutagenicity: The test item induced up to 6.0- and 2.5-fold dose related increases in the number of revertant colonies compared to the solvent control in the tester strains TA98 and WP2uvrA, respectively. The increases observed in tester strain TA98 were above the historical control data range, whilst the increases observed in tester strain WP2uvrAwere within the historical control data range.

In tester strain TA1537, an increase above the laboratory historical control data range was observed. However, this increase was only 1.7-fold the concurrent control.

In tester strain TA100, up to 1.9-fold dose related increases in the number of revertant colonies were observed. However, the increases were within the historical control data range.

Applicant's summary and conclusion

Conclusions:
Test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The test was performed according to the most recent OECD guideline (1997). Strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and Escherichia coli (E. coli) strain WP2uvrA were treated with the test material 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. An additional pre-incubation assay was performed in the absence of S9-mix. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Since 1.7- to 6.0-fold increases were observed in the absence of S9-mix in the tester strains TA1537, TA98, TA100 and WP2uvrA, and the results were reproducible in a follow up experiment, these increases are considered to be biologically relevant and the test item is considered to be mutagenic.

 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimuriumreverse mutation assay and in the Escherichia colireverse mutation assay.