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 2017-5-19 to 2017-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S2(R1). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human use. CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline (Into Operation June 2012).
Version / remarks:
2012
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 No.: 16023LS4B5
Purity: 98.3%

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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
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, both with and without S9-mix, 52, 164, 512, 1600 and 5000 μg/plate.
Second Experiment: TA1535, TA1537, TA98, TA100 and WP2uvrA, both with and without S9-mix, 17, 52, 164, 512, 1600 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Through solubility test based on visual assessment, the test item was dissolved in dimethyl sulfoxide.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA1535, without metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
For TA1537, without metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA1537 and TA98, without metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
For WP2uvrA, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle 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 experimental conditions:
METHOD OF APPLICATION: Dose-Range Finding Test and Experiment 1: in agar (plate incorporati on); Experiment 2: preincubation
- Cell density at seeding (if applicable): 1.0 ± 0.1 at 700 nm (1E+09 cells/mL).

DURATION
- Preincubation period: 30 minutes for Experiment 2.
- Exposure duration:48 ± 4 h both for dose range finding test, 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 xperiment 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 / choice of top concentrations:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
Vehicle 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:
cytotoxicity
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Dose range finding test/first mutation experiment

-Precipitate: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate. No precipitation was observed at the end of the incubation period.

-Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in the tester strains TA1537, TA98 and TA100 in the absence and presence of S9-mix. In the tester strains TA1535 and WP2uvrA, no toxicity was observed at any of the dose levels tested.

-Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with test item under all conditions tested.

Second Experiment

-Precipitate: Precipitation of the test item on the plates was observed at the start of the incubation period at

the concentration of 5000 μg/plate. No precipitation 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.

-Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:
The test item is not 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 ICH guideline (2012). 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.

 

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrAin the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence and presence of S9-mix at the highest dose level tested. 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 item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1537 and TA98 in the absence and presence of S9-mix. In tester strain TA1535 no toxicity was observed at any of the dose levels tested.

 

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level.

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.

 

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 item 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 WP2uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

In conclusion, based on the results of this study it is concluded that test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichiacoli reverse mutation assay.