Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ICH. S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use
Version / remarks:
CHMP/ICH/126642 ICH Consensus Guideline, Step 5 Guideline (Into Operation June 2012)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)
Specific details on test material used for the study:
- The characterisation of the test item was conducted in a GMP environment.

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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 S9-mix induced by Aroclor 1254
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 direct plate assay
Dose range finding test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study: TA1535, TA1537 and TA98; Without and with S9-mix; 17, 52, 164, 512 and 1600 µg/plate

Experiment 2 pre-incubation assay
All strains; without and with S9-mix; 17, 52, 164, 512 and 1600 µg/plate

The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test and based on the results of the dose range finding test, the dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix.

To obtain more information about the possible mutagenicity of Diona-R, the second experiment was a pre-incubation experiment performed in all strains in the absence and presence of S9-mix.
Vehicle:
- Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle:
A solubility test was performed. The test item could not be dissolved in water, dimethyl sulfoxide, ethanol, THF, hexane or acetone. A homogeneous suspension was obtained with propylene glycol after treatment with the blender and with ultra-sonic waves. Therefore propylene glycol was used as vehicle in this project.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 5 µg/plate in saline for TA1535
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9 in DMSO 2.5 µg/plate (direct plate) for TA1537
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9 in DMSO 10 µg/plate for TA98 and 15 µg/plate (pre-incubation) for TA1537
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 in DMSO 650 µg/plate for TA100
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 in DMSO 10 µg/plate for WP2uvrA
Negative controls:
no
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 in DMSO 2.5 µg/plate TA1535,TA1537; 1 µg/plate TA98, TA100(direct plate); 5 µg/plate TA100(pre-incubation), 15 µg/plate WP2uvrA
Details on test system and conditions:
METHOD:
Experiment 1: direct plate assay
Experiment 2: pre-incubation assay. Test item and bacterial culture were pre-incubated for 30 minutes by 70 rpm at 37°C, either with or without S9-mix.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Rationale for test conditions:
The test item was initially tested in the tester strains TA100 and WP2uvrA as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. 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: 17, 52, 164, 512, and 1600 μg/plate.
To obtain more information about the possible mutagenicity of Diona-R, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 1600 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Evaluation criteria:
Acceptability of the assay
The 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.

In addition to the criteria stated below, 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.

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 in tester 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.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
First experiment: Direct plate assay
- Precipitation: At the start of the incubation period at concentrations of 164 μg/plate and upwards and at 512 μg/plate and above at the end of the incubation period.
- Toxicity: No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed.

In strain TA1537 (absence of S9-mix), a fluctuations in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 52 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely that this reduction was caused by an incidental fluctuation in the number of revertant colonies.

Second experiment: Pre-incubation assay
- Precipitation: at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 164 μg/plate and above at the end of the incubation period.
- Toxicity: no reduction in 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 strain TA1537 (absence of S9-mix), a fluctuations in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 52 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction was caused by an incidental fluctuation in the number of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control values were within the laboratory historical control data ranges, except the responses for TA1537 (presence of S9-mix, first experiment) and TA98 (absence of S9-mix, second experiment). However since the mean number of revertant colonies showed a characteristic number of revertant colonies (2 and 9 revertant colonies, respectively) when compared against relevant historical control data (3 and 10 relevant colonies, respectively), the validity of the test was considered to be not affected.
The 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, except the responses for TA98 (absence of S9-mix in the first and second experiment). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the values were more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive controls had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:
In the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay, performed according to ICH guidelines and GLP principles, Diona-R was found not to be mutagenic with or without metabolic activation.
Executive summary:

An evaluation of the mutagenic activity of Diona-R was performed using the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay according to ICH guideline and GLP principles. All bacterial strains showed negative responses up to precipitating concentrations, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity of the test substance was observed. 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.

Based on the results of this study it is concluded that Diona-R is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.