Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-01-20 to 2016-03-04
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strainopen allclose all
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 (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Six (+S9 Mix) and seven (-S9 Mix) concentrations of the test item were tested in the main study. The test concentrations were:
-S9 Mix: 5000; 4000; 2000; 800; 320; 128 and 51.2 μg/plate;
+S9 Mix: 5000; 2000; 800; 320; 128 and 51.2 μg/plate.

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The suitability of the solvent had been determined in the preliminary Solubility Test.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Strain: Salmonella TA 98; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strain: Salmonella TA 100 and TA 1537; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain: Salmonella TA 1537; without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Strain: E. coli WP2 uvrA; 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 (2AA)
Remarks:
Strain: all strains used; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
Rationale for test conditions:
According to guidelines
Evaluation criteria:
The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

HISTORICAL CONTROL DATA
- Positive historical control data: see "Any other information on results" table 1
- Negative historical control data: "Any other information on results" table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Inhibitory effects of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

Any other information on results incl. tables

Table 1 Historical control values for revertants/plate (for the period of 2008-2015)

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

20.9

101.4

10.3

7.9

24.9

SD

3.5

26.2

1.4

2.5

4.9

Minimum

10

65

3

2

11

Maximum

39

150

23

20

44

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

27.1

114.7

12.0

8.8

34.2

SD

4.0

19.3

1.5

2.1

5.2

Minimum

15

71

4

3

16

Maximum

48

161

24

20

56

Historical control data of positive control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

255.6

958.9

842.1

467.4

712.3

SD

30.7

149.+

134.0

105.7

57.5

Minimum

123

522

354

109

320

Maximum

647

1927

1871

1498

1283

+S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

1224.8

1431.9

165.4

148.0

264.7

SD

293.8

339.9

35.1

21.3

74.2

Minimum

409

581

85

68

141

Maximum

2587

2923

507

407

487

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay shows that under the experimental conditions reported, the test item (acting as direct mutagen, in absence of exogenous metabolic activation) induced gene mutations by base-pair substitution in the genome of the Salmonella typhimurium TA100 tester strain investigated. Therefore, the test item is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the following concentrations were examined: -S9 Mix: 5000; 4000; 2000; 800; 320; 128 and 51.2 μg/plate; +S9 Mix: 5000; 2000; 800; 320; 128 and 51.2 μg/plate. An Additional Plate Incorporation Test was performed (in parallel with the pre-incubation test) with S. typhimurium TA100 in the absence of metabolic activation (-S9 Mix). In this additional experiment the corresponding Initial Mutation Test part was repeated with the same concentration levels. Bacteria In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were used. In the Additional Plate Incorporation Test the Salmonella typhimurium TA100 strain was investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In parallel with the Confirmatory Mutation Test an Additional Plate Incorporation Test (partial) was performed with Salmonella typhimurium TA100 in the absence of metabolic activation (-S9 Mix). In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. In the Initial Mutation Test following treatment with the test item significant, biological relevant revertant colony number increases, positive result was noticed at the concentration of 5000 μg/plate in S. typhimurium TA100 in the absence of exogenous metabolic activation (-S9 Mix). The revertant colony number increases showed clear dose-relationship at the further, lower concentration levels. The dose related tendencies, the unequivocal positive results were repeated in subsequent experiments: in the Additional Plate Incorporation Test, and in the Confirmatory Mutation Test. In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.