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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2017- 9 February 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Toxi-Coop ZRT, 8230 Balatonfüred, Arácsi út 97, Hungary
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid: Clear colourless
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: VPS9016001
- Expiration date of the lot/batch: 27.06.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (15-25°C)

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 / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain characteristics:
other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
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:
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test.
±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate in Experiment I (Initial Mutation Test) and Experiment II (Confirmatory Mutation Test)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO; ultrapure water

Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, TA1537, 50 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, TA100 and TA1535, 2 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, E.coli WP2 uvrA, 2 µL
Negative controls:
yes
Solvent controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with metabolic activation, all of Salmonella strains (2 µg), E.coli strain (50 µg)
Negative controls:
yes
Solvent controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
without metabolic activation, TA98, 4 µg
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number
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 induction of mutations with 3 plates each.

Evaluation criteria:
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined by manual counting, the mean values, standard deviations and the mutation rates were calculated.

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 Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium 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 item 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.
Statistics:
None

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 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 98
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 100
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:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No. In the main experiments the pH of the test system varied between 6.21 and 7.45.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration levels (±S9 Mix).

RANGE-FINDING/SCREENING STUDIES:
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 induction of mutations with 3 plates each. In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Slightly lower revertant colony numbers (within the biological variability range of the applied test system) were obtained in S. typhimurium TA98 at 5000, 1600, 160 μg/plate in the absence and presence (±S9 Mix) and at 500 μg/plate, in the presence of exogenous metabolic activation (+S9 Mix).
The revertant colony numbers were above the vehicle control data range (within the historical control data and biological variability range) in S. typhimurium TA100 at 5000 and 16 μg/plate (-S9 Mix) and in TA98 at 16 μg/plate (+S9 Mix).

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1

strain

TA 98

strain

TA 100

Strain

TA 1535

strain

TA 1537

E. coli WP2 uvrA

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Untreated control

19.0

23.3

81.7

101.0

11.7

13.7

10.3

8.0

25.7

31.3

DMSO Control

15.0

23.3

99.0

10.7

7.7

8.7

28.7

Water control

23.3

24.0

76.0

110.0

11.7

11.7

8.3

8.7

23.7

30.0

5000

24.7

21.0

90.7

80.0

12.0

10.0

9.7

6.3

25.0

30.7

1600

23.7

20.0

92.0

114.7

11.0

11.0

9.7

8.3

31.3

28.3

500

25.0

23.0

90.7

105.3

10.3

13.0

8.0

10.3

26.0

39.3

160

16.7

23.0

88.0

108.0

10.7

10.0

9.7

7.3

35.0

33.7

16

23.0

24.3

84.7

99.3

12.0

13.7

9.0

7.7

28.3

40.7

NPD (4µg)

17.7

24.7

90.0

110.0

9.7

12.7

9.0

10.0

27.0

27.3

SAZ (2µg)

344.0

9AA (50 µg)

1072.0

1037.3

MMS (2µL)

450.0

2AA (2µg)

810.7

2AA (50 µg)

1597.3

3014.7

171.3

153.3

Table 2: Number of revertants per plate (mean of three plates), Experiment 2

strain

TA 98

strain

TA 100

Strain

TA 1535

strain

TA 1537

E. coli WP2 uvrA

conc. [µg]

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

Untreated control

15.7

28.7

81.7

106.7

11.7

11.7

8.0

9.0

30.0

33.7

DMSO Control

17.0

25.3

111.3

13.3

8.3

8.3

38.7

Water control

19.3

30.0

82.3

104.0

12.0

14.3

8.0

8.7

34.3

51.0

5000

15.0

17.3

81.7

99.3

11.3

17.7

5.7

9.7

30.3

41.3

1600

17.7

22.3

90.3

93.0

11.3

10.0

9.3

9.3

35.3

49.3

500

18.0

25.3

120.3

90.0

10.7

12.0

6.7

9.7

40.0

49.0

160

19.3

21.3

90.3

94.7

12.7

11.7

10.3

12.0

34.3

37.7

50

19.7

22.3

94.3

90.3

10.3

12.3

9.3

10.7

33.0

38.3

16

16.3

24.0

90.3

102.7

10.7

19.3

9.0

10.7

24.7

36.7

NPD (4µg)

187.3

SAZ (2µg)

607.3

984.0

9AA (50 µg)

170.7

MMS (2µL)

898.7

2AA (2µg)

2496.0

2546.7

229.3

333.0

2AA (50 µg)

280.0

 

Applicant's summary and conclusion

Conclusions:
The test item did not increase the frequency of revertant colonies in bacteria in the presence and absence of the metabolic activation system and thus is considered to be non-mutagenic in bacteria.
Executive summary:

The mutagenic potential of the test item was investigated according OECD TG 471 in Salmonella typhimurium strains TA 98, TA100, TA 1535, TA 1537, and TA 1538 and in E. coli WP2 uvrA. A plate incorporation test and a pre-incubation test were conducted with and without metabolic activation using S9 fraction of Phenobarbital and β-naphthoflavone (BNF) induced rat liver. The following concentrations were examined in triplicate: ±S9 Mix: 5000, 1600, 500; 160; 50 and 16 μg/plate. Positive and negative (vehicle) controls were run concurrently.

No substantial increases were observed in revertant colony numbers following treatment with the test item, neither in the presence nor in the absence of metabolic activation. Sporadic increases in revertant colony numbers compared to the vehicle control remained within the corresponding historical control data ranges. Control plates without mutagen showed no increase in revertant colonies. The positive controls showed the expected, biological relevant increases in revertant colonies in all tests. No cytotoxicity was observed in either tested strains with or without metabolic activation.

In conclusion, it can be stated that the test item is not mutagenic in these bacterial test systems either with or without metabolic activation at any dose level investigated.