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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:
2017-08-16 to 2017-10-26
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:
21st July, 1997
Deviations:
yes
Remarks:
only one strain tested
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
yes
Remarks:
only one strain tested
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
yes
Remarks:
only one strain tested
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:
yes
Remarks:
only one strain tested
Principles of method if other than guideline:
Only one strain was tested as requested by ECHA. An older study with Disodium peroxodisulphate (SPS) does include 5 bacterial strains; however non of these strains does detect cross-linking mutagens. Therefore an additional bacterial reverse mutation assay was performed using only Escherichia coli WP2 uvrA. This study was designed to fill the data gap for the detection of cross linking mutagens.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
The Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- → trp+ reversions. The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).
Species / strain
Species / strain / cell type:
E. coli WP2 uvr A
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 (Informatory Toxicity Test).
±S9 Mix: 5000; 160; 500; 160; 50 and 16 μg/plate (Experiment I - plate incorporation method)
±S9 Mix: 5000; 160; 500; 160; 50 and 16 μg/plate (Experiment II - pre-incubation method)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ultrapure water (ASTM Type I) was applied as vehicle of the test item and the positive control substance MMS (Methyl methanesulfonate); and DMSO was applied as vehicle for positive control substances 2AA.

- Justification for choice of solvent/vehicle: In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2AA (2-Aminoanthracene)
Remarks:
With metabolic activation (+S9), 50 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without Metabolic activation (-S9), 2 µL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The toxicity of the test item was determined with Escherichia coli WP2 uvrA in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.
Evaluation criteria:
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 (the number of reversions is at least three times higher than the reversion rate of the vehicle control) for at least one of the dose groups occurs in the examined strain with or without metabolic activation.

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.

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- The Escherichia coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at the tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

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
- clearing or diminution of the background lawn (reduced background lawn development occurs).
Statistics:
none

Results and discussion

Test results
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:
valid
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 strain at any examined concentration level (±S9 Mix) throughout the study.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with Escherichia coli WP2 uvrA 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.
The revertant colony numbers of vehicle control plates in the examined strain with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies.
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 increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates in the examined bacterial strain at any examined concentration levels (±S9 Mix).


HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
In the Initial Mutation Test all of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges, were far below the biologically relevant threshold for being positive (3 fold higher mutation rate of control) and were considered to reflect the biological variability of the applied test system. In the Initial Mutation Test inhibitory effect of the test item was not observed; the colony and background lawn development was not affected in any case.
In the Confirmatory Mutation Test all of the decreased revertant colony numbers (when compared to the revertant colony numbers of the vehicle control) remained in the corresponding historical control data ranges of the ultrapure water vehicle control, and were without any biological significance. The background lawn development was not affected in any case.
In the performed experiments the revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the different experimental phases were slightly higher or lower than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges.

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Concentration Range Finding Test

Concentration Range Finding Test

(Informatory Toxicity Test)

Concentrations (µg/plate)

Escherichia coli WP2 uvrA

-S9

+S9

Mean values of revertants per plate and Mutation rate (MR)

Mean

MR

Mean

MR

Untreated Control

32.0

1.09

29.0

0.83

DMSO Control

-

-

34.7

1.00

Ultrapure Water Control

29.3

1.00

35.0

1.00

5000

29.3

1.00

30.7

0.88

1600

39.0

1.33

38.3

1.10

500

27.0

0.92

26.7

0.76

160

34.3

1.17

34.3

0.98

50

30.7

1.05

30.7

0.88

16

29.3

1.00

27.3

0.78

5

23.7

0.81

30.7

0.88

MMS (2 µL)

773.3

26.36

-

-

2AA (50 µg)

-

-

220.7

6.37

MR: Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test

(Plate Incorporation Test)

Concentrations (µg/plate)

Escherichia coli WP2 uvrA

-S9

+S9

Mean values of revertants per plate and Mutation rate (MR)

Mean

MR

Mean

MR

Untreated Control

29.0

1.02

37.3

0.94

DMSO Control

-

-

38.0

1.00

Ultrapure Water Control

28.3

1.00

39.7

1.00

5000

29.3

1.04

30.0

0.76

1600

26.0

0.92

39.3

0.99

500

27.3

0.96

42.3

1.07

160

34.7

1.22

36.7

0.92

50

35.0

1.24

29.3

0.74

16

32.3

1.14

30.7

0.77

MMS (2 µL)

768.0

27.11

-

-

2AA (50 µg)

-

-

174.0

4.58

MR:Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

Table 3: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (µg/plate)

Escherichia coli WP2 uvrA

-S9

+S9

Mean values of revertants per plate and Mutation rate (MR)

Mean

MR

Mean

MR

Untreated Control

29.3

0.86

38.7

0.84

DMSO Control

-

-

50.7

1.00

Ultrapure Water Control

34.0

1.00

46.0

1.00

5000

20.0

0.59

52.3

1.14

1600

23.0

0.68

52.3

1.14

500

33.7

0.99

49.3

1.07

160

36.7

1.08

46.7

1.01

50

27.7

0.81

43.3

0.94

16

32.0

0.94

30.3

0.66

MMS (2 µL)

750.7

22.08

-

-

2AA (50 µg)

-

-

180.7

3.57

MR:Mutation Rate

Remarks:Ultrapure water was applied as vehicle of the test item and the positive control substance: MMS and the DMSO was applied as vehicle for positive control substance: 2AA. The mutation rate of the test item, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial stain

Historical control data for untreated control

-S9

 

E. coli

Average

25.4

SD

5.2

Minimum

11

Maximum

45

+S9

 

E. coli

Average

33.9

SD

5.2

Minimum

17

Maximum

56

 

Bacterial Strain

Historical control data of DMSO control

-S9

 

E. coli

Average

24.7

SD

4.6

Minimum

11

Maximum

45

+S9

 

E. coli

Average

33.7

SD

5.0

Minimum

16

Maximum

57

 

Bacterial Strain

Historical control data of Water control

-S9

 

E. coli

Average

26.1

SD

5.5

Minimum

12

Maximum

48

+S9

 

E. coli

Average

34.9

SD

4.9

Minimum

18

Maximum

57

Historical control data of positive controls

-S9

 

E. coli

Average

724.5

SD

65.0

Minimum

320

Maximum

1313

+S9

 

E. coli

Average

257.7

SD

72.5

Minimum

140

Maximum

477

 Abbreviations:E. coli:Escherichia coliWP2uvrA;SD: Standard deviation

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by base-pair substitution in the genome of the Escherichia coli WP2 uvrA used. Therefore, Dipotassium peroxodisulphate (KPS) is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

A bacterial reverse mutation assay according OECD guideline 471, EU method B.13/14 and EPA OPPTS 789.5100 was performed to investigate the mutagenic potential of Dipotassium peroxodisulphate (KPS) in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).
The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: 5000, 1600, 500, 160, 50 and 16 μg/plate. 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 the performed experiments all of the validity criteria, regarding the investigated strain, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of the investigated Escherichia coli WP2 uvrA tester strain following treatment with KPS at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in the independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. 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 strain at any examined concentration level (±S9 Mix) throughout the study.
The reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by base-pair substitution in the genome of the Escherichia coli WP2 uvrA used. Therefore, KPS is considered non-mutagenic in this bacterial reverse mutation assay.