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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:
From 04 Nov 2011 to 10 may 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP Study, OECD 471 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2010-12-16
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate
EC Number:
911-467-3
Molecular formula:
C2O2F3K and CO2F3KS
IUPAC Name:
Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate
Constituent 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Test material form:
liquid
Remarks:
aqueous solution

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Cofactor-supplemented post-mitochondrial fraction (S9 fraction) prepared by CiToxLAB and obtained from the liver of male Wistar rats treated with with phenobarbital and B-naphthoflavone at 80 mg/kg/day by oral gavage for three consecutive days.
Test concentrations with justification for top dose:
5000; 1581, 500; 158.1, 50; 15.81 and 5 μg active ingredients/plate.

A preliminary solubility test was performed. The solubility of the test item was examined in Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). The test item was soluble in Distilled water at 100 mg/mL concentration; while the use of DMSO or DMF resulted an opalescent formulation at the same concentration. Based on these solubility results, Distilled water was selected for solvent of the study.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled Water
- Justification for choice of solvent/vehicle: The test item was soluble in Distilled water at 100 mg/mL concentration; while the use of DMSO or DMF resulted an opalescent formulation at the same concentration.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); with or without preincubation (for initial mutation test and confirmatory mutation test respectively)

POSITIVE CONTROLS :
-Without activation :
-Salmonella TA98 : 4 µg/plate of 4-nitro-1,2-phenylene-diamine (NPD) in DMSO
-Salmonella TA100 and TA1535 : 2 µg/plate of Sodium azide (SAZ) in Distilled water
-Salmonella TA1537 : 50 µg/plate of 9-aminoacridine (9AA) in DMSO
-E.coli WP2 uvrA : 2 µL/plate of Methyl-methanesulfonate (MMS) in Distilled water
-With activation :
-all Salmonella strains : 2 µg/plate of 2-aminoanthracene (2AA) in DMSO
-E.coli WP2 uvrA : 50 µg/plate of 2-aminoanthracene (2AA) in DMSO

DURATION
- Preincubation period: none for the initial mutation test. For confirmatory mutation test, the test item solution (or solvent or reference control) (50 µL), the bacterial culture (100 µL) and the S9 mix or phosphate buffer (500 µL) were incubated for 20 min at 37ºC in a shaking incubator before the overlaying
- Exposure duration: 48 hours
- Expression time (cells in growth medium): the number of revertants is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertants per plates are counted

OTHER EXAMINATIONS:
- Other: Visual examination of the plates was performed : precipitation or signs of growth inhibition were reported

The plate incorporation method was performed as follow : the test item solution (or solvent or reference control) (50 µL), the bacterial culture (100 µL) and the S9 mix or phosphate buffer (500 µL) and the molten top agar (2000 µL) were mixed in individual tubes at 45°C and poured on the surface of minimal agar plates.
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
-The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
-at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
-a dose–related increase in the number of revertants occurred and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
-in Salmonella typhimurium TA100 strain the number of reversion is more than two times higher than the spontaneous reversion rate of the solvent control plates;
-in Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains the number of reversions is more than three times higher than the spontaneous reversion rate of the solvent control plates.

Criteria for a Negative Response:
A test article was considered non-mutagenic 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:
No statistical analysis was performed

Results and discussion

Test resultsopen allclose all
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:
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A Preliminary Range Finding Test was performed with the plate incorporation method. The test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. The concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.

The observed numbers of revertant colonies were mostly in the normal range. Minor difference (slightly higher number of revertant colonies compared to the solvent control plates) was observed in one case. However, it had no biological significance and was considered as reflecting the variability of the test system.

No insolubility or signs of cytotoxicity were observed in the preliminary experiment.


COMPARISON WITH HISTORICAL CONTROL DATA:
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analyzable concentrations were presented in all strains of the main tests.

The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Slight inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain at the highest examined concentration (5000 μg active ingredients/plate ) with and without metabolic concentration.

Any other information on results incl. tables

Summary Table of the Initial Mutation Test

Concentrations

(µg active ingredients/
plate)

Mean
values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

25.3

35.3

85.7

109.7

8.0

9.3

11.0

9.0

26.3

37.3

MF

1.17

1.16

1.00

0.99

1.50

1.22

1.74

1.17

0.71

0.80

DMSO
control

Mean

24.7

35.7

--

112.3

--

8.3

12.3

10.7

--

41.7

MF

1.14

1.18

--

1.02

--

1.09

1.95

1.39

--

0.89

Distilled water control

Mean

21.7

30.3

86.0

110.3

5.3

7.7

6.3

7.7

37.3

46.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

24.7

33.3

81.3

89.3

7.3

9.7

5.7

9.7

31.7

37.3

MF

1.14

1.10

0.95

0.81

1.38

1.26

0.89

1.26

0.85

0.80

1581

Mean

25.3

34.3

89.0

113.0

9.3

11.7

6.7

11.7

30.0

38.0

MF

1.17

1.13

1.03

1.02

1.75

1.52

1.05

1.52

0.80

0.81

500

Mean

26.0

32.0

82.0

114.3

6.3

8.7

7.3

9.3

24.7

28.7

MF

1.20

1.05

0.95

1.04

1.19

1.13

1.16

1.22

0.66

0.61

158.1

Mean

24.7

40.0

85.0

105.0

8.3

12.7

6.3

9.7

24.3

41.3

MF

1.14

1.32

0.99

0.95

1.56

1.65

1.00

1.26

0.65

0.89

50

Mean

25.3

37.3

80.3

107.7

10.3

11.0

8.0

7.0

25.0

33.7

MF

1.17

1.23

0.93

0.98

1.94

1.43

1.26

0.91

0.67

0.72

15.81

Mean

29.7

37.7

81.3

99.0

9.7

10.7

10.3

7.3

28.0

35.7

MF

1.37

1.24

0.95

0.90

1.81

1.39

1.63

0.96

0.75

0.76

5

Mean

21.0

32.7

77.7

118.0

8.3

11.3

6.3

10.0

25.7

38.3

MF

0.97

1.08

0.90

1.07

1.56

1.48

1.00

1.30

0.69

0.82

NPD (4mg)

Mean

200.0

--

--

--

--

--

--

--

--

--

MF

8.11

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2172.0

--

2186.7

--

202.7

--

222.0

--

--

MF

--

60.90

--

19.47

--

24.32

--

20.81

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

208.0

MF

--

--

--

--

--

--

--

--

--

4.99

SAZ (2mg)

Mean

--

--

1452.0

--

1465.3

--

--

--

--

--

MF

--

--

16.88

--

274.75

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

308.0

--

--

--

MF

--

--

--

--

--

--

24.97

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1236.7

--

MF

--

--

--

--

--

--

--

--

33.13

--

Applicant's summary and conclusion

Conclusions:
The Reaction mass of TFSK/TFAK had no mutagenic activity in the tested strains with or without activation up to 5000 µg (active ingredient)/plate.
Executive summary:

The test item Reaction mass of TFSK/TFAKwas tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

The examined test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were: 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg active ingredients/plate.

In the Initial Mutation Test and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect, both with and without activation, in any of the five strains. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item Reaction mass of TFSK/TFAK did not show mutagenic activity this study.