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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:
2017-05-30 - 2017-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to an OECD guideline

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
June 2015
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Barium fluoride
EC Number:
232-108-0
EC Name:
Barium fluoride
Cas Number:
7787-32-8
Molecular formula:
BaF2
IUPAC Name:
barium fluoride
Test material form:
solid: crystalline

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:
- Preliminary concentration range finding test (Informatory toxicity test): 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
- Test item concentrations in the Mutagenicity tests (Initial Mutation test and Confirmatory Mutation test): 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-Dimethylformamide (DMF). Homogeneous suspension with quickly sedimentation was observed at 100 mg/mL concentration using DMF. At the same concentration homogeneous suspension with slower sedimentation was detected using DMSO. DMSO was selected as vehicle (solvent) for the study.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine [1] and 2-aminoanthracene [2]
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for the Initial Mutation Test; pre-incubation method fro the Confirmatory Mutation Test

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours (Confirmatory Mutation Test)
- Incubation: 37°C
- 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 recorded and reported.
Rationale for test conditions:
Concentration selection: Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used.
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:
- the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

CRITERIA FOR A NEGATIVE RESPONSE:
The test item was considered to have shown no mutagenic activity in this study 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:
other: S. typhimurium TA 1535, TA 1537, TA 98, 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:
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:
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect

Precipitate was observed in the Preliminary Concentration Range Finding Test in the both tester strains without metabolic activation at the concentration of 5000 μg/plate.

Inhibitory, cytotoxic effect of the test item was not detected in the Preliminary Concentration Range Finding Test, in the Initial Mutation Test and Confirmatory Mutation Test.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Any other information on results incl. tables

Table 1: Summary Table of the Initial Mutation Test

 

Concentrations

(mg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella thyphimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

19.3

24.3

93.0

97.3

10.0

12.7

10.7

11.3

45.7

51.3

MF

0.78

0.90

0.92

1.04

0.71

1.23

1.07

0.94

0.90

1.03

DMSO control

Mean

24.7

27.0

100.7

93.7

14.0

10.3

10.0

12.0

50.7

49.7

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

Distilled water control

Mean

--

--

92.0

--

12.7

--

--

--

47.3

--

MF

--

--

0.91

--

0.90

--

--

--

0.93

--

5000

Mean

20.7

26.0

94.3

99.0

16.0

10.3

14.0

14.3

54.0

51.0

MF

0.84

0.96

0.94

1.06

1.14

1.00

1.40

1.19

1.07

1.03

1581

Mean

21.7

23.7

92.3

95.0

10.7

10.0

15.3

15.7

52.7

56.3

MF

0.88

0.88

0.92

1.01

0.76

0.97

1.53

1.31

1.04

1.13

500

Mean

28.3

22.3

91.7

97.0

12.3

12.3

11.7

11.0

51.3

48.0

MF

1.15

0.83

0.91

1.04

0.88

1.19

1.17

0.92

1.01

0.97

158.1

Mean

19.0

25.7

95.3

98.0

11.0

10.3

14.0

12.0

49.0

51.7

MF

0.77

0.95

0.95

1.05

0.79

1.00

1.40

1.00

0.97

1.04

50

Mean

20.7

27.7

93.3

94.3

13.3

12.0

14.0

12.0

50.3

45.0

MF

0.84

1.02

0.93

1.01

0.95

1.16

1.40

1.00

0.99

0.91

15.81

Mean

21.3

26.7

92.3

92.3

9.7

13.7

14.0

12.3

39.7

50.7

MF

0.86

0.99

0.92

0.99

0.69

1.32

1.40

1.03

0.78

1.02

NPD (4mg)

Mean

382.7

--

--

--

--

--

--

--

--

--

MF

15.51

--

--

--

--

--

--

--

--

--

2AA (2mg)

Mean

--

2426.7

--

2432.0

--

222.7

--

220.0

--

--

MF

--

89.88

--

25.96

--

21.55

--

18.33

--

--

2AA (50mg)

Mean

--

--

--

--

--

--

--

--

--

262.0

MF

--

--

--

--

--

--

--

--

--

5.28

SAZ (2mg)

Mean

--

--

1090.7

--

1114.7

--

--

--

--

--

MF

--

--

11.86

--

88.00

--

--

--

--

--

9AA (50mg)

Mean

--

--

--

--

--

--

422.7

--

--

--

MF

--

--

--

--

--

--

42.27

--

--

--

MMS (2mL)

Mean

--

--

--

--

--

--

--

--

1065.3

--

MF

--

--

--

--

--

--

--

--

22.51

--

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that Barium Fluoride is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Barium fluoride was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to the OECD 471 Guideline and under GLP. The experiments were carried out using four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and a tryptophan-requiring strain of Escherichia coli WP2uvrA in the presence and absence of a metabolic activation system (phenobarbital/B-naphthoflavone-induced rat liver S9-mix). Based on the results of a solubility test, the test item was formulated in DMSO.

ln a dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA98 and TA100. The test item concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81 and 5 (Confirmatory only) μg/plate.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Barium fluoride did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that Barium fluoride is not mutagenic in the Salmonella typhimurium and in the Escherichia coli reverse mutation assay.