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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to internationally accepted guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Boron trifluoride dihydrate
- Physical state: Colourless liquid
- Analytical purity: 66%
- Composition of test material, percentage of components: boron trifluoride 66%, water 44%
- Purity test date: November 2014
- Lot/batch No.: R.F.L.A.P. 154.19
- Expiration date of the lot/batch: November 2014
- Storage condition of test material: Room temperature

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y mouse lymphoma (3.7.2c) cells were obtained from American Type Culture Collection (ATCC), Virginia. These cells are heterozygous at the thymidine kinase locus, TK0+/-. Spontaneous thymidine kinase deficient mutants, TK0 -/-, were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver fraction S9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.9, 1.8, 3.5, 7, 14.1, 28.1, 56.3, 112.5, 225 and 450 µg/mL
Mutation tests: 3 hours with and without S9 mix 14.06, 28.13, 56.25, 112.5, 225 and 450 µg/mL
24 hours without S9 mix 28.13, 56.25, 112.5, 225, 250, 300, 350, 400 and 450 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance soluble at 67.8 mg/mL in water. A solution of 67.8 mg/mL, dosed at 1% in medium, showedno precipitate in the culture medium. The osmolality of the test substance in medium was tested at concentrations of 450 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control. Fluctuations in pH of the medium were observed at 500, 550, 600 and 678 µg/mL of more than 1.0 unit compared with the vehicle control. The maximum concentration tested in the preliminary toxicity test was 450 µg/mL in order to test up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
methylmethanesulfonate 10 µg/mL (3 hr exposure), 5 µg/mL (24 hr exposure)
Remarks:
Absence of S9 mix
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Presence of S9 mix
Details on test system and experimental conditions:
Preliminary toxicity test:
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix. For 3 hour exposures, cultures contained a total of 6 x 10E6 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24 hour exposures, cultures contained a total of 1.5 x 10E6 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition.
The test substance was formulated and serially diluted in the solvent. Aliquots of 50 µL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static humidified incubator, at 37°C, 5% (v/v) CO2). At the end of the 3 hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss), incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. At the end of the 24 hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 105 cells/mL with R10p where necessary.
The relative suspension growth (RSG) was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce relative total growth (RTG) to approximately 10 to 20% of the concurrent vehicle control value. There was evidence of toxicity in the preliminary toxicity test, so the maximum concentration tested in the 3 hour exposure in the absence and presence of S9 mix, and in the 24 hour exposure in the absence of S9 mix was 450 µg/mL. The formulations being added at 1% final volume in medium.

Main mutation test - 3 hour exposure with and without S9 mix:

The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10E7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37ºC. At least four serial dilutions of the test substance were tested.
Following the 3 hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10E5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10E3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96 well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentration assessed for mutant frequency in the first main test was 450 µg/mL in the absence and presence of S9 mix.

Main mutation test 24 exposure without S9 mix:

A second test was carried out, with a 24 hour exposure in the absence of S9 mix. Duplicate 10 mL cultures containing 3 x 10E6 cells were treated for 24 hours with 100 µL of test substance, solvent or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p, the intention being to retain at least 1 x 10E7 cells. Following this, the procedure was the same as in the 3 hour treatment. The maximum concentration assessed for mutant frequency in the second main test was 300 µg/mL.


Evaluation criteria:
Tests were accepted on the basis of the following criteria:

Acceptance criteria for test substance:

The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth (RTG) reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:

The mean vehicle control value for mutant frequency was between 50 to 170 x 10E-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment. Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:

Positive controls showed an absolute increase in mean total mutant frequency (MF) above the mean concurrent vehicle control MF of at least 300 x 10E-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics are only reported if the Global Evaluation Factor is exceeded, and this was accompanied by a significant positive linear trend.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
No Precipitate (observed by eye at the end of treatment) was observed in the absence and presence of S9 mix, following a 3 hour exposure. Exposureto Boron Trifluoride Dihydrate at concentrations from 0.9 to 450 µg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values from 107 to 71% and from 113 to 68% respectively.
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed. Exposure to concentrations from 0.9 to 450 µg/mL resulted in RSG values from 114 to 1%. Concentrations used in the main test were based upon these data.

3 hr treatment in absence of S9 mix:

No precipitate was observed by eye at the end of treatment. Cultures exposed to Boron Trifluoride Dihydrate at concentrations from 28.13 to 450 µg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 109 to 51% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

3 hour treatment in presence of S9 mix:

No precipitate was observed by eye at the end of treatment. Cultures exposed to Boron Trifluoride Dihydrate at concentrations from 28.13 to 450 µg/mL were assessed for determination of mutation frequency. RTG values from 132 to 46% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
The results obtained in response to the exposure of cultures to Boron Trifluoride Dihydrate in the presence of S9 mix did not demonstrate mutagenic potential. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical solvent control values. Therefore it was considered unnecessary to perform a direct repeat of the assay.

24 hour treatment in absence of S9 mix:

No precipitate was observed by eye at the end of treatment. Cultures exposed to Boron Trifluoride Dihydrate at concentrations from 56.25 to 300 µg/mL were assessed for determination of mutation frequency. RTG values from 100 to 11% were obtained relative to the vehicle control. There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table2             Main mutation test 1 – 3 hour treatment in the absence of S9 mix

Treatment / Concentration

Cell Concentration (x105/mL)

Viability Plate Counta

Mutant Plate Counta

Mean RTG

Mean MF

(µg/mL)

Replicate ID

24 h

48 h

Day 2

Day 2

(%)

(x10-6)

Vehicle Controlb

A

3.15

11.35

35

(192)

154

(192)

100

84

 

B

2.95

13.34

31

(192)

158

(192)

 

 

 

C

3.56

13.76

30

(192)

163

(192)

 

 

 

D

4.12

13.67

38

(192)

165

(192)

 

 

Boron Trifluoride Dihydrate

A

3.37

11.77

23

(192)

152

(192)

109

91

28.13

B

3.85

12.55

32

(192)

156

(192)

 

 

Boron Trifluoride Dihydrate

A

3.17

11.79

26

(192)

146

(192)

106

87

56.25

B

3.86

12.50

28

(192)

164

(192)

 

 

Boron Trifluoride Dihydrate

A

3.21

13.74

29

(192)

150

(192)

103

105

112.5

B

3.43

13.71

36

(192)

154

(192)

 

 

Boron Trifluoride Dihydrate

A

3.21

14.14

29

(192)

157

(192)

92

78

225

B

2.95

13.05

41

(192)

168

(192)

 

 

Boron Trifluoride Dihydrate

A

2.07

10.30

30

(192)

147

(192)

51

121

450

B

2.12

11.11

34

(192)

146

(192)

 

 

MMS

A

3.04

12.55

51

(192)

78

(192)

81

534

10

B

3.46

14.08

40

(192)

69

(192)

 

 

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = Water (1% v/v)

 

MMS - Methyl methanesulphonate

Table3             Main mutation test 1 – 3 hour treatment in the absence of S9 mix, colony size analysis

Treatment / Concentration

Mutant Plate Counta

Total Mutant Colonies

Large Mutant Colonies

Small Mutant Colonies

% Small Colony Mutants

Mean % Small Colony Mutants

(µg/mL)

Replicate ID

Day 2

Vehicle Controlb

A

154

(192)

41

12

29

71

61

 

B

158

(192)

38

18

20

53

 

 

C

163

(192)

33

17

16

48

 

 

D

165

(192)

28

8

20

71

 

MMS

A

78

(192)

130

30

100

77

76

10

B

69

(192)

130

33

97

75

 

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = Water (1% v/v)

 

MMS - Methyl methanesulphonate

Table4             Main mutation test 1 – 3 hour treatment in the presence of S9 mix

Treatment / Concentration

Cell Concentration (x105/mL)

Viability Plate Counta

Mutant Plate Counta

Mean RTG

Mean MF

(µg/mL)

Replicate ID

24 h

48 h

Day 2

Day 2

(%)

(x10-6)

Vehicle Controlb

A

3.30

11.08

30

(192)

163

(192)

100

75

 

B

3.33

11.06

33

(192)

159

(192)

 

 

 

C

3.27

10.04

29

(192)

161

(192)

 

 

 

D

3.17

10.06

22

(192)

160

(192)

 

 

Boron Trifluoride Dihydrate

A

3.49

8.62

13

(192)

154

(192)

130

70

28.13

B

3.73

11.00

21

(192)

157

(192)

 

 

Boron Trifluoride Dihydrate

A

3.18

9.75

13

(192)

166

(192)

132

52

56.25

B

4.28

10.11

24

(192)

164

(192)

 

 

Boron Trifluoride Dihydrate

A

3.66

9.25

22

(192)

158

(192)

120

66

112.5

B

3.71

9.89

19

(192)

161

(192)

 

 

Boron Trifluoride Dihydrate

A

4.20

10.31

24

(192)

159

(192)

115

86

225

B

3.64

9.60

33

(192)

154

(192)

 

 

Boron Trifluoride Dihydrate

A

1.79c

8.03

30

(192)

155

(192)

46

100

450

B

2.01

8.87

34

(192)

152

(192)

 

 

BaP

A

3.24

9.37

32

(192)

45

(192)

94

533

1

B

3.33

9.60

22

(192)

59

(192)

 

 

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = Water (1% v/v)

c. Cell concentration not adjusted due to insufficient growth

 

BaP - Benzo[a]pyrene

Table5             Main mutation test 1 – 3 hour treatment in the presence of S9 mix, colony size analysis

Treatment / Concentration

Mutant Plate Counta

Total Mutant Colonies

Large Mutant Colonies

Small Mutant Colonies

% Small Colony Mutants

Mean % Small Colony Mutants

(µg/mL)

Replicate ID

Day 2

Vehicle Controlb

A

163

(192)

30

13

17

57

59

 

B

159

(192)

36

13

23

64

 

 

C

161

(192)

33

16

17

52

 

 

D

160

(192)

40

15

25

63

 

BaP

A

45

(192)

197

40

157

80

80

1

B

59

(192)

182

36

146

80

 

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = Water (1% v/v)

 

BaP - Benzo[a]pyrene

 

Table6             Main mutation test 2 – 24 hour treatment in the absence of S9 mix

Treatment / Concentration

Cell Concentration (x105/mL)

Viability Plate Counta

Mutant Plate Counta

Mean RTG

Mean MF

(µg/mL)

Replicate ID

0 h

24 h

48 h

Day 2

Day 2

(%)

(x10-6)

Vehicle Controlb

A

13.51

5.23

13.91

42

(192)

160

(192)

100

101

 

B

13.26

5.12

15.15

43

(192)

159

(192)

 

 

 

C

13.24

5.38

13.57

44

(192)

160

(192)

 

 

 

D

13.28

5.36

14.14

54

(192)

162

(192)

 

 

Boron Trifluoride Dihydrate

A

11.83

5.92

14.20

43

(192)

172

(192)

100

66

56.25

B

12.33

5.52

13.76

44

(192)

168

(192)

 

 

Boron Trifluoride Dihydrate

A

11.43

5.67

12.82

38

(192)

171

(192)

94

70

112.5

B

9.46

6.42

13.76

38

(192)

162

(192)

 

 

Boron Trifluoride Dihydrate

A

6.15

4.62

13.56

40

(192)

170

(192)

38

82

225

B

5.39

3.82

14.29

38

(192)

156

(192)

 

 

Boron Trifluoride Dihydrate

A

4.31

3.20

15.19

41

(192)

157

(192)

21

131

250

B

4.21

2.91

14.89

44

(192)

143

(192)

 

 

Boron Trifluoride Dihydrate

A

3.43

2.18

13.83

43

(192)

128

(192)

11

215

300

B

3.53

2.81

14.65

59

(192)

141

(192)

 

 

MMS

A

9.92

4.87

13.43

69

(192)

47

(192)

43

1200

5

B

10.05

4.82

12.70

77

(192)

43

(192)

 

 

a. Number of non-colony bearing wells (total number of wells)

b. Vehicle control = Water (1% v/v)

 

MMS - Methyl methanesulphonate

Applicant's summary and conclusion

Conclusions:
It was concluded that Boron Trifluoride Dihydrate did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimentalconditions described.
Executive summary:

Boron Trifluoride Dihydrate was tested for mutagenic potential in an in vitro mammalian cell mutation assay (OECD 476, GLP)

The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

Boron Trifluoride Dihydrate was found to be soluble at 67.8 mg/mL in water. A final concentration of 450 mg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit.

Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Boron Trifluoride Dihydrate at concentrations from 0.9 to 450 mg/mL, relative suspension growth (RSG) was reduced from 107 to 71% and from 113 to 68% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 114 to 1%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG), or to assess exposure up to the highest concentration that does not cause a fluctuation in pH of more than 1.0 unit.

Following 3 hour treatment in the absence and presence of S9 mix, there were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix was 450mg/mL. In the absence and presence of S9 mix RTG was reduced to 51 and 46% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 300 mg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed at concentrations up to 300 mg/mL, where RTG was reduced to 11%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded thatBoron Trifluoride Dihydratedid not demonstrate mutagenic potential in this in vitrocell mutation assay, under the experimental conditions described.