Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity (2015), GLP, OECD 471, Ames-Test, up to 5000 µg, with and without metabolic activation, negative
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct. 2014 - Feb. 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Gouvernment of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
histidine-, tryptophan-
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: rfa-, uvrB-, R factor plasmid pKM101 (TA98 and TA100)
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomal preparation (S9-mix)
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: uvrA-
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomal preparation (S9-mix)
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for experiment 1
50, 150, 500, 1500 and 5000 µg/plate for experiment 2
Vehicle:
dimethyl sulphoxide
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
dimethyl sulphoxide
Positive controls:
yes
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP)
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP)
Details on test system and conditions:
Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (5%) of the test item. Prior to use, the solvent was dried to remove water using molecular sieves.
The S9 Microsomal fraction was prepared in house from male rats. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
Experiment 1:
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed with and without S9 mix using triplicate plates. All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours.
Experiment 2:
Five concentrations of the test item (50, 150, 500, 1500 and 5000 µg/plate), appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed with and without S9 mix using triplicate plates. All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours, pre-incubation time was 20 min.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Experiment 1:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to bubbles in the base agar slightly distorting the actual plate count.
Experiment 2:
All of the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to interference in the base agar e.g. marks on the base of the plates slightly distorting the counts.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first experiment (plate incorporation method) there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) and consequently the same maximum dose level was used in the second mutation test. Second experiment (pre-incubation method) results once again showed no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of S9-mix. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Experiment 1 – Without Metabolic Activation

Test Period

From: 03 February 2015

To: 06 February 2015

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

108

107

107

(107)

0.6#

11

15

15

(14)

2.3

31

45

19

(32)

13.0

31

32

31

(31)

0.6

11

13

15

(13)

2.0

1.5 µg

112

98

106

(105)

7.0

16

15

17

(16)

1.0

27

24

24

(25)

1.7

37

32

33

(34)

2.6

16

16

11

(14)

2.9

5 µg

98

116

98

(104)

10.4

11

12

15

(13)

2.1

33

21

21

(25)

6.9

15

20

16

(17)

2.6

8

16

16

(13)

4.6

15 µg

104

111

106

(107)

3.6

15

16

13

(15)

1.5

25

28

28

(27)

1.7

25

29

17

(24)

6.1

16

9

8

(11)

4.4

50 µg

104

99

95

(99)

4.5

8

16

17

(14)

4.9

28

32

35

(32)

3.5

27

29

17

(24)

6.4

4

16

16

(12)

6.9

150 µg

84

99

106

(96)

11.2

16

12

12

(13)

2.3

25

23

27

(25)

2.0

32

35

29

(32)

3.0

16

11

5

(11)

5.5

500 µg

102

122

108

(111)

10.3

17

17

13

(16)

2.3

39

25

19

(28)

10.3

36

25

31

(31)

5.5

15

11

5

(10)

5.0

1500 µg

120

92

90

(101)

16.8

12

13

12

(12)

0.6

24

20

23

(22)

2.1

16

31

32

(26)

9.0

8

16

12

(12)

4.0

5000 µg

90

118

112

(107)

14.7

17

12

16

(15)

2.6

25

29

29

(28)

2.3

16

32

35

(28)

10.2

7

12

15

(11)

4.0

Positive controls

S9-Mix

(-)

Name

ENNG

ENNG

ENNG

4NQO

9AA

Dose Level

3 µg

5 µg

2 µg

0.2 µg

80 µg

No. of Revertants

345

373

377

(365)

17.4

351

325

222

(299)

68.2

373

421

437

(410)

33.3

164

162

156

(161)

4.2

869

1017

794

(893)

113.5

Experiment 1 – With Metabolic Activation

Test Period

From: 03 February 2015

To: 06 February 2015

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

110

115

118

(114)

4.0#

12

15

13

(13)

1.5

37

41

40

(39)

2.1

24

24

27

(25)

1.7

11

11

9

(10)

1.2

1.5 µg

104

119

110

(111)

7.5

13

8

11

(11)

2.5

41

24

35

(33)

8.6

29

24

25

(26)

2.6

8

12

16

(12)

4.0

5 µg

119

108

109

(112)

6.1

13

13

8

(11)

2.9

27

15

40

(27)

12.5

25

25

13

(21)

6.9

11

8

11

(10)

1.7

15 µg

98

114

107

(106)

8.0

11

9

11

(10)

1.2

43

40

40

(41)

1.7

23

25

28

(25)

2.5

12

12

7

(10)

2.9

50 µg

98

96

110

(101)

7.6

13

12

9

(11)

2.1

23

32

37

(31)

7.1

29

24

19

(24)

5.0

12

7

9

(9)

2.5

150 µg

120

94

92

(102)

15.6

8

8

15

(10)

4.0

25

25

31

(27)

3.5

24

29

24

(26)

2.9

15

9

8

(11)

3.8

500 µg

119

82

122

(108)

22.3

12

7

11

(10)

2.6

40

39

39

(39)

0.6

24

29

32

(28)

4.0

8

13

8

(10)

2.9

1500 µg

105

122

119

(115)

9.1

12

15

11

(13)

2.1

25

25

29

(26)

2.3

24

24

29

(26)

2.9

12

9

13

(11)

2.1

5000 µg

123

99

116

(113)

12.3

8

14

11

(11)

3.0

32

35

37

(35)

2.5

29

33

29

(30)

2.3

16

9

11

(12)

3.6

Positive controls

S9-Mix

(+)

Name

2AA

2AA

2AA

BP

2AA

Dose Level

1 µg

2 µg

10 µg

5 µg

2 µg

No. of Revertants

966

1010

1155

(1044)

98.9

253

204

219

(225)

25.1

170

151

148

(156)

11.9

152

147

148

(149)

2.6

238

273

254

(255)

17.5

Experiment 2 – Without Metabolic Activation

Test Period

From: 09 February 2015

To: 12 February 2015

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

86

64

78

(76)

11.1#

15

17

23

(18)

4.2

20

19

16

(18)

2.1

25

25

24

(25)

0.6

15

19

13

(16)

3.1

50 µg

64

84

64

(71)

11.5

17

13

21

(17)

4.0

23

23

21

(22)

1.2

8

16

19

(14)

5.7

9

27

19

(18)

9.0

150 µg

72

102

83

(86)

15.2

12

24

27

(21)

7.9

23

13

12

(16)

6.1

31

12

29

(24)

10.4

15

21

16

(17)

3.2

500 µg

80

78

76

(78)

2.0

12

20

13

(15)

4.4

16

11

13

(13)

2.5

16

17

15

(16)

1.0

8

15

24

(16)

8.0

1500 µg

72

78

68

(73)

5.0

11

9

12

(11)

1.5

11

24

28

(21)

8.9

19

27

19

(22)

4.6

7

8

13

(9)

3.2

5000 µg

80

80

94

(85)

8.1

21

7

11

(13)

7.2

17

19

17

(18)

1.2

12

12

13

(12)

0.6

12

13

7

(11)

3.2

Positive controls

S9-Mix

(-)

Name

ENNG

ENNG

ENNG

4NQO

9AA

Dose Level

3 µg

5 µg

2 µg

0.2 µg

80 µg

No. of Revertants

1947

1489

1070

(1502)

438.6

1511

1303

1113

(1309)

199.1

597

524

520

(547)

43.3

202

186

230

(206)

22.3

461

730

819

(670)

186.4

Experiment 2 – With Metabolic Activation

Test Period

From: 09 February 2015

To: 12 February 2015

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

79

108

107

(98)

16.5#

15

13

15

(14)

1.2

32

17

19

(23)

8.1

19

19

24

(21)

2.9

16

8

8

(11)

4.6

50 µg

84

90

112

(95)

14.7

9

12

12

(11)

1.7

32

35

31

(33)

2.1

8

13

17

(13)

4.5

8

16

12

(12)

4.0

150 µg

72

96

87

(85)

12.1

9

7

9

(8)

1.2

33

32

20

(28)

7.2

19

16

16

(17)

1.7

17

12

11

(13)

3.2

500 µg

80

86

91

(86)

5.5

11

12

9

(11)

1.5

32

31

37

(33)

3.2

17

20

24

(20)

3.5

12

8

9

(10)

2.1

1500 µg

90

95

68

(84)

14.4

9

9

9

(9)

0.0

32

21

21

(25)

6.4

16

13

17

(15)

2.1

16

12

13

(14)

2.1

5000 µg

94

95

83

(91)

6.7

8

9

15

(11)

3.8

25

23

25

(24)

1.2

24

16

17

(19)

4.4

7

12

5

(8)

3.6

Positive controls

S9-Mix

(+)

Name

2AA

2AA

2AA

BP

2AA

Dose Level

1 µg

2 µg

10 µg

5 µg

2 µg

No. of Revertants

1088

1057

1010

(1052)

39.3

182

176

176

(178)

3.5

95

94

151

(113)

32.6

104

136

138

(126)

19.1

246

293

283

(274)

24.8

Conclusions:
Interpretation of results (migrated information):
negative

Based on the study result, (benzylamine)trifluoroboron is considered to be non-mutagenic and therefore classification is not warranted according to the criteria of the Regulation (EC) No. 1272/2008.
Executive summary:

Salmonella typhimuriums trains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 50 to 5000 µg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method). 

Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 50 to 5000 µg/plate.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method). 


Justification for selection of genetic toxicity endpoint
well documented study with GLP certificate

Justification for classification or non-classification

Based on the study result, (benzylamine)trifluoroboron is considered to be non-mutagenic and therefore classification is not warranted according to the criteria of the Regulation (EC) No. 1272/2008.