(benzylamine)trifluoroboron

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Gouvernment of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(benzylamine)trifluoroboron

EC Number:

211-802-7

EC Name:

(benzylamine)trifluoroboron

Cas Number:

696-99-1

Molecular formula:

C7H9BF3N

IUPAC Name:

(benzylamine)trifluoroboron

Test material form:

solid: particulate/powder

Remarks:

agglomerated powder

Details on test material:

- Lot/batch No.: 05-23851

Method

Target gene:

histidine-, tryptophan-

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: rfa-, uvrB-, R factor plasmid pKM101 (TA98 and TA100)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type 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 / solvent:

dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle 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 experimental 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.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.