4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

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:

2017-09-19 to 2017-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

EC Number:

929-209-3

Cas Number:

1123312-95-7

Molecular formula:

C14 H13 B F2 O2

IUPAC Name:

4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9 (experiment I) non-induced hamster liver (experiment II)

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Experiment IIa:
Strain TA 100 without S9 mix: 0.3; 1; 3; 10; 33; 100; and 333 µg/plate

No correction for purity was made.

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains used

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used.

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 VV Plate	Date Plated: 19.09.2017
Assay Conditions:	Date Counted: 22.09.2017

Metabolic Activation	Test Group		TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		13 ± 4	10 ± 2	22 ± 3	151 ± 7	31 ± 2
Activation	Untreated		22 ± 8	10 ± 4	20 ± 4	150 ± 6	31 ± 6
	Test item	3 µg	12 ± 4	7 ± 1	26 ± 5	132 ± 15	30 ± 8
		10 µg	13 ± 6	8 ± 2	19 ± 3	135 ± 6	31 ± 6
		33 µg	11 ± 1	9 ± 3	24 ± 4	107 ± 7	31 ± 7
		100 µg	9 ± 2	8 ± 3	19 ± 6	42 ± 9	41 ± 10
		333 µg	7 ± 2^P	8 ± 1^P	13 ± 3^P M	9 ± 2^P M	25 ± 9^P
		1000 µg	5 ± 2^P M	4 ± 1^P M	5 ± 1^P M	4 ± 1^P M	13 ± 2^P M
		2500 µg	4 ± 1^P M	3 ± 1^P M	3 ± 2^P M	2 ± 1^P M	13 ± 1^P M
		5000 µg	1 ± 1^P M	1 ± 1^P M	2 ± 1^P M	1 ± 1^P M	8 ± 2^P M
	NaN3	10 µg	1181 ± 33			2113 ± 150
	4-NOPD	10 µg			293 ± 29
	4-NOPD	50 µg		78 ± 11
	MMS	2.0 µL					994 ± 85
With	DMSO		15 ± 3	13 ± 3	27 ± 6	130 ± 1	38 ± 2
Activation	Untreated		9 ± 2	14 ± 4	35 ± 7	126 ± 13	41 ± 10
	Test item	3 µg	14 ± 3	16 ± 6	28 ± 10	118 ± 10	32 ± 11
		10 µg	11 ± 5	15 ± 5	29 ± 3	123 ± 17	48 ± 6
		33 µg	14 ± 4	11 ± 2	31 ± 10	127 ± 6	34 ± 6
		100 µg	11 ± 1	12 ± 6	27 ± 4	132 ± 8	48 ± 12
		333 µg	8 ± 2^P	18 ± 3^P	23 ± 4^P	42 ± 11^P	30 ± 7^P
		1000 µg	5 ± 1^P M	7 ± 1^P M	12 ± 3^P M	7 ± 2^P M	19 ± 4^P M
		2500 µg	4 ± 1^P M	4 ± 1^P M	5 ± 1^P M	3 ± 1^P M	14 ± 1^P M
		5000 µg	2 ± 1^P M	2 ± 1^P M	3 ± 1^P M	2 ± 1^P M	12 ± 0^P M
	2-AA	2.5 µg	410 ± 30	166 ± 26	3187 ± 195	4245 ± 136
	2-AA	10.0 µg					415 ± 59

Summary of Experiment II

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 HV2 Pre	Date Plated: 04.10.2017
Assay Conditions:	Date Counted: 11.10.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		9 ± 1	12 ± 3	30 ± 7	155 ± 9	32 ± 5
Activation	Untreated		9 ± 3	13 ± 2	32 ± 6	162 ± 1	39 ± 8
	Test item	3 µg	7 ± 2	15 ± 3	23 ± 5	139 ± 5	36 ± 10
		10 µg	5 ± 2	15 ± 6	29 ± 5	130 ± 7	34 ± 11
		33 µg	6 ± 1	12 ± 4	20 ± 3	61 ± 5	32 ± 6
		100 µg	6 ± 1	10 ± 3	12 ± 2	36 ± 11	25 ± 2
		333 µg	6 ± 1^P	9 ± 3^P M	2 ± 1^P M	2 ± 1^P M	18 ± 4^P
		1000 µg	3 ± 2^P M	4 ± 1^P M	0 ± 1^P M	0 ± 1^P M	18 ± 4^P
		2500 µg	2 ± 1^P M	3 ± 1^P M	0 ± 1^P M	0 ± 0^P M	10 ± 2^P M
		5000 µg					6 ± 2^P M
	NaN3	10 µg	1213 ± 71			1961 ± 62
	4-NOPD	10 µg			314 ± 6
	4-NOPD	50 µg		89 ± 9
	MMS	2.0 µL					858 ± 90
With	DMSO		14 ± 5	17 ± 3	33 ± 3	140 ± 13	45 ± 7
Activation	Untreated		11 ± 5	17 ± 7	40 ± 8	193 ± 35	54 ± 12
	Test item	3 µg	15 ± 4	16 ± 8	36 ± 10	144 ± 17	47 ± 2
		10 µg	14 ± 3	20 ± 2	35 ± 7	145 ± 14	39 ± 5
		33 µg	18 ± 4	14 ± 6	39 ± 4	127 ± 12	44 ± 8
		100 µg	13 ± 4	15 ± 6	41 ± 8	100 ± 9	42 ± 6
		333 µg	10 ± 4^P	15 ± 5^P	29 ± 8^P	8 ± 3^P M	26 ± 9^P
		1000 µg	9 ± 3^P	6 ± 2^P M	7 ± 2^P M	2 ± 1^P M	26 ± 5^P
		2500 µg	7 ± 3^P M	6 ± 1^P M	1 ± 1^P M	0 ± 1^P M	15 ± 4^P M
		5000 µg					13 ± 1^P M
	2-AA	2.5 µg	378 ± 11	104 ± 17	4405 ± 188	3595 ± 36
	2-AA	10.0 µg					532 ± 25

Summary of Experiment IIa

Study Name: 1862000	Study Code: Envigo 1862000
Experiment: 1862000 HV2a Pre	Date Plated: 19.10.2017
Assay Conditions:	Date Counted: 23.10.2017

Metabolic Activation	Test Group	Dose Level (per plate)	TA 100 Revertant Colony Counts (Mean ±SD)
Without	DMSO		145 ± 17
Activation	Untreated		192 ± 14
	Test item	0.3 µg	137 ± 7
		1 µg	145 ± 12
		3 µg	152 ± 6
		10 µg	130 ± 9
		33 µg	71 ± 9
		100 µg	1 ± 1^P M
		333 µg	0 ± 0^P M
	NaN3	10 µg	2092 ± 34

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in three independent experiments with and without liver microsomal activation (S9 mix). Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:          3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:

Strain WP2uvrA:                              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The remaining strains:                       3; 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:

Strain TA 100 without S9 mix:         0.3; 1;3; 10; 33; 100; and 333 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in experiment I, from 1000 to 5000 µg/plate in experiment II and at 333 µg/plate in experiment IIa. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and II and from 100 to 333 µg/plate in experiment IIa. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I without S9 mix	Experiment I with S9 mix	Experiment II without S9 mix	Experiment II with S9 mix	Experiment IIa without S9 mix
TA 1535	1000 – 5000	1000 – 5000	1000 – 2500	/	-
TA 1537	1000 – 5000	1000 – 5000	1000 – 2500	1000 – 2500	-
TA 98	1000 – 5000	2500 – 5000	100 – 2500	1000 – 2500	-
TA 100	100 – 5000	333 – 5000	33 – 2500	333 – 2500	100 – 333
WP2uvrA	1000 – 5000	2500 – 5000	2500 – 5000	2500 – 5000	-

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

- = not performed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.