3-Fluorphenylboric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Nov 2016 - 06 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-720-8

EC Name:

-

Cas Number:

768-35-4

Molecular formula:

C6 H6 B F O2

IUPAC Name:

(3-fluorophenyl)boronic acid

Test material form:

solid

Method

Target gene:

histidine or tryptophan

Species / strain

Species / strain / cell type:

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

Remarks:

(E.coli WP2 uvrA)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by ß-Naphthoflavone/phenobarbital
Type and composition of metabolic activation system:
- source of S9
Liver of male Wistar rats, Crl:WI (HAN) (age: 6-8 weeks)
Animals are pretreated with ß-Naphthoflavone/phenobarbital. One day after last administration animals were sacrificed. The livers were removed and homogenized in ice-cold 0.15 M KCI. The homogenate was spun for 10 minutes at 9000 rpm at 4 °C. The supernatant fluid was decanted, transferred to sterile tubes and stored in liquid nitrogen at -196 °C.
- method of preparation of S9 mix
Liver homogenate (0.1 mL), MgCl2/KCI aqueous solution (0.02 mL), Glucose-6-phosphate disodium salt (5 µmol), NADP disodium salt (4 µmol), Sodium phosphate buffer (0.5 mL) and Ultra pure water (0.38 mL) are used to produce 1 mL of S9 mix.
- concentration or volume of S9 mix and S9 in the final culture medium
0.5 mL of S9 mix (containing 10 % S9) were added per plate, if applicable.
- quality controls of S9
S9-batch was tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene. and 3-methylcholanthrene was thus determined once for the S9-batch.

Test concentrations with justification for top dose:

5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: Comparing the standard vehicles for this assay indicated that the test item showed best solubility performance in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

10 µL/plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene 2-10 µg/plate, daunomycin 1 µg/plate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: The incubation of plates was performed at 36 - 38 °C for 2 to 3 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition, counting numbers of revertants

Evaluation criteria:

A test material was to be defined as negative or non-mutagenic in this assay if
• the assay was to be considered valid, and
• "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)
For valid data, the test material was considered to be positive or mutagenic if:
• a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
• "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

Statistics:

Not performed as not mandatory for this test system.

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: There were clear dose dependent increases in revertant numbers after test item exposure observed in TA 100, TA 1537, and WP2 uvrA in the absence and presence of S9 mix

Ames test:
- Signs of toxicity : No toxicity was observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
TA 98: -S9: 362+/-201.9 (DAUN), +S9: 769+/-257.4 (2-AA)
TA 100: -S9: 1360+/-305 (NaN3), +S9: 1365+/-314.9 (2-AA)
TA 1535: -S9: 867+/-172.3 (NaN3), +S9: 262+/-51.8 (2-AA)
TA 1537: -S9: 977+/-429.9 (9-AA), +S9: 378+/-142.3 (2-AA)
WP2 uvrA: -S9: 1606+/-488.8 (NQO), +S9: 394+/-142.2(2-AA)
- Negative (solvent) historical control data:
TA 98: -S9: 37+/-4.3, +S9: 42+/-5.6
TA 100: -S9: 111+/-12.2, +S9: 120+/-11.3
TA 1535: -S9: 31+/-6.9, +S9: 28+/-4.4
TA 1537: -S9: 24+/-3.4, +S9: 25+/-4.7
WP2 uvrA: -S9: 33+/-5.3, +S9: 38+/-5.3

Any other information on results incl. tables

Table 1: Summary of Experiment 1

Metabolic Activation	Test material	Concentr. (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO		37 +/- 5	119 +/- 8	22 +/- 3	25 +/- 5	21 +/- 5
	Test item	5	44 +/- 18	115 +/- 10	21 +/- 3	25 +/- 3	19 +/- 2
		15.8	35 +/- 4	126 +/- 6	18 +/- 3	32 +/- 5	19 +/- 5
		50	35 +/- 13	144 +/- 13	28 +/- 4	30 +/- 2	26 +/- 8
		158	28 +/- 4	172 +/- 10	26 +/- 11	27 +/- 2	31 +/- 7
		500	39 +/-4	168 +/- 24	17 +/- 8	39 +/- 7	40 +/- 8
		1580	43 +/- 7	211 +/- 16	24 +/- 8	50 +/- 2	84 +/- 21
		5000	29 +/- 25	149 +/- 44	20 +/- 3	39 +/- 6	71 +/- 74
	DAUN	1	402 +/- 96
	NaN3	2		437 +/- 36	583 +/- 34
	9-AA	50				324 +/- 87
	NQO	2					582 +/- 75
With Activation	DMSO		40 +/- 7	121 +/- 19	19 +/- 7	32 +/- 9	24 +/- 11
	Test item	5	33 +/- 9	141 +/- 17	25 +/- 1	24 +/- 6	23 +/- 1
		15.8	41 +/- 12	134 +/- 7	23 +/- 3	25 +/- 5	23 +/- 9
		50	39 +/- 11	126 +/- 19	21 +/- 5	29 +/- 5	27 +/- 2
		158	39 +/- 3	151 +/- 9	21 +/- 4	32 +/- 4	24 +/- 1
		500	41 +/- 5	162 +/- 25	24 +/- 6	37 +/- 3	32 +/- 9
		1580	42 +/- 2	170 +/- 23	24 +/- 5	57 +/- 6	74 +/- 31
		5000	35 +/- 10	127 +/- 56	27 +/- 6	67 +/- 18	89 +/- 4
	2-AA	2	543 +/- 54	896 +/- 70	92 +/- 19
	2-AA	5				493 +/- 43
	2-AA	10					176 +/- 8

Key to Positive Controls

NaN₃                Sodium azide

2-AA                2-Aminoanthracene

9-AA                9-Aminoacridine

DAUN              Daunomycin

NQO                4-Nitroquinoline-N-oxide

Applicant's summary and conclusion

Conclusions:

The test item is considered mutagenic under the test conditions.

Executive summary:

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA in a study according to OECD 471. The plate incorporation test with and without addition of liver S9 mix from Phenobarbital / p-Naphthoflavone pre-treated rats was used. In this study, one experimental series was performed. In the one series with S9 mix, 10 % S9 mix was used.

The test item was applied at concentrations of 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate.

Vehicle (DMSO) and positive control (dependent on tester strain) treatments were included for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test item treatments of all the tester strains in the absence and presence of S9 mix, clear dose dependent increases in revertant numbers were observed in TA 100, TA 1537 and WP2 uvrA. No cytotoxicity and no precipitation of the test item were detected. 

It was concluded that the test item was mutagenic with and without addition of S9 mix as the external metabolizing system under the experimental conditions.