3-Fluorphenylboric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was positive in an Ames test - both with and without metabolic activation (reference 7.6.1 -1).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

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

Target gene:

histidine or tryptophan

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.

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed similar to OECD TG 471. A read across justification is provided in the attached document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The read-across substance was negative in an in vivo Micronucleus assay in rats (reference 7.6.2 -1) and in an combined in vivo Comet + micronucleus assay in rats (reference 7.6.1 -2).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD Guidelines 489 and 474. A read across justification is provided in the attached document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Comet assay: Negative for inducing genotoxicity in the liver and glandular stomach. The duodenum was assessed as an inappropriate tissue to test with this experimental design.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

Micronucleus assay: Equivocal in the bone marrow of treated animals when dosed up to the toxic dose.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GRP and the methods applied are fully compliant with OECD TG 474. A read across justification is provided in the attached document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The mean percentage of PCE was less than 20 % of the historical vehicle control (males 49.5 ± 5.27 ‰; females 43.9 ± 9.72 ‰), indicating that no relevant bone marrow toxicity was present.

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames test

Key study (reference 7.6.1 -1)

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.

 

Supporting (reference 7.6.1 -2)

The mutagenic potential of the read-across substance was examined in a screening assay equivalent to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100 and Escherichia coli WP2 uvrA. The plate incorporation test (two parallel plates per condition) with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. The read-across substance was dissolved in DMSO and tested at concentrations ranging from 5 - 5000 μg/plate.

Precipitation of the read-across substance on the agar plates occurred at concentrations of >= 1580 μg/plate and toxicity to bacteria at 5000 µg/plate. The treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following read-across substance treatments in the absence and presence of S9, the read-across substance was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA.

With and without addition of S9 mix as the external metabolizing system, the read-across substance was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA.

 

 

In vivo

No in vivo study on genetic toxicity using the test item is available. Therefore, a read-across to the substance with a very similar chemical structure and comparable physico-chemical parameters is used to evaluate the in vivo genetic toxicity potential of the test item.

 

Micronucleus test (reference 7.6.2 -1)

The objective of this study was to investigate the potential of the read-across substance to induce micronuclei formation in the bone marrow of rats.

Therefore, a micronucleus test, according to OECD 474 was performed. Femurs from male and female animals from a 14-day oral dose range finding toxicity study in rats were used. Both femurs were taken from animals treated in that study, 24 hours after the last administration, processed and evaluated microscopically as in a standard acute micronucleus study. The read-across substance was given orally by gavage at doses of 100, 300 or 600 mg/kg body weight/day. No animals in this study were treated with a negative or positive control compound. However, positive control slides from a previous study were coded together with the slides of this study in order to check accurate slide reading.

Bone marrow smears were prepared from two femurs of each animal and stained with Giemsa's solution. A total of 30 animals (5 for each main group and sex) were used for slide preparation. For microscopic investigation from five animals per group (30 animals in total) one out of three slides prepared from each animal was selected and coded. The number of polychromatic (immature) erythrocytes (PCE) containing micronuclei (MN-PCE) per 4000 polychromatic erythrocytes was determined.

The proportion of PCE among total erythrocytes was determined based on the analysis of 1000 erythrocytes per animal. The micronucleated nonnochromatic erythrocytes (NCE) were registered also when scoring the polychromatic erythrocytes.

The slide reading control (positive control slides of male and female rats treated with 10 mg/kg bw cyclophosphamide) showed the expected increase in the number of MN-PCE. The values obtained were perfectly within the historical positive control (males: 16.4 +/- 5.8 ‰; females: 10.7 +/- 3.9 ‰).For each animal, the appropriate number of cells (4000 PCE) was analysed. Hence, the study was accepted as valid.

In the 14 -day oral dose range finding toxicity, the daily dosing with 600 mg/kg bw/d for 14 days was considered to be the maximum tolerated dose and therefore appropriate for micronucleus investigations.

In none of the groups treated with the read-across substance, the mean percentage of PCE was less than 20 % of the historical vehicle control (males: 49.5 +/- 5.27 ‰; females: 43.9 +/- 9.72 ‰), indicating that no relevant bone marrow toxicity was present.

No relevant increases in the number of MN-PCE were observed in any of the female or male groups treated with the read-across substance compared to the historical negative controls (males: 1.7 +/- 0.7 ‰; females: 1.3 +/- 0.7 ‰).

According to the predetermined criteria for the interpretation of results, the read-across substance was not mutagenic in the micronucleus test in bone marrow of male and female rats treated with a daily oral dose of 100, 300 or 600 mg/kg bw/d for 14 days under the conditions described.

 

Comet assay and micronucleus test (reference 7.6.2 -2)

This study was intended to evaluate the potential of the read-across substance to induce genotoxicity in the liver, duodenum, glandular stomach and bone marrow of male Sprague Dawley rats following oral administration (gavage). Among the various mechanistic-based techniques for evaluating the genotoxic activity of a test article, the comet assay can detect DNA damage (strand breaks, alkali-labile sites, cross-linking, adduct formation) in individual cells from any target organ and regardless of mitotic activity. Combined with or supplemental to the in vivo micronucleus (MN) assay which can detect clastogens and aneugens in the bone marrow and peripheral blood, this study provided the best method for determining the genotoxicity of the test article. The study was conducted in accordance with OECD test guidelines 489 and 474.

Groups of 5 male Sprague Dawley rats were dosed orally once daily via gavage with 0, 30, 100, 300 or 1000 mg/kg bw/d over 3 consecutive days. The sample time was 4 hours after last dosing and the evaluated samples in the comet assay were liver, glandular stomach, duodenum and in the micronucleus assay the bone marrow. A histopathological examination in liver, glandular stomach and duodenum was performed.

In the comet assay, there was no response in the liver or glandular stomach. In the MN assay, a significant and dose dependent increase in MN-PCEs was induced in the bone marrow at 1000 mg/kg bw/d. However, since liver toxicity and a significant decrease in body weight gain was also detected at this dose level, the increase in micronuclei may be due to toxicity. With the exclusion of the toxic dose from statistical analysis, there was no dose-dependent increase in MN-PCEs. A significant increase in MN-PCEs at a dose level of 30 mg/kg bw/d was within the historical control range for the vehicle control and is most likely a statistical artefact.

Under the experimental conditions of this study, the read-across substance was negative for inducing genotoxicity in the liver and glandular stomach and equivocal in the bone marrow of treated animals when dosed up to the toxic dose. Since the duodenum was an inappropriate tissue to test with this experimental design and a valid test was achieved in the glandular stomach as the site of contact, a repeat test in the duodenum is not recommended.

 

 

Overall conclusion

The test item showed an increase in revertant numbers in the Ames test for some of the used strains (Salmonella typhimurium tester strains TA 100, TA 1537 and Escherichia coli WP2 uvrA) indicating a genetic mutation potential by base (pair) substitution and frame shifts.

Due to this finding two in vivo micronucleus tests and a comet assay conducted with the read-across substance, possessing a very similar chemical structure and comparable physico-chemical parameters as well as demonstrating a similar behavior in the Ames test, were employed to further assess the genetic toxicity potential of the test item in vivo. No increase in formation of micronuclei and no response in the comet assay for the read-across substance were detected at non-toxic dose levels in the appropriate tissues. Therefore it was determined the read-across substance and the test item are considered to be non-mutagenic and non-clastogenic in mammalian cells.

Considering all available data it was concluded that the test item is not to be classified for genetic toxicity.

 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available data for genetic toxicity is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be not classified for genetic toxicity (UN GHS: No category) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EC) 2019/521.