4-Butoxy-2,3-difluorophenylboronic Acid (contains varying amounts of Anhydride)

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:

Mar 06 - Jun 29, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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-butoxy-2,3-difluorophenyl)boronic acid

EC Number:

692-793-0

Cas Number:

156487-12-6

Molecular formula:

C10H13BF2O2

IUPAC Name:

(4-butoxy-2,3-difluorophenyl)boronic acid

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver homogenate from male Wistar rats, Crl: WI (HAN) (Charles River, Germany), aged 6 - 8 weeks that were pretreated with ß-Naphthoflavone (100 mg/kg body weight) and Phenobarbital (80 mg/kg body weight).
- method of preparation of S9 mix: livers removed and homogenized in ice-cold 0.15 M KCl (3 mL
KCl per g liver wet-weight). The homogenate was spun for 10 minutes at 9000 rpm (8784 x g, Biofuge 28 RS) and +4°C. The supernatant fluid (S9) was decanted, transferred to sterile tubes and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 20 % S9 in the S9 mix were used in the 1st and 2nd test series; 0.5 mL were used in the final culture medium
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates.

Test concentrations with justification for top dose:

5, 15.8, 50, 158, 500, 1580, 5000 μg/plate ± S9 mix

Vehicle / solvent:

- Vehicle used: DMSO

DMSO was selected as solvent for the current experiment and 5000 µg/plate was chosen as the appropriate maximum test concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

20 μg/plate (TA98); 60 μg/plate (TA 1537) without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 μg/plate (WP2 uvrA) without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 μg/plate (TA 100, TA 1535) without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

2 µg/plate (TA98, TA 100); 5 μg/plate (TA1535, TA 1537); 10 μg/plate (WP2 uvrA) with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98,
TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is
observed
- an increase exceeding the threshold at only one concentration is considered as biologically
meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exce
eded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met
Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

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

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevanted by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
Following treatment of all bacteria tester strains with the test substance in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.
According to the criteria for negative and positive results predetermined in the study plan, the test substance was not mutagenic under the described experimental conditions.

Any other information on results incl. tables

1^st Series

 

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO			34 ± 12	123 ± 7	16 ± 7	10 ± 3	27 ± 4
	Test item	5.00		40 ± 2	117 ± 3	21 ± 1	9 ± 5	24 ± 5
		15.8		38 ± 6	107 ± 26	17 ± 10	9 ± 3	28 ± 9
		50.0		29 ± 8	106 ± 8	20 ± 6	10 ± 2	32 ± 3
		158		37 ± 5	124 ± 13	12 ± 2	10 ± 6	29 ± 6
		500		30 ± 4^B	112 ± 10^B	18 ± 8^B	7 ± 2^B	33 ± 2^B
		1580		35 ± 7^E	116 ± 12^T E	11 ± 3^T E	1 ± 1^T E	32 ± 8^E
		5000		37 ± 7^E	90 ± 17^T E	12 ± 5^T E	0 ± 0^T E	34 ± 8^E
	DAUN	2.00		397 ± 39
	NaN3	2.00			1525 ± 75	668 ± 71
	9-AA	50.0					1380 ± 541
	NQO	2.00						1558 ± 159
With Activation	DMSO			36 ± 11	125 ± 10	13 ± 4	10 ± 5	32 ± 5
	Test item	5.00		31 ± 6	125 ± 12	9 ± 3	9 ± 3	42 ± 5
		15.8		36 ± 6	126 ± 13	13 ± 1	11 ± 3	41 ± 6
		50.0		36 ± 6	112 ± 3	16 ± 6	7 ± 4	36 ± 4
		158		33 ± 6	147 ± 11	11 ± 5	12 ± 0	31 ± 2
		500		36 ± 1^B	151 ± 5^B	13 ± 1^B	7 ± 2^B	32 ± 1^B
		1580		37 ± 4^E	147 ± 13^T E	5 ± 4^T E	1 ± 1^T E	30 ± 4^E
		5000		37 ± 8^E	117 ± 12^T E	10 ± 2^T E	1 ± 1^T E	28 ± 10^E
	2-AA	2.00		1120 ± 162	2099 ± 137
	2-AA	5.00				105 ± 2	291 ± 21
	2-AA	10.0						303 ± 59

 

2^nd Series

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	DMSO			29 ± 6	123 ± 11	22 ± 5	8 ± 4	33 ± 7
	Test item	15.8		-	-	-	7 ± 5	-
		50.0		33 ± 9	123 ± 9	23 ± 7	12 ± 4	35 ± 6
		158		26 ± 10	131 ± 2	23 ± 7	7 ± 4	34 ± 6
		500		31 ± 12^B	130 ± 14^B	19 ± 3^B	8 ± 2^B	33 ± 4^B
		1580		24 ± 6^T E	74 ± 19^T E	12 ± 4^T E	2 ± 1^T E	35 ± 2^E
		5000		22 ± 3^T E	40 ± 64^T E	11 ± 5^T E	2 ± 1^T E	33 ± 1^E
	DAUN	2.00		223 ± 81
	NaN3	2.00			1333 ± 43	760 ± 28
	9-AA	50.0					538 ± 228
	NQO	2.00						1742 ± 116
With Activation	DMSO			35 ± 4	128 ± 21	17 ± 3	14 ± 7	38 ± 6
	Test item	15.8		-	-	-	12 ± 5	-
		50.0		35 ± 2	130 ± 17	15 ± 3	13 ± 1	40 ± 17
		158		36 ± 12	128 ± 10	14 ± 5	8 ± 5	41 ± 5
		500		35 ± 5^B	140 ± 19^B	17 ± 4^B	10 ± 6^B	38 ± 8^B
		1580		31 ± 7^T E	101 ± 27^T E	13 ± 2^T E	5 ± 3^T E	36 ± 2^E
		5000		26 ± 11^T E	46 ± 32^T E	9 ± 2^T E	1 ± 1^T E	33 ± 5^E
	2-AA	2.00		231 ± 18	492 ± 105
	2-AA	5.00				89 ± 6	135 ± 3
	2-AA	10.0						176 ± 12

 

 

 

Key to Positive controls	Key to Plate Postfix Codes
2-AA 2-Aminoanthracene 4-NOPD 4-Nitro-o-phenylendiamin NaN3 Sodium azide NQO 4-Nitroquinoline-N-oxide	E Precipitation until end of experiment M manual count T Toxicity = red. bact. background lawn

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing System, the test item was not mutagenic in this Salmonella typhimurhim and Escherichia coli reverse mutation test.