Boronic acid, (4-ethylphenyl)-

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:

2006-12-04 to 2007-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000-06-08

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4-ethylphenyl)boronic acid

EC Number:

613-145-5

Cas Number:

63139-21-9

Molecular formula:

C8 H11 B O2

IUPAC Name:

(4-ethylphenyl)boronic acid

Test material form:

solid

Method

Target gene:

- HIS operon (Salmonella typhimurium strains)
- TRP operon (Escherichia coli WP2 uvrA);

The strains are constructed to differentiate between base pair (E.coli WP2, TA 1535, TA 100, TA 102) and frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor-induced male Wistar rats

- Method of preparation of S9 mix: Male Wistar rats were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun al 9000 x g for 10 minutes al about 4°C and the supernatant fluid was decanted and transferred into sterilized and prccooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.

- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix in the final medium (total: 2.61 mL), 10% of S9 in the S9 mix (1st series) and 30% of S9 in the S9 mix (2nd series)

- Quality controls of S9: Every S9-batch is 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 is thus determined once for every S9-batch.

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg per plate
2nd series: 158, 500, 889, 1580, 2810 µg per plate;

A maximum concentration of 5000 µg/plate was selected as the maximum recommended concentration according to current regulatory guidelines (OECD, EEC).

Vehicle / solvent:

- Vehicles/solvents used: DMSO (test item, cumene hydroperoxide, 2-aminoanthracene, benzo[a]pyrene, 4-nitroquinoline-N-oxide), ethanol (9-aminoacridine), ultrapure water (daunomycin, sodium azide)

- Justification for choice of solvent/vehicle: Solubility up to 1580 ug/plate

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of solvent will dilute the top agar, the maximum amount of solvent was limited to 100 µL per plate for water and DMSO. For ethanol, acetone and other organic solvents, 10 µL per plate was not exceeded.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, used for TA 102 (10 µg/plate)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, used for TA 98, TA100 and TA 1535 (2 µg/plate), TA 1537 (5 µg/plate) E.coli WP2 uvrA (10 µg/plate)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, used for E.coli WP2 uvrA (2 µg/plate)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, used for TA 1537 (50 µg/plate)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9, used for TA 102 (200 µg/plate)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

H2O

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, used for TA 100 and TA1535 (2 µg/plate)

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

H2O

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9, used for TA 98 (1 µg/plate)

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:
- Plate incorporation method

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Background growth inhibition
- Reduction in the number of spontaneous revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Scoring of revertants colonies using an Artek MiniCount colony counter or manually

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. Criteria, which are based upon the historical controls of the laboratory and statistical considerations are shown in table 1.

Statistics:

n.a.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 1580 µg/plate until the end of the experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: Cytotoxic effects to the bacteria were observed at concentrations > 2810 µg/plate
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"


HISTORICAL CONTROL DATA
- Positive historical control data: See "Any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: See "Any other information on results incl. tables"

Any other information on results incl. tables

Table 2: According to the publications of Levin et al. (1982) and Kier et al. (1986) and the historical controls of the laboratory, usually the following mean numbers of revertants are acceptable as negative (or solvent) controls for the bacterial strains used:

 

TA 98:	15-60	TA 1535:	3-37
TA 100:	75-200	TA 1537:	4-31
TA 102:	200-450	E.coli WP2 uvrA:	10-70

 

 

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coti WP2 uvrA, The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1^st and 2^nd series, respectively. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations > 1580 µg/plate. Cytotoxic effects to the bacteria were observed at concentrations > 2810 µg/plate. Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide and cu-mene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each 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. With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.