Boronic acid, (4-ethylphenyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay according to OECD guideline 471 and in a in vitro mammalian cell gene mutation assay according to OECD guideline 476, the test item was not mutagenic with and without metabolic activation (S9 mix) under the experimental conditions described (reference 7.6.1-1 and 7.6.1-2).

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:

2006-12-04 to 2007-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000-06-08

Deviations:

no

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

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 / cell type:

E. coli WP2 uvr A

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.

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)

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)

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)

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)

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)

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)

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.

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

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

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

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

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

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"

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

 

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-11-27 to 2007-04-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2000-06-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK locus (mutations + chromosome aberrations)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK (+/-) mouse lymphoma cells, obtained from Dr. W. Muster, Hoffmann-La Roche, Basel, Switzerland
- Suitability of cells: L5178Y cells and the TK gene locus are well-validated and widely used

For cell lines:
- Absence of Mycoplasma contamination: Each batch of frozen cells was checked for absence of mycoplasma.
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, temperature:
Media: RPMI 1640 with 1%penicillin/streptomycin and 5% (exposure medium)/10% (culture medium) or 20% (survivor- and selection medium) heat inactivated horse serum
CO2 concentration: 5%
Temperature: 37°C

Additional strain / cell type characteristics:

not specified

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 PBS-HEPES. 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 precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.

Test concentrations with justification for top dose:

1st and 2nd series (- S9): 8.89, 28.1, 88.9, 281, 889 and 2810 µg/mL medium;

1st series (+ S9): 8.89, 28.1, 88.9, 281, 889 and 2810 µg/mL medium;
2nd series (+ S9): 8.89, 28.1, 88.9, 281 µg/mL medium

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item + positive control)

- Justification for choice and percentage of solvent in the final culture medium: Analysis of the historical data of the laboratory and experience of other research groups showed that such amounts of the selected solvents have no influence on the mutation frequency in this test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

0.1 and 0.2 µg/mL

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:

0.5, 1, 2, 3 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^7 cells
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours or 24 hours
1st series: 3 hours (+ S9), 24 hours (- S9)
2nd series: 3 hours (+ S9), 3 hours (- S9)

FOR GENE MUTATION:
- Expression time : 2 days
- Selection time: 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 10-14 days
- Selective agent: trifluorothymidine (TFT), 3 µg/mL
Duration and period of cell exposure to TFT: Plates were incubated until scorable (day 10 to day 14)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for viability: 1.6 cells per well, staining with MTT and identification + counting by eye
Plating for TFT restistance: 2x10^3 cells per well, staining with MTT and identification + counting by eye

METHODS FOR MEASUREMENT OF CYTOTOXICITY
relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
At least for the negative and positive controls and, in case of a positive test material-induced effect, also for those cultures that showed the highest test material-induced effect, the mutation frequency is determined separately for small and large colonies in addition to the total mutation frequency.

Rationale for test conditions:

Range-finding test:
In a preceding range finding test, the relative survival was determined after exposure to various test material concentrations ranging between 2.81 and 2810 ug/mL. A reduction in the relative survival of the cells occurred at concentrations > 2810 ug/mL in the absence and 889 ug/mL and presence of S9 mix, respectively. Precipitation of the test item in the cell culture medium was seen at concentrations > 281 ug/mL. A relevant change in the pH and the osmolarity of the culture medium was not detected in the concentration range tested in the main study.

Justification of the concentrations used:
At least four concentrations over an adequate concentration range should be employed. The highest concentration should precipitate, in the culture medium or exhibit cytotoxicity, The cytotoxicity of the lowest concentration should usually correspond to that of the negative controls. Soluble test materials, if not toxic, should be tested up to a maximum concentration of 5000 ug/mL, 5 uL/mL, or 10 mM, respectively.

If cytotoxicity is the limiting factor for the selection of test material concentrations, usually five concentrations are established for the main study because toxicity may alter due to biological variability. Depending on the degree of toxicity in the main study, the highest or lowest concentration is omitted in the course of the mutagenicity experiment.

Evaluation criteria:

Evaluation criteria:
The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.
Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.
Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.

Statistics:

n.a.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but 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:

mouse lymphoma L5178Y cells

Metabolic activation:

with

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
- Data on pH: A relevant change in the pH of the culture medium was not detected in the concentration range tested in the main study.
- Data on osmolality: A relevant change in osmolarity of the culture medium was not detected in the concentration range tested in the main study.
- Precipitation and time of the determination: Precipitation of the test material in the cell culture medium occurred at concentrations > 281 ug/mL.

RANGE-FINDING/SCREENING STUDIES: A reduction in the relative survival of the cells occurred at concentrations > 2810 µg/mL in the absence and 889 µg/mL and presence of S9 mix, respectively.

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

For all test methods and criteria for data analysis and interpretation: See "Any other information on materials and methods incl. tables"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 10^7 cells were treated. After the incubation period, 2x10^5 cells/mL were transferred to flasks for growth through the expression period or were diluted to be plated for survival. During the expression period, subculturing was performed as required with the aim of not exceeding 1x10^6 cells/mL and, where possible, retaining at least 1x10^7 cells/flask.
o Number of cells plated in selective and non-selective medium: 2x10^3 cells per well were incubated in selective/non-selective medium.
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"


HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

The test item was non-mutagenic in this in vitro mammalian cell gene mutation test.

Executive summary:

The test item was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in L5178Y mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). Dimethyl sulfoxide (DMSO) was used as the solvent for the test item. The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively. Various test item concentrations ranging from 8.89 to 2810 µg/mL were tested in the absence or presence of S9 mix. Precipitation of the test material in the incubation medium was observed at concentrations > 281 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations > 281 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine (TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz-[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. No biological relevant increases in mutant frequency were observed following treatment with the test item in the two experimental series in the absence and presence of S9 mix. It is therefore concluded that the test item is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay, key study

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.

 

In vitro mammalian cell gene mutation test, key study

The test item was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in L5178Y mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). Dimethyl sulfoxide (DMSO) was used as the solvent for the test item. The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively. Various test item concentrations ranging from 8.89 to 2810 µg/mL were tested in the absence or presence of S9 mix. Precipitation of the test material in the incubation medium was observed at concentrations > 281 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations > 281 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine (TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz-[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. No biological relevant increases in mutant frequency were observed following treatment with the test item in the two experimental series in the absence and presence of S9 mix. It is therefore concluded that the test item is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

 

Conclusion: The test item was found to be non-mutagenic in both tests.

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.