Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD TG 471: Negative

OECD TG 490: Negative

OECD TG 487: Negative

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:

22 Jul 2006 to 22 Aug 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Official Notification, February 8, 1999) and "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemicals Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No. 031121002 of the Environmental Health Department, MOE (November 21, 2003)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- Dose range finding test 1 (without and with S9 mix): 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
- Dose range finding test 2 (without S9 mix): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Dose range finding test 2 (with S9 mix): 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
- Main test (without S9 mix): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Main test (with S9 mix): 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was insoluble in distilled water at 50 mg/mL but it was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates were used for the solvent control and duplicate plate per dose for the test substance treatment and positive control groups.

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

Evaluation criteria:

The test substance was judged positive when the number of revertant colonies increased to twice or more than in the solvent control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases, it was judged negative.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, 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

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- Dose range finding test 1: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed in groups exposed to more than 78.1 µg/plate in all test strains without S9 mix and more than 313 µg/plate in all test strains with S9 mix. The precipitation of the test substance was observed at 5000 µg/plate without S9 mix.
- Dose range finding test 2: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed in groups exposed to more than 39.1 µg/plate in TA 100 and TA 1535 without S9 mix and at 78.1 µg/plate in WP2uvrA, TA 98 and TA 1537 without S9 mix and more than 156 µg/plate in all test strains with S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY MAIN TEST:
The bacterial growth inhibition was observed in groups exposed to more than 39.1 µg/plate in TA 100 and TA 1535 without S9 mix and at 78.1 µg/plate in WP2uvrA, TA 98 and TA 1537 without S9 mix and more than 156 µg/plate in all test strains with S9 mix.

Conclusions:

The substance is not mutagenic in the Salmonella Typhimurium and Escherichia Coli reverse mutation assay performed equivalent to OECD 471.

Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECDTG 471 (1997) and according to GLP principles. A pre-incubation assay was performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxicity in two dose range finding studies. The test substance was considered to be toxic between 10 – 200 µg/plate depending on the strain and with and without metabolic activation. Solvent controls and positive controls were included. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Apr 2016 to 17 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 - - Environment Protection Agency; Eisei No. 127 - - Ministry of Health and Welfare; Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited, Sharlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

Thymidine Kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test (7.42 to 1900 µg/mL).

The dose levels plated for viability and expression of mutant colonies were as follows:
- 4-hour without S9: 5, 10, 20, 40 µg/mL
- 4-hour with S9 (2%): 10, 20, 40, 60, 80 µg/mL
- 24-hour without S9: 1.25, 2.5. 5, 10, 20, 30 µg/mL

The maximum dose level used was limited by Test item induced toxicity.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-hour exposure groups both in the absence and presence of metabolic activation. 24-hour exposure group in the absence of metabolic activation
- Expression time: 2 days
- Selection time: 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Evaluation criteria:

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

For acceptance criteria see 'Any other information on material and methods incl. tables'

Statistics:

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality; No
- Precipitation: No precipitate was observed during the main test.

RANGE-FINDING/SCREENING STUDIES:
The dose range of the test item used in the preliminary toxicity test was 7.42 to 1900 μg/mL. In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity curve was very steep in all three exposure groups. A precipitate of the test item was observed at and above 475 μg/mL in the 4-hour and 24-hour exposures in the absence of metabolic activation and at and above 237.5 μg/mL in the 4-hour exposure in the presence of metabolic activation. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity.

HISTORICAL CONTROL DATA
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity test item dose levels 80, 100 and 120 μg/mL in the 4-hour exposure in the absence of metabolic activation, 120 μg/mL in the 4 hour exposure in the presence of metabolic activation, and 40 μg/mL in the 24-hour exposure absence of metabolic activation were not plated out for 5- TFT resistance and viability due to excessive toxicity. The dose levels 60 μg/mL in the 4- hour exposure in the absence of metabolic activation and 100 μg/mL in the presence of metabolic activation were plated out for 5-TFT resistance and viability but later discarded from analysis due to excessive toxicity.

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug 2013 to 15 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2010

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED:
Peripheral blood lymphocytes were obtained from a healthy non-smoking 25-year-old adult male on 20 August 2013 for the preliminary toxicity assay and from the same donor on 03 September 2013 for the initial assay and on 30 September 2013 for the repeat assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs, and had abstained from alcohol for 12 or more hours prior to donation. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals.

PREPARATION OF TARGET CELLS:
Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Cytokinesis block (if used):

6 µg/mL Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st EXPERIMENT
- with and without S9 mix, 4 h treatment time: 25, 50, 100, 200, 300, 400, 600, 800, 1000 µg/mL
- without S9 mix, 24 h treatment time: 5, 10, 20, 25, 30, 35, 50 µg/mL

2nd EXPERIMENT
- with and without S9 mix, 4 h treatment time: 25, 50, 75, 100, 125, 150, 175, 200, 250, 300 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine (VB)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and may be stored overnight or longer at 2-8°C. To prepare slides, the cells were collected by centrifugation and if necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. After this the slides were stained.

NUMBER OF CELLS EVALUATED: at least 500 cells in the preliminary toxicity test and at least a 1000 cells were evaluated in the main test to determine the cytokinesis-blocked proliferation index (CBPI).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 2000 binucleated cells

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Micronuclei in a binucleated cell (MN-BN) were recorded if they meet the following criteria:
• the micronuclei should have the same staining characteristics as the main nucleus;
• the diameter of the micronuclei must be approximately 1/3 or less the diameter of a main nucleus;
• the micronuclei must be non-refractile, located in the cytoplasm, no overlapping with nucleus and not connected by cytoplasmic bridges;
• the micronuclei are not linked or connected to the main nuclei;
• the micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary.

DETERMINATION OF CYTOTOXICITY
- Method: The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

- VALIDITY:
The frequency of cells with micronucleus induction in the vehicle control must be within the historical control range. The percentage of cells with micronucleus induction must be statistically increased (p ≤ 0.05, Fisher's exact test) in the positive control condition relative to the vehicle control.

- EVALUATION OF RESULTS:
The test substance was considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.

Statistics:

Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest concentration of test substance in treatment medium was 7.0 in both the 1st and 2nd experiment.
- Precipitation: Visible precipitate was observed in the 1st experiment in treatment medium at dose levels ≥ 200 μg/mL, while dose levels ≤ 100 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 800 μg/mL, while dose levels ≤ 600 μg/mL were soluble in treatment medium. Visible precipitate in the 2nd experiment was observed in treatment medium at dose levels ≥ 175 μg/mL, while dose levels ≤ 150 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period all dose levels were soluble in the treatment medium in both treatment groups.
- Other confounding effects: hemolysis: In the 1st experiment hemolysis was observed at dose levels ≥ 300 μg/mL in the non-activated and S9-activated 4-hour exposure groups. In the 2nd experiment hemolysis was observed at dose levels ≥ 250 μg/mL in the non-activated 4-hour exposure group and at 300 μg/mL in the S9-activated 4-hour exposure group.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive micronucleus assay. HPBL cells were first exposed to nine concentrations of the test substance ranging from 0.19 to 1900 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 24 hours in the absence of S9 activation. The test substance was soluble in DMSO at all concentrations tested. Visible precipitate was observed in treatment medium at dose levels ≥ 190 μg/mL, while dose levels ≤ 57 μg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at 1900 μg/mL, while dose levels ≤ 570 μg/mL were soluble in treatment medium. Also at the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 570 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 190 μg/mL in the non-activated 24-hour exposure group. The osmolality in treatment medium of the highest concentration tested, 1900 μg/mL, was 393 mmol/kg. The osmolality in treatment medium of the lowest precipitating concentration, 190 μg/mL, was 415 mmol/kg. The osmolality in treatment medium of the highest soluble concentration, 57 μg/mL, was 431 mmol/kg. The osmolality of the vehicle (DMSO) in the treatment medium was 427 mmol/kg. The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20%. The pH of the highest concentration of test substance in treatment medium was 7.0.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial micronucleus assay (1st experiment), substantial cytotoxicity was observed at dose levels ≥ 35 μg/mL in the non-activated 24-hour exposure group. Due to unusual cytotoxicity in the non-activated and S9-activated 4-hour exposure groups, the micronucleus assay was repeated at doses ranging from 25 to 300 μg/mL in both groups. In the repeat micronucleus assay (2nd experiment), substantial cytotoxicity was observed at dose levels ≥ 100 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 150 μg/mL in the S9-activated 4-hour exposure group.

Conclusions:

Based on the findings of this study the test substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Executive summary:

In a GLP compliant study, in accordance with OECDTG 487, the test substance, was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In both assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Based on the findings in a preliminary toxicity assay, the doses chosen for the micronucleus assay ranged from 25 to 1000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 5 to 50 μg/mL for the non-activated 24-hour exposure group. In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 35 μg/mL in the non-activated 24-hour exposure group. Due to unusual cytotoxicity in the non-activated and S9-activated 4-hour exposure groups, the micronucleus assay was repeated at doses ranging from 25 to 300 μg/mL in both groups. In the repeat micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 100 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 150 μg/mL in the S9-activated 4-hour exposure group. The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei. The percentage of cells with micronucleated binucleated cells in the test-substance treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met. Based on the findings of this study the test substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genemutation in bacterial cells (Ames)

The mutagenic activity of the test substance was evaluated in a study equivalent to OECDTG 471 (1997) and according to GLP principles. A pre-incubation assay was performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxic two dose range finding studies. The test substance was considered to be toxic between 10 – 200 µg/plate depending on the strain and with and without metabolic activation. Adequate solvent and positive controls were included. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

Genemutations in mammalian cells (MLA)

A GLP compliant study was conducted, in accordance with OECDTG 490, according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. In this main test, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (DMSO media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The maximum dose level used was limited by test item induced toxicity. The toxicity levels up to 10 -20% could be scored except for the 4 -h without S9 because of the very steep dose response (40 ug/ml insufficient toxicity and 60 ug/ml too much toxicity). No precipitate was observed during the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10 ^-6, consequently it is considered to be non-mutagenic in this assay.

In vitro micronucleus

In a GLP compliant study, in accordance with OECDTG 487, the test substance, was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In both assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Based on the findings in a preliminary toxicity assay, the doses chosen for the micronucleus assay ranged from 25 to 1000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 5 to 50 μg/mL for the non-activated 24-hour exposure group. In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 35 μg/mL in the non-activated 24-hour exposure group. Due to unusual cytotoxicity in the non-activated and S9-activated 4-hour exposure groups, the micronucleus assay was repeated at doses ranging from 25 to 300 μg/mL in both groups. In the repeat micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 100μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 150 μg/mL in the S9-activated 4-hour exposure group. The highest dose analyzed under each treatment condition produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei. The percentage of cells with micronucleated binucleated cells in the test-substance treated groups was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met. Based on the findings of this study the test substance was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

Justification for classification or non-classification

Based on the negative results of the Ames test, the mouse lymphoma assay, and micronucleus assay, the test substance does not need to be classified for genotoxicity in vitro according to EU CLP (EC No. 1272/2008 and its updates).