3-(p-methoxyphenyl)-2-methylpropionaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in bacterial cells (Ames) according to OECD TG 471: Negative

Genetic toxicity in mammalian cells (MLA) according to OECD TG 490: Negative

Micronucleus assay in mammalian cells according to 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:

06 Jul 2004 to 15 Jul 2004

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:

July 21 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

Cytotest Cell Research GmbH (RCC-CCR), In den Leppsteinswiesen 19, D-64380, Rossdorf

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

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:

rat liver S9 induced with phenobarbital and ß-Napthoflavone

Test concentrations with justification for top dose:

Pre-experiment
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
The pre-experiment is reported as main experiment l, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Main experiment I (plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Main experiment II (pre-incubation assay) without S9 mix: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
Main experiment II (pre-incubation assay) with S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

ethanol (>99%)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

100 µL ethanol

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:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION:
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

OTHER EXAMINATIONS:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to reduced background growth, the colonies were partly counted manually.

Evaluation criteria:

Acceptability of the Assay:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not required

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment is reported as main experiment l, since the following criteria are met: Evaluable plates >0 colonies) at five concentrations or more in all strains used.

HISTORICAL CONTROL DATA
- Positive historical control data: The historical range of positive controls was exceeded in strain TA 1537 (experiment I) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.
- Negative (solvent/vehicle) historical control data: In experiment I, with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in the solvent control of strain TA 102. Since this deviation is rather small, this effect is judged to be based upon biological fluctuations and has no detrimental impact on the outcome of the study.

Distinct toxic effects, evident as a reduction in the number of revertants (below the factor of 0.5) were observed at the following concentrations (µg/plate):

 

Strain	Experiment I		Experiment Il
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000 - 5000	1000 - 5000	1000, 2500	2500, 5000
TA 1537	1000 - 5000	5000	1000, 2500	2500, 5000
TA 98	2500, 5000	5000	2500	2500, 5000
TA 100	1000- 5000	5000	1000, 2500	2500, 5000
TA 102	333 - 5000	333 - 5000	1000, 2500	1000 - 5000

Conclusions:

The test substance had, under the present test conditions, no ability to induce mutations in the bacterial reverse mutation assay according to OECD 471.

Executive summary:

The mutagenic activity of the test substance was evaluated in accordance with OECD 471 (1997) and GLP principles. The test was performed according to the plate incorporation (experiment 1) and pre-incubation method (experiment 2), in the absence and presence of S9-mix. In the pre-experiment the concentration range of the test item was 3- 5000 µg/plate. The pre-experiment is reported as experiment I since evaluable plates (> 0 colonies) were obtained at five concentrations or more in all strains used. In both experiment 1 and 2, reduced background growth was observed with and without S9 mix at higher concentrations in all strains (Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102). No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Adequate vehicle and positive controls were included. Based on the results of this study it is concluded that the test substance is not mutagenic in the bacterial 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:

05 Sep 2016 to 04 Oct 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:

adopted 29 July 2016

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Envigo Research Limited., Shardlow 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):

CELLS USED
Source of cells: 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. The cells have a generation time of approximately 12 hours and were subcultured accordingly.

MEDIA USED
Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at approximately 37°C with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study.
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

PB/ßNF induced rat liver S9

Test concentrations with justification for top dose:

A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 6.95 to 1780 µg/mL for all three of the exposure groups.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
- The upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
- Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
- In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002)

In the subsequent mutagenicity test the maximum dose was limited by test item induced toxicity. The following doses were selected for the main test:
4h -S9: 27.75, 55.5, 111, 148, 185, 222 µg/mL
4h +S9: 27.75, 55.5, 111, 222, 296, 370 µg/mL
24h -S9: 2.5, 5, 10, 15, 20, 25 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours in absence and presence of metabolic activation and 24 hours in absence of metabolic activation.
- Expression time: At the end of the exposure period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL.The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
- Selection time: 10 to 12 days.

SELECTION AGENT
4 μg/mL 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
Duplicates

DETERMINATION OF CYTOTOXICITY
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium in 96-well microtitre plates. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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: In prescreen test only. No precipitate of the test item was observed at any of the dose levels in the main test

RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups when compared to the concurrent vehicle control groups, with the most marked reductions observed in the 24- hour exposure group in the absence of metabolic activation. The toxicity curve was very steep in all three exposure groups. Precipitate of the test item was observed at and above 890 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at 1780 μg/mL in the 24-hour exposure group in the absence of metabolic activation. In the subsequent mutagenicity test the maximum dose was limited by test item-induced toxicity.

Conclusions:

A mouse lymphoma assay (MLA) with the substance was conducted according to OECD TG 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD guideline 490 and GLP, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance. Based on a preliminary cytotoxicity study, concentrations of 27.75, 55.5, 111, 148, 185, 222 µg/mL were used for the 4 hour exposure experiment in absence of metabolic activation, 27.75, 55.5, 111, 222, 296, 370 µg/mL for the 4 hour experiment in presence of metabolic activation and 2.5, 5, 10, 15, 20, 25 µg/mL for the 24 hour experiment in absence of metabolic activation. A vehicle (DMSO) control and concurrent positive controls were included. Incubations were performed in duplicate and PB/ ßNF induced rat S9 was used as metabolic system. After a two-day expression period cells were exposed to 4 µg/mL trifluorothymidine (TFT) for 10 to 12 days for mutant selection.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 satisfactory, and that the metabolic activation system was functional. The toxicity was around 10 -20%: 4h-S9 reduced RTG to 23%; 4h+S9 to 31% and 24h-S9 to 7% at the highest concentrations tested. The test item did not induce any toxicologically significant increases in the mutant frequency in any of the three exposure groups.Therefore, under the test conditions, the test substance is negative in the L5178Y TK+/- mouse lymphoma mutagenesis assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Nov 2014 to 15 Mar 2015

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:

adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B, Guideline B.49 “InVitro Mammalian Cell Micronucleus Test". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193,

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking male volunteers (age 18 to 35 years). Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.
- Cell cycle length, doubling time or proliferation index: The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2014): Dose range finding study: AGT = 12.8 h, First cytogenetic assay: AGT = 12.8 h, Second cytogenetic assay: AGT = 12.6 h, Cytogenetic assay 2A: AGT = 12.8 h, Cytogenetic assay 2B: AGT = 13.0 h
- Sex and age of blood donors: male, Dose range finding study: age 32, First cytogenetic assay: age 23, Second cytogenetic assay: age 27, Cytogenetic assay 2A: age 23 and Cytogenetic assay 2B: age 26
- The lymphocytes were cultered for 46 ± 2 hours

MEDIA USED
- Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

ENVIRONMENTAL CONDITIONS
- All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 49 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 37.2°C).

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

RANGE FINDER
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed to selected doses for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. A vehicle control was included at each exposure time. The highest tested concentration was determined by the solubility of the substance in the culture medium. Cytotoxicity of the substance in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).

DOSE CONSIDERATIONS FOR RANGE FINDER
- At a concentration of 512 μg/mL the substance precipitated in the culture medium.
- 17, 52, 164, 512 and 1600 μg/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

DOSE LEVELS IN CYTOGENIC ASSAYS
1st cytogenic assay: Without and with S9: 10, 150, 200, 250, 300, 350, 400 and 450 μg/mL
2nd cytogenic assay: Without S9 10, 50, 70, 90, 110, 130 and 150 μg/mL
Since the 2nd experiment did not result in analysable results assay 2A was performed at 10, 75, 90, 95, 100, 105, 110, 115 and 120 μg/mL without S9
Since experiment 2A did not reveal analysable concentrations assay 2B was performed at 10, 75, 80, 85, 90, 95, 100, 110 and 120 μg/mL without S9

Vehicle / solvent:

- dimethyl sulfoxide (DMSO)
- Hanks’ Balanced Salt Solution (HBSS), without calcium and magnesium for positive controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the absence and presence of S9-mix (first cytogenic assay) or 24 hours in the absence of S9-mix (second cytogenic assay)

SPINDLE INHIBITOR:
cytochalasin B (5 μg/mL)

STAIN
5% (v/v) Giemsa solution in Sörensenbuffer pH 6.8

NUMBER OF REPLICATIONS:
Duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated coverslipper

NUMBER OF CELLS EVALUATED:
Three to four analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
two slides

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- %Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)} where t=test substance or control treatment culture and c= vehicle control culture
- CBPI= (No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/ Total number of cells

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
- It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
- A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
- None of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
- The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics with a confidence interval of 95%.

Key result

Species / strain:

lymphocytes: human peripheral

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 512 μg/mL test substance precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test blood cultures were treated with 17, 52, 164, 512 and 1600 μg test substance/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

NUMBER OF CELLS WITH MICRONUCLEI
- First cytogenetic assay:
The following dose levels were selected for scoring of micronuclei, without S9-mix : 10, 150 and 250 μg/mL culture medium (3 hours exposure time, 27 hours harvest time) and with S9-mix : 10, 200 and 300 μg/mL culture medium (3 hours exposure time, 27 hours harvest time). Both in the absence and presence of S9-mix, the substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei
- Second cytogenetic assay:
Without S9-mix : 10, 50, 70, 90, 110, 130 and 150 μg/mL culture medium 24 hours exposure time, 24 hours harvest time). No appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 90 μg/ml not enough cytotoxicity was observed (47%), whereas the next higher concentration of 110 μg/mL was too toxic for scoring (65%). The experiment was repeated in cytogenetic assay 2A.
- Cytogenetic assay 2A:
Without S9-mix : 10, 75, 90, 95, 100, 105, 110, 115 and 120 μg/mL culture medium (24 hours exposure time, 24 hours harvest time). No appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 75 μg/ml not enough cytotoxicity was observed (43%), whereas the next higher concentration of 90 μg/ml was too toxic for scoring (64%). The experiment was repeated in cytogenetic assay 2B.
-Cytogenetic assay 2B:
The following dose levels were selected for the scoring of micronuclei: Without S9-mix : 10, 75, 80 and 85 μg/mL culture medium (24 hours exposure time, 24 hours harvest time). The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei

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 in both the absence and presence ofphenobarbital and ß-naphthoflavone induced rat liver S9-mix.The possible clastogenicity and aneugenicity of the test substance was tested in two independent experiments. In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. At a concentration of 512 μg/ml the substance precipitated in the culture medium. In the dose range finding test blood cultures were treated with 17, 52, 164, 512 and 1600 μg/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control. In the first cytogenetic assay, the substance was tested up to 250 μg/mL and 300 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, which was repeated twice at slightly different dose levels (cytogenic assays 2A and 2B) due to the absence of appropriate cytotoxicity, the substance was tested up to 85 μg/mL (assay 2B) for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore it is concluded that the substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

OECD TG 471 (Ames test)

The mutagenic activity of the test substance was evaluated in accordance with OECD 471 (1997) and GLP principles. The test was performed according to the plate incorporation (experiment 1) and pre-incubation method (experiment 2), in the absence and presence of S9-mix. In the pre-experiment the concentration range of the test item was 3- 5000 µg/plate. The pre-experiment is reported as experiment I since evaluable plates (> 0 colonies) were obtained at five concentrations or more in all strains used. In both experiment 1 and 2, reduced background growth was observed with and without S9 mix at higher concentrations in all strains (Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102). No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Adequate vehicle and positive controls were included. Based on the results of this study it is concluded that the test substance is not mutagenic in the bacterial reverse mutation assay.

OECD TG 490 (mouse lymphoma assay)

In a mammalian cell gene mutation assay conducted according to OECD guideline 490 and GLP, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance. Based on a preliminary cytotoxicity study, concentrations of 27.75, 55.5, 111, 148, 185, 222 µg/mL were used for the 4 hour exposure experiment in absence of metabolic activation, 27.75, 55.5, 111, 222, 296, 370 µg/mL for the 4 hour experiment in presence of metabolic activation and 2.5, 5, 10, 15, 20, 25 µg/mL for the 24 hour experiment in absence of metabolic activation. A vehicle (DMSO) control and concurrent positive controls were included. Incubations were performed in duplicate and PB/ ßNF induced rat S9 was used as metabolic system. After a two-day expression period cells were exposed to 4 µg/mL trifluorothymidine (TFT) for 10 to 12 days for mutant selection. 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 satisfactory, and that the metabolic activation system was functional. The toxicity was around 10 -20%: 4h-S9 reduced RTG to 23%; 4h+S9 to 31% and 24h-S9 to 7% at the highest concentrations tested. The test item did not induce any toxicologically significant increases in the mutant frequency in any of the three exposure groups. Therefore, under the test conditions, the test substance is negative in the L5178Y TK+/- mouse lymphoma mutagenesis assay.

OECD TG 487 ( In vitro mammalian cell micronucleus test)

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 in both the absence and presence of phenobarbital and ß-naphthoflavone induced rat liver S9-mix.The possible clastogenicity and aneugenicity of the test substance was tested in two independent experiments. In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. At a concentration of 512 μg/ml the substance precipitated in the culture medium. In the dose range finding test blood cultures were treated with 17, 52, 164, 512 and 1600 μg/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control. In the first cytogenetic assay, the substance was tested up to 250 μg/mL and 300 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, which was repeated twice at slightly different dose levels (cytogenic assays 2A and 2B) due to the absence of appropriate cytotoxicity, the substance was tested up to 85 μg/mL (assay 2B) for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments. Therefore it is concluded that the substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions used.

Justification for classification or non-classification

Based on the negative results of the Ames test, the mouse lymphoma assay, and the in vitro micronucleus assay, classification of the test substance for genetic toxicity is not warranted according to EU CLP (EC No. 1272/2008 and its updates).