Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item has been shown to be not mutagenic/not genotoxic in a battery of different in vitro genotoxicity assays both with and without metabolic activation including the Ames test, the mouse lymphoma assay (MLA) and the in vitro chromosomal aberration test (in vitro CA). All in vitro tests were performed according to international accepted guidelines and under GLP regulation, i.e. these studies are of very high quality.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-09-10 to 2005-03-17

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:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

08 Jume 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium), TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor (pKM101)

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor (pKM101)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa +R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

trp-, uvrB

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 15.8, 50.0, 158, 500, 1580 µg per plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

cumene hydroperoxide

other: Daunomycin

Remarks:

without S9.mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-naphthoflavone/phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 30% S9 in the 2nd series.

Rationale for test conditions:

according to Guidelines.

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 in-crease in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control (Test Material)
<=10 <=9 >=30
<=30 <=19 >= 40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: "No Increase" "Clear Increase"

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".
Interpretations:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main ex¬periment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and 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:

S. typhimurium TA 100

Metabolic activation:

with and 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:

S. typhimurium TA 102

Metabolic activation:

with and 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:

S. typhimurium TA 1535

Metabolic activation:

with and 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:

S. typhimurium TA 1537

Metabolic activation:

with and 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:

E. coli WP2 uvr A

Metabolic activation:

with and 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

Conclusions:

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

Executive summary:

Purpose

The objective of this study was to evaluate the mutagenic activity of the test item by examining its ability to revert five histidine-requiring strains of Salmonella typhimurium and in a tryptophane-requiring E.coli strain in the absence and in the presence of a rat liver metabolising system (S-9).

Study Design

The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997).

Results

The test material 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 > 500 µg/plate. Toxicity to the bacteria was not observed.
Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene 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.

Conclusion

The test item is not mutagenic under the test conditons employed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-26 to 2014-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

08 June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human lymphocytes from peripheral blood of healthy volunteers

Metabolic activation:

with and without

Metabolic activation system:

- Type and composition of metabolic activation system: S9 mix
- source of S9 : Aroclor 1254-induced male Wistar rats
- method of preparation of S9 mix : according to internal SOP.
- concentration or volume of S9 mix and S9 in the final culture medium : 900µL Cofactor solution and 100µL S9 in 10ML test culture
- quality controls of S9: yes (metabolic activity)

Test concentrations with justification for top dose:

First series: (+/- S9 mix): 281, 889 and 2810 µg/mL
Second series: (-S9): 28.1, 88.9, 281, 889 and 2810 µg/mL
Due to the limited solubility of the test item in solvents used for such in vitro tests, concentrations ranging from 8.89 to 2810 µg/mL were applied. The test item precipitated in the culture medium at concentrations >/= 889 µg/mL in the absence and >/= 1580 µg/mL in the presence of S9 mix. As only slight concentration-independent toxicity was seen in the absence of metabolic activation, 2810 µg/mL was chosen as the highest concentration to be evaluated.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solubility of test item
- Justification for percentage of solvent in the final culture medium: 0.1%, as recommended by test guideline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 1
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 and 29 hours (-S9 mix)
5 hours (+S9 mix)
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): colchicine, 0.1 µg/mL, 3 hours
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 x 150 metaphases
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): well spread metaphases
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: no

Evaluation criteria:

A total of 100 well spread metaphases were examined per culture (slide) at a magnification of 1250 x, using an oil immersion phase contrast objective.
A test material is positive or clastogenic in this test system if
• a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
• a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.

Statistics:

Exact Fisher Test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and 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

Conclusions:

In conclusion, treatment of human lymphocyte cultures with the test item did not increase the proportion of cells with aberrant chromosomes under the experimental conditions. The test substance was thus not clastogenic in this in vitro test system up to the limit of solubility.

Executive summary:

The test material was investigated in three independent experiments for induction of chromosomal aberrations in human peripheral blood lymphocytes in vitro. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in endoreduplications or polyploidy.

The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254):

 

No. of slides per concentration: Solvent control: Others:	4 2
No. of metaphases evaluated per slide:	100 (for structural aberrations) 1000 (for polyploidy)
Preparation times                - S9 mix:                +S9 mix:	77 hours 77 hours
Exposure times                - S9 mix:                +S9 mix:	5 and 29 hours 5 hours
Solvent for the test item:	Dimethylsulfoxide (DMSO), 0.1% final concentration
Concentrations evaluated: test material,         first series (-/+ S9 mix):                             second series (-S9 mix): Positive control,  - S9 mix:                           +S9 mix:	281, 889 and 2810 µg/mL 28.1, 88.9, 281, 889 and 2810 µg/mL 0.150 and 0.300 µg Mitomycin C (MMC)/mL 5.00 µg Cyclophosphamide (CPA) /mL

The positive control test materials, MMC and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations.

The test item precipitated in the culture medium at concentrations >/= 1580 µg/mL in the absence and the presence of S9 mix until end of exposure. No clear cytotoxic effects, i.e. reduction in mitotic index, were induced by the test material in the absence or presence of metabolic activation (S9 mix). Hence, solubility of the test item was the limiting factor for top concentration selection.

The mean values of aberrant metaphases (gaps excluded) in the negative controls of all experiments (– and + S9 mix) ranged from 1.00 % to 1.75 %. The test item did not show any relevant increase in the number of aberrant metaphases. Furthermore, no relevant increase in endoreduplications or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected. In conclusion, treatment of human lymphocyte cultures with the test material did not increase the proportion of cells with aberrant chromosomes under the experimental conditions. The test item was thus not clastogenic in this in vitro test system up to the limit of solubility.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-09-29 to 2005-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

08 June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK gene

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; original cultures were from Hoffmann-La Roche, Basel, Switzerland.
- Suitability of cells: yes

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: max. 20
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
Growth media
Three media, supplementing RPMI 1640-medium with Glutamax 1 with different serum concentrations were used:

Exposure medium:
RPMI- 5 (RPM 1640 with 5% heat inactivated horse serum) 470 mL RPMI 1640
25 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Culture medium:
RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 1640
50 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Survivor- and selection medium:
RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640
100 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Liver from Aroclor 1254-pretreated Wistar rats.
- method of preparation of S9 mix : Male rats, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw) dissolved in Miglyol 812 oil. The animals received drinking water and a standard diet ad libitum. About 16 hours before sactifice, the rats remained without food. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing PBS-HEPES. The livers were homogenized in a sterila glass potter homogenizer with a Teflon pestle containing 3 mL PBS-HEPES per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000xg for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was frozen and stored in liquid nitrogen at -196°C
- concentration or volume of S9 mix and S9 in the final culture medium : 3% (1st experiment) 1% (2nd experiment)
- quality controls of S9: metabolic activity

Test concentrations with justification for top dose:

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 µg/mL, 5 µL/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.
In addition to the biologic effects mentioned, the effects of the test material on the pH and the osmolarity of the cell culture medium were assessed as fluctuations in the pH of more than one unit and in osmolarity of more than 50 mOsm/kg can be responsible for genotoxic effects (Brusick 1986).

According to these criteria and considering the results of the dose range finder, the following concentrations were selected for the 1st series of the main study in the presence and absence of S9 mix:
-S9 mix: 5.00, 15.8, 50.0, 88.9 and 158 µg / mL medium
+S9 mix: 15.8, 50.0, 158, 500, 1580 and 5000 µg / mL medium

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix, conc.:0.1 and 0.2 µg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix; conc.: 1.0, 2.0 and 3.0 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: 500.000/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: +S9-mix: 3 hours; -S9-mix: 3 and 24 hours


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): at least 7 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure: Trifluorothymidine, 3 µg/mL (final concentration), at least 7 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies: visible inspection

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth (RTG)

further details see below.

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.

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
The test material did not lead to a relevant fluctuation in both, the pH and the osmolarity of the cell culture medium. Precipitation of the test material in the incubation medium was observed at concentrations 500 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 >=50.0, >=500 or >=1580 µg/mL, depending upon the experimental condition used.


Mutation
The analysis of the individual plate counts observed in each experiment and the historical data of the laboratory show that:
- the acceptance criteria of the assay were met
- the relation of small to large colony mutant frequencies for the negative and positive controls were in the range acceptable for this cell line and assay (Applegate et al. 1990, Moore et at. 1985).

The study was therefore accepted as valid.

In the absence of S9 mix, the individual mutation frequency of the solvent controls ranged from 183 x 10^-6 to 220 x 10^-6 in both experimental series performed. The mutation frequencies of cells treated with NQO were clearly elevated to values ranging from 581 x 10^-6 to 1200 x 10^-6. The cultures treated with the different test material concentrations showed mutation frequencies between 144 x 10^-6 and 224 x 10^-6. The mean mutation frequencies for the treatment groups and the solvent controls were not relevantly different.

In the presence of S9 mix, the individual mutation frequency of the solvent controls ranged from 84.1 x 10^-6 to 251 x 10^-6 in both experimental series performed. The mutation frequencies of cells treated with DMBA were clearly elevated to values ranging from 448 x 10^-6 to 2950 x 10^6. The cultures treated with the different test material concentrations showed mutation frequencies between 98.8 x 10^-6 and 339 x 10^-6. Once more, the mean mutation frequencies for the treatment groups and the solvent controls were not relevantly different.

In conclusion, no relevant increase in the mutation frequency induced by the test item could be detected.

Conclusions:

It is concluded that the test substance is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Executive summary:

Purpose

The objective of this study was to evaluate the mutagenic activity of the test material by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix).

Study design
The principle of the mouse lymphoma L5I78Y TK assay is the following: L5178Y cells are heterozygous for the enzyme thymidine kinase (TK+/-) which metabolizes the thymidine analogue 5-trifluorothymidine (TFT) to toxic intermediates. In consequence, non-mutant cells do not survive in TFT-containing media. Test materials inducing mutations in the TK gene may inactivate the TK enzyme activity. Hence, the mutants (TK -/-) are unable to use the toxic analogue TFT and survive in its presence. Quantification of the number of L5178Y cells growing in TFT containing media after test material exposure thus gives a measure of the mutagenic potential of the test material.

The TK locus is autosomal. Two types of TFT-resistant colonies are selected: large colonies and small slow-growing colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations (Applegate et al., 1990; Moore et al., 1985; Thacker, 1985). Thus, in this system, gene mutations within the TK gene (11-13 kilobases) and chromosomal events involving the gene may be detected. The TK system has a high spontaneous mutant frequency and high numbers of cells can be treated and sampled. The application of a fluctuation protocol which has been developed for use with the assay (Cole et al., 1983) is, therefore, reasonable.

The objective of this study was therefore to evaluate the mutagenic activity of the test item by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix). The methodology in this study complies with the ICH guidelines, the OECD Principles of Good Laboratory Practice , the OECD Test Guideline 476 and the EEC Directive.

Results
Various test item concentrations ranging from 5.0 to 5000 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 larger and equals to 500 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 of >=50, >=500, or >=1580 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). In addition, the relation of small to large colony mutant frequencies for the negative and positive controls were in the range acceptable for this cell line and assay. Therefore, the study was accepted as valid.

Neither relevant increases in mutant frequency nor alterations of the relation of small to large colony mutant frequencies were observed following treatment with the test item in the two experimental series in the absence and presence of S9 mix.

Conclusions
Based on the results it is 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

Mode of Action Analysis / Human Relevance Framework

The substance has no structural alerts for mutagenicity. Furthermore, all in vitro studies were GLP compliant and of high quality (Klimisch score = 1). Therefore, there is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

Based on the results on three in vitro genotoxicity assays, the test item is considered to be not genotoxic and must not be classified for mutagenicity according to EU Directive 67/548/EC amended and repealed by EU Regulation No. 1272/2008 and CLP.