2-({2-[4-(dimethylamino)benzoyloxy]ethyl}(methyl)amino)ethyl 4-(dimethylamino)benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

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:

18 April 2006 to 08 May 2006

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:

2lst July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 2000/32, L 136, Annex 4D, 19 May 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: LFC 2098
Product/common name: LFC 2098
Purpose: Industrial chemical
Colour: white
Physical state: solid, powder
Purity: 99.69%
Storage: at room temperature, protected from light
Molecular formula: C23H3104N3
Molecular weight: 413
Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Target gene:

The Salmonella Uphimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and B-Naphtoflavone (100 mg/kg bw) for 3 consecutive days by oral route.

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate
For soluble, non-toxic test compounds the recommended maximum test concentration is 5 mg/plate or 5 µL/plate

Vehicle / solvent:

Test substance dissolved in dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Details on test system and experimental conditions:

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.

For the pre-incubation method 100 µL of the test item preparation was pre—incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Rationale for test conditions:

According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, reveitant colonies are counted.
At least five different amounts of the test item are tested with approximately half log (i.e. √10) intervals between test points for an initial test. More narrow spacing between dose levels may be appropriate when a dose response is investigated. For soluble, non-toxic test compounds the recommended maximum test concentration will be 5 mg/plate or 5 µL/plate.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):

TA 98: 18-63
TA 100: 79-197
TA 1535: 5-30
TA 1537: 4-32
TA 102: 173-396

- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the spontaneous reversion rate.

According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment l and II precipitation of the test item was found at a dose of 316 µg/plate and higher (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with LFC 2098 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, LFC 2098 did not cause gene mutations by base pair changes or frameshifts in the genome of the
tester strains used.
Therefore, LFC 2098 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of LFC 2098 for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella thphimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I and Experiment II:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment l and II precipitation of the test item was found at a dose of 316 µg/plate and higher (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with LFC 2098 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 September to 17 September 2009

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

European Community (EC). Commission regulation (EC) Np. 440/2008. Official Journal of the EUropean Union No. L142, 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Workshop on Genotoxicity Tests WOrkgroup (the IWGT)

Version / remarks:

Published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Identification ESACURE A198
Molecular formula C23H31N304
Molecular weight 413.52
Description White powder
Purity 100%
Test substance storage At room temperature protected from light
Stability under storage conditions Stable

Target gene:

thymidine-kinase locus (TK gene)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

rat liver SQ-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

100 pg/ml in the absence of SQ-mix
Up to concentrations of 66 pg/ml in the presence of SQ-mix

ESACURE A198 was tested up to concentrations of 66 pg/ml in the absence and presence of SQ-mix. The incubation time was 3 hours. ESACURE A198 precipitated in the exposure medium at this dose level.
In the second experiment, ESACURE A198 was tested up to concentrations of 100 pg/ml in the absence of SQ-mix with an incubation time of 24 hours. ESACURE A198 precipitated in the exposure medium and the cytotoxicity was 77%. In the presence of SQ-mix, ESACURE A198 was tested up to concentrations of 66 pg/ml with an incubation time of 3 hours. ESACURE A198 precipitated in the exposure medium at the dose level of 66 pg/ml.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

with/without s9 mix

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

L5178Y/TK (+/-) 3.7.2C mouse lymphoma cells.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC) and literature (see chapter 9 for references).

Evaluation criteria:

A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controis) + 126 in a dose—dependent manner; or
b) in case a repeat experiment is performed when a positive response is observed in one of the
tester strains, the positive response should be reproducible in at least one independently repeated experiment.

An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(contmls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the lWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

77% in absence of S9

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
ESACURE A198 precipitated in the exposure medium at concentrations of 33 µg/ml and above.

Dose range finding test
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 1 to 66 µg/ml in the absence of SQ-mix with a 3 and 24-hour treatment period and in the presence of SQ-mix with a 3 hour treatment period. ESACURE A198 precipitated at the highest dose level tested.
Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of ESACURE A198 and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.
Both in the absence and presence of SQ-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 66 µg/ml compared to the suspension growth of the solvent control.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of ESACURE A198 and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
In the absence of SQ-mix, the relative suspension growth was 73% at the test substance concentration of 66 µg/ml compared to the relative suspension growth of the solvent control.

First mutagenicity test
Table 3 shows the percentages of cell survival and the mutation frequencies for various concentrations of ESACURE A198. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 5-7 of Appendix I.
The following dose range was selected for the first mutagenicity test in the absence and presence of SQ-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 66 µg/ml exposure medium.

Evaluation of toxicity
No toxicity was observed and all dose levels were evaluated in the absence and presence of SQ-mix.

Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with ESACURE A198 either in the absence or in the presence of SQ-mix. The numbers of small and large colonies in the ESACURE A198 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test
Table 4 shows the percentages of cell survival and the mutation frequencies for various concentrations of ESACURE A198. Individual colony counts of cloning and selective plates, and cell counts during subculturing are listed in Tables 8-10 of Appendix I.
To obtain more information about the possible mutagenicity of ESACURE A198, a second mutation experiment was performed in the absence of 89-mix with a 24 hour treatment period and in the presence of SQ-mix with a 3 hour treatment period.

Based on the results of the dose range finding test and experiment 1, the following dose levels were selected for mutagenicity testing.
Without 89-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33, 66 and 100 µg/ml exposure medium.
With S9-mix: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 66 µg/ml exposure medium.

Evaluation of toxicity
In the absence of SQ-mix, the dose levels of 0.03 to 33 µg/ml showed no cytotoxicity. Therefore, the dose level of 0.03 µg/ml was not regarded relevant for mutation frequency measurement. In the presence of SQ-mix, all dose levels were evaluated. The dose levels selected to measure mutation frequencies at the TK-locus were:
Without SQ—mix: 0.1, 0.3, 1, 3, 10, 33, 66 and 100 µg/ml exposure medium.
With SQ—mix: 0.03, 0.1, 0.3, 1, 3, 10. 33 and 66 µg/ml exposure medium.
ln the absence of SQ-mix (Table 4), the relative total growth of the highest test substance was reduced by 73% compared to the total growth of the solvent controls.
In the presence of SQ-mix, no reduction in the relative total growth was observed up to up to and including the highest test substance concentration of 66 µg/ml.

Evaluation of mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with ESACURE A198 either in the absence or in the presence of SQ-mix. The numbers of small and large colonies in the ESACURE A198 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

The growth rate over the two-day expression period for cultures treated with DMSO was between 21 and 28 (3 hours treatment) and 46 and 55 (24 hours treatment).

Mutation frequencies in cultures treated with positive control v chemicals were increased by 13- and 94-fold for MMS in the absence of SQ-mix, and by 17- and 12-fold for CP in the presence of SQ-mix, in the first and second experiment respectively (See table 3 and 4). It was therefore concluded that the test conditions, both in the absence and presence of SQ-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (SQ-mix) functioned properly. In addition, the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.
in the absence of SQ-mix, ESACURE A198 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of SQ-mix, ESACURE A198 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment.

In conclusion, ESACURE A198 is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

Evaluation of the mutagenic activity of ESACURE A198 in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

This report describes the effects of ESACURE A198 on the induction of forward mutations at the thymidine-kinase locus (TK-Iocus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of SQ-mix (rat liver SQ-mix induced by a combination of phenobarbital and B-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC, guidelines.

The ESACURE A198 was a white powder with a purity of 100%. The test substance was dissolved in dimethyl sulfoxide.

In the first experiment, ESACURE A198 was tested up to concentrations of 66 µg/ml in the absence and presence of SQ-mix. The incubation time was 3 hours. ESACURE A198 precipitated in the exposure medium at this dose level.

In the second experiment, ESACURE A198 was tested up to concentrations of 100 µg/ml in the absence of SQ-mix with an incubation time of 24 hours. ESACURE A198 precipitated in the exposure medium and the cytotoxicity was 77%. In the presence of SQ-mix, ESACURE A198 was tested up to concentrations of 66 µg/ml with an incubation time of 3 hours. ESACURE A198 precipitated in the exposure medium at the dose level of 66 µg/ml.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 9.4-fold for MMS in the absence of SQ-mix, and by 17- and 12-fold for CP in the presence of SQ-mix. It was therefore concluded that the test conditions, both in the absence and presence of SQ-mix. were appropriate and that the metabolic activation system (SQ-mix) functioned properly.

in the absence of SQ-mix, ESACURE A198 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of SQ-mix, ESACURE A198 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment.

It is concluded that ESACURE A198 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 August 2006 - 23 November 2006

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:

21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 2000/32, L 136, Annex 4A, 19 May 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Testing Methods of New chemical sSubstances

Version / remarks:

21 November 2003 (Yakushokuhatsu No. 1121002, Heisei 14.11.13 Seikyoku No. 2 and Kanpokihatsu No. 031121002).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: LFC 2098
Colour: White
Physical state: solid, powder
Purity: 99.69%
Storage: Room temperature, protected from light

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells in vitro were widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells were chosen because of their relatively small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rate (doubling time of the ESL BIOSERVICE V79 done in stock cultures: 12 — 14 h) and a high plating efficiency of untreated cells (normally more than 50%). These facts were necessary for the appropriate performance of the study.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Duplicate cultures are treated at each concentration. The selection of the concentrations used in experiment I and 11 based on data from the pre—experiment. The following concentrations were used in the main experiments:

Experiment I:
with and without metabolic activation:
125, 250, 500, 1000, 2500 and 5000 ug/mL

Experiment II:
without metabolic activation:
31.25, 62.5, 125, 250, 500, 1000, 2000 and 3000 ug/mL
with metabolic activation:
250, 500, 1000, 2000, 3000, 4000 and 5000 ug/mL

The cells were treated in experiment I (with and without metabolic activation) for 4 h with the test item. The metaphases were prepared 20 h after the treatment. In experiment 11 with metabolic activation the cells
were treated for 4 h and prepared 20 h afier the treatment. In experiment II without metabolic activation the cells were treated for 20 h and prepared at the end of the treatment. The dose group selection for microscopic analyses of chromosomal aberrations based on the mitotic index in accordance with the guidelines.

The following concentrations were selected in the main experiment for the microscopic analyses:

Experiment I:
with and without metabolic activation: 1000, 2500 and 5000 ug/mL

Experiment II:
without metabolic activation: 1000, 2000 and 3000 ug/mL
with metabolic activation: 2000, 4000 and 5000 ug/mL

Untreated negative controls:

yes

Remarks:

Solvent controls, consisting of solvent or vehicle alone and treated in the same way as the treatment groups were included. Concurrent negative and/or solvent controls were performed.

Negative solvent / vehicle controls:

yes

Remarks:

Solvent controls, consisting of solvent or vehicle alone and treated in the same way as the treatment groups were included. Concurrent negative and/or solvent controls were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures:
Three or four days old stock cultures (in exponential growth) with higher than 50% confluency were trypsinised at 37 °C for 5 min. by adding a trypsine solution in Ca-Mg-free PBS. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca—Mg-free PBS prior to the tiypsine treatment.

The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). hito each chamber 1E4 - 5E4 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.

Treatment:
Experiment 1: Short time exposure:
Two days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 uL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without metabolic activation. 4 h after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.

There is no requirement for verification of a clear positive response (OECD guideline 473).

Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation):
The treatment with metabolic activation was perfomied as described above for experiment I. In the experiment without metabolic activation, two days after seeding the cells were incubated with the test item in complete medium (MEM with 10% FCS) for 20 h. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.

All cultures are incubated at 37 °C in a humidified atmosphere with 5.0% CO2 (95.0% air).

Preparation of the Cultures:
17.5 h (4 h and 20 h treatment) after the start of the treatment Colcemid® was added to the cultures (0.2 ug/mL culture medium). 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. All the steps were carried out on precision hot plates. After fixation the cells were stained with Giemsa.

Analysis of Metaplzase Cells:
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the cultures was performed [according to standard protocol of the "Arbeitsgruppe der lndustrie, Cytogenetik" (9)] using microscopes with 100x oil immersion objectives. As structural chromosomal aberrations breaks, fragments, deletions, exchanges and chromosomal disintegration were recorded. Gaps were recorded as well but not included in the calculation of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally. At least 200 well spread metaphases per concentration and negative/positive controls were scored for cytogenetic damage (for exceptions see tables).

The cells scored contained 22 ± 1centromeres. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined by counting the number of mitotic cells in 1000 cells. Additionally the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

Analysis of Relative Cell Density:
As an additional parameter for cytotoxic effects of the test item the relative cell density was calculated as the mean of twenty cell counts per test group (cells within the visual field at a 400-fold magnification, tables 2 and 5).

Rationale for test conditions:

Guideline study

Evaluation criteria:

There are several criteria for determining a positive result:
— a clear and dose—related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary (3)(10). However, for the interpretation of the data, both biological and though evaluated statistical significance should be considered together.

A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation at 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Pre-Experiment for Toxicity

According to the relevant guidelines the highest recommended dose is 5000 ug/mL. The test item was diluted in cell culture medium. No precipitation of the test item was noted. The highest dose group evaluated in the pre-experiment was 5000 ug/mL. The relative mitose index and the relative cell density were used as parameter for toxicity. A decrease below 70% relative cell density indicates a biologically relevant cytotoxicity. A decrease below 70% relative mitose index indicates a

biologically relevant cytostatic effect which can lead to a delayed cytotoxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre—experiment (table 1).

The test item LFC 2098 was investigated for a possible potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation with S9 homogenate.

The selection of the concentrations used in experiment I and II based on data from the solubility test and the pre-experiment according to the guidelines.

In experiment I with and without metabolic activation 5000 ng/mL was selected as highest dose group for the microscopic analysis of chromosomal aberrations. In experiment II with and without metabolic activation 5000 ug/mL and 3000 uglinL were respectively selected as highest dose groups for the microscopic analysis of chromosomal aberrations.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment 11). Two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosomal aberrations (for exceptions, see tables).

The following concentrations were evaluated for microscope analysis:

Experiment I:

with and without metabolic activation: 1000, 2500 and 5000 ng/mL

ExperimentII:

without metabolic activation: 1000, 2000 and 3000 pg/mL

with metabolic activation: 2000, 4000 and 5000 ug/mL

Precipitation:

The test item was suspended in culture medium (MEM medium). Precipitate of the test item was noted in all dose groups evaluated.

Toxicity:

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 2500 ug/mL and higher (54% at 2500 ug/mL and 32% at 5000 ug/mL, table 2). The cell density was also decreased below 70% at 5000 ug/mL (38%). These results stated a cytostatic effect of the test item at 2500 ug/mL and both biologically relevant decreases indicated a cytotoxic effect of the test item at 5000 ng/mL. With metabolic activation no decrease of the relative mitose index below 70% was noted up to the highest dose evaluated. However, the cell density was also slightly decreased below 70% at 1000 ng/mL (68%) and this decrease was biologically relevant at 2500 ug/mL and 5000 pg/mL (54%), which is a hint of cytotoxicity.

In experiment II without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 2000 ug/mL and higher (54% at 2000 ug/mL and 45% at 3000 ug/mL, table 5). The cell density was also slightly decreased below 70% at 2000 ug/mL (68%). These results stated a cytostatic effect of the test item. With metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 4000 ug/mL and higher (54% at 4000 ug/mL and 66% at 5000 pg/mL, table 5). No decrease of the cell density was noted up to the highest dose evaluated.

Clastogenicity:

In experiment I without metabolic activation the aberration rate of the negative control (2.5%) was within the historical control data of the negative control (0.0% — 4.0%, table 10). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 3.5% (1000 ug/mL), 2.5% (2500 ug/mL) and 3.8% (5000 pg/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment I with metabolic activation the number of aberrant cells noted for the negative control (3.0%) was within the historical control data of the negative control (0.0% — 4.5%, table 10). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control, except at 5000 ng/mL (4.8%).However, this value was not statistically significant (p=0.3893). For the other evaluated concentrations, the mean values noted were 2.0%(1000 ug/mL and 2500 ug/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment II without metabolic activation the aberration rate of the negative control (2.5%) was within the historical control data of the negative control (0.0% — 4.0%, table 10). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 2.5% (1000 ug/mL), 2.9% (2000 ug/rnL) and 0.0% (3000 ug/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment II with metabolic activation the aberration rate of the negative control (3.0%) was within the historical control data of the negative control (0.0% — 4.5%, table 10). The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. The mean values noted were 2.5% (2000 ug/mL), 4.0% (4000 ug/mL) and 1.5% (5000 ug/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

Concerning the dose group 6 with metabolic activation in both 1^st and 2^nd experiments, a difference in scored aberrant cells between the 2 parallel cultures was noted. It could be explained by the state of the test item in solution which was distributed to cultures as suspension. This, however, has no influence on the validity of the study.

Polyploid cells

Table 2 and 5 show the occurrence of polyploid metaphases. No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

EMS (400 and 900 ug/mL) and CPA (0.83 ug/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item LFC 2098 did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line,.

Therefore, the test item LFC 2098 is considered to be non-clastogenic.

Executive summary:

In order to investigate a possible potential of LFC 2098 for its ability to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro a chromosome aberration assay was carried out.

The chromosomes were prepared 20h after start of treatment with the test item. The treatment interval was 4h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4h with and 20h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations (for exceptions, see tables).

Experiment I:

with and without metabolic activation: 1000, 2500 and 5000 μg/mL

Experiment II:

without metabolic activation: 1000, 2000 and 3000 μg/mL

with metabolic activation: 2000, 4000 and 5000 μg/mL

In experiment I and II, precipitate of the test item was noted with and without metabolic activation in all dose groups evaluated.

In experiment I with and without metabolic activation, toxic effects of the test item were noted at 2500 μg/mL and higher.

In experiment II without metabolic activation, toxic effects of the test item were noted at 2000 μg/mL and higher. With metabolic activation, toxic effects of the test item were noted at 4000 μg/mL and higher.

In experiment I no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of aall dose groups treated with the test item were within the historical control data of the negative control, except with metabolic activation at the highest dose evaluated. However, no statistical significance was noted.

In experiment II no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II with and without metabolic activation no increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 900 μg/mL) and CPA (0.83 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

All in vitro genetic toxicity testing showed no adverse effects, thus the substance EC 466-080-8 is not classified under the CLP regulation.