Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in-vitro studies were conducted on the substance:

- Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli

- Chromosome aberration test in human lymphocytes

- Mouse lymphoma assay

All 3 studies gave negative results.

Link to relevant study records

Referenceopen allclose all

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:
17 August 2005 to 04 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies , which do not affect the quality of relevant results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Target gene:
Not applicable
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
- Type and identity of media:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated 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.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Dose range-finding test: 0.3, 1.0, 3.0, 10, 33 and 100 µg/ml
First cytogenetic assay: 3, 10 and 33 µg/ml (with and without S9-mix)
Second cytogenetic assay: 10, 33 and 100 µg/ml (without S9-mix)
3, 10 and 33 µg/ml (with S9-mix)
Vehicle:
- Vehicle(s)/solvent(s) used: Ethano.
EA-3098 was suspended in ethanol absolute. The final concentration of the solvent in the culture medium amounted to 1.0% (v/v).
Negative controls:
no
Solvent controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control )
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation (-S9-mix)
Negative controls:
no
Solvent controls:
yes
Remarks:
(solvent treatment groups were used as the vehicle control )
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- First cytogenetic assay: With and without S9-mix: 3 h exposure time, 24 h fixation time.
- Second cytogenetic assay: Without S9 mix: 24 h and 48 h exposure time, 24 and 48 h fixation time).
With S9 mix: 3 h exposure time, 48 h fixation time.


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium).
STAIN (for cytogenetic assays): 5% (v/v/) Giemsa solution in tap water

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells . To analyse slides for chromosome aberrations, 100 metaphase chromosome spreads per culture were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy



Evaluation criteria:
The test substance was considered positive (clastogenic) if:
a) It induced a dose-related statistically significant (Chi-square test), P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absnece of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromsome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using chi-square statistics.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
(> 33 µg/ml)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
DOSE RANGE FINDING TEST:
At a concentration of 33 µg/ml EA-3098 precipitated in the culture medium. In the dose range finding study, at the 3 h exposure time, blood cultures were treated with 0.3, 1, 3, 10 and 33 µg EA-3098/ml culture medium with and without S9-mix. At the 24 hand 48 h continuous exposure time blood cultures were treated with 0.3,1,3,10,33 and 100 µg EA-3098/ml culture medium without S9-mix. EA-3098 was tested beyond the limit of solubility to obtain adequate toxicity data.

Table 1 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the negative control substance.

FIRST CYTOGENETIC ASSAY:
Based on the results of the dose range finding test, the following dose levels were selected for the cytogenetic assay:
WIth and without S9-mix: 3, 10 and 33 µg/ml

Table 2 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 3, 4 - attached backgroud material).

Both in the absence and presence of S9-mix EA-3098 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY:
To obtain more information about the possible clastogenicity of EA-3098, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to EA-3098 in the absence of S9 mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to EA-3098. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix: 10, 33 and 100 µg/ml
With S9-mix: 3, 10 and 33 µg/ml

Table 5 (attached background material) shows the mitotic index of cultures treated with various EA-3098 concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Tables 6-8- attached backgroud material).

Both in the absence and presence of S9-mix EA-3098 did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.















Evaluation of the results

The mitotic indices of cultures treated with various EA-3098 concentrations or with the negative control substances are presented in Tables 1, 2 and 5 (see attached background material). The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of EA-3098 are presented in Tables 3, 4 and 6-8 (see attached background material). Duplicate cultures are indicated by A and B.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploidy cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9 -mix EA-3098 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of EA-3098 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that EA-3098 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that EA-3098 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

The test material is not clastogenic in human lymphocytes under the experimental conditions in the study.
Executive summary:

The ability of EA-3098 to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted to the following guidelines: OECD Guideline 473 and EU Method B.10. The possible clastogenicity was tested in two independent experiments.

In the first cytogenetic assay, EA-3098 was tested up to 33 µg/ml for a 3 h exposure time with a 24 hour fixation time in the absence and presence of 1.8 % (v/v) S-9 fraction. EA-3098 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, EA-3098 was tested up to 100 µg/ml for a 24 -h and 48 -h continuous exposure time with a 24 -h and 48 -h fixation time in the absence of S9 -mix. In the presence of S9 -mix EA-3098 was tested up to 33 µg/ml for a 3 -h exposure time with a 48 -h fixation time. EA-3098 precipitated in the culture medium at these dose levels.

Positive control chemicals, mytomycin-C and cyclophosphamide, both produced statistically significant increases in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

EA-3098 did not induce a statistically significant or biologically relvenat increase in the number of cells with chromosomeome aberrations in the absence or presence of S9 -mix, in two independently repeated experiments.

No effects of EA-3098 on the number of polyploid cells and cells with andoreplicated chromosomes were observed both in the absence and presence of S9 -mix. Therefore it can be concluded that EA-3098 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditins described in this report.

It is concluded that this test is valid and that EA-3098 is not clastogenic to human lymphocytes under the experimental conditions in the study.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 19 August 2011 and 21 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. 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 37 °C with 5% CO 2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

- Properly maintained: yes

- Periodically "cleansed" against high spontaneous background: yes
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/ml), Hypoxanthine (15 μg/ml), Methotrexate (0.3 μg/ml) and Glycine (22.5 μg/ml). For the following 24 hours the cells were cultured in THG medium
(i.e. THMG without Methotrexate) before being returned to R10 medium.





Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthoflavone induced rat liver (S9-mix)
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/ml.

Mutagenicity Tests:
Experiment 1:
39.06 to 1250 μg/ml in the absence of metabolic activation: (39.06, 78.13, 156.25, 312.5, 468.75, 625, 937.5, 1250 μg/ml)
78.13 to 2500 μg/ml in the presence of metabolic activation: (78.13, 156.25, 312.5, 625, 937.5, 1250, 1975, 2500 μg/ml)

Experiment 2:
25 to 600 μg/ml in the absence of metabolic activation: (25, 50, 100, 200, 300, 400, 500, 600 μg/ml)
19.53 to 1875 μg/ml in the presence of metabolic activation: (19.53, 39.06, 78.13, 156.25, 312.5, 468.75, 625, 937.5, 1250, 1975 μg/ml)
Vehicle:
- Vehicle(s)/solvent(s) used: The test item was accurately weighed, suspended in dimethyl sulfoxide (DMSO) and serial dilutions prepared.

- Justification for choice of solvent/vehicle:
With a molecular weight greater than 500 a maximum dose level of 5000 μg/ml, the maximum recommended dose level, was investigated initially in
a solubility test. However, due to formulation difficulties, the maximum dose was reduced to 2500 μg/ml, the maximum achievable dose level, and this was used in the preliminary toxicity test. The purity of the test item was 100 % and, therefore, not accounted for when formulating the dosing solutions. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.
Negative controls:
no
Solvent controls:
yes
Remarks:
Solvent (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate (EMS), at 400 μg/ml and 150 μg/ml for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation.
Negative controls:
no
Solvent controls:
yes
Remarks:
Solvent (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide (CP) at 2 μg/ml was used as the positive control in the presence of metabolic activation.
Details on test system and conditions:
Preliminary Toxicity Test:
A preliminary toxicity test was performed on cell cultures at 5 x 10+5 cells/ml, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10+5 cells/ml using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10+5 cells/ml.

The cultures were incubated at 37 °C with 5% CO 2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10+5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.

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:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).


Mutagenicity Test:
Experiment 1:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10+6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (39.06 to 1250 μg/ml in the absence of metabolic activation, and 78.13 to 2500 μg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix (if required), 0.2 ml of the treatment dilutions, 0.2 ml for the positive control, and sufficient R0 medium to bring the total volume to 20 ml.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2:
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10+6 cells/ml in 10 ml duplicate cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours; therefore 0.3 x 10+6 cells/ml in 10 ml duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 ml of S9-mix was added (if required), 0.2 ml of the treatment dilutions, 0.2 ml for the positive control, and sufficient R0 medium to give a final volume of 20 ml (R10 is used for the 24-hour exposure group). The dose range of the test item was 25 to 600 μg/ml in the absence of metabolic activation, and 19.53 to 1875 μg/ml in the presence of metabolic activation.

The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Measurement of Survival, Viability and Mutant Frequency:
At the end of the treatment 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% CO 2 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.

On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

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

Plate Scoring:
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.
















Evaluation criteria:
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely,
when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media (pH 7.29 - 7.32)
- Effects of osmolality: The osmolality did not increase bu more than 50 mOsm when the test item was dosed into media.
- Precipitation: Precipitate of test item was observed in the preliminary toxictiy study and mutagenicity tests 1 and 2 (see any other information on results incl. tables for full details).


COMPARISON WITH HISTORICAL CONTROL DATA:
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Preliminary Toxicity Test

The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 μg/ml. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose Level (µg/ml)

% RSG (-S9) 4-Hour Exposure

% RSG (+S9) 4-Hour Exposure

% RSG (-S9) 24-Hour Exposure

0

100

100

100

9.77

98

97

87

19.53

97

103

98

39.06

91

105

96

78.13

87

110

88

156.25

105

107

93

312.5

72

102

72

625

23

75

11

1250

4

29

0

2500

5

9

0

In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The greatest reductions were observed in the 24-hour exposure groups. Precipitate of the test item was observed at and above 78.13 μg/ml in 4-hour and 24-hour exposure groups in the absence of metabolic activation, and at and above 156.25 μg/ml in the 4-hour exposure group in the presence of metabolic activation. In addition, an increase in intensity was associated with an increase in dose concentration. Based on %RSG values observed, the maximum dose level in the subsequent Experiment 1 was set at 1250 μg/ml by toxicity for the 4-hour exposure group in the absence of metabolic activation, and the maximum achievable dose level of 2500 μg/ml for 4-hour exposure group in the presence of metabolic activation.

Mutagenicity Test

A summary of the results from the test is presented in Table 1 (see attached background material).

Experiment 1

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7 (see attached background material).

There was once again evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was no evidence of any significant dose related

reductions in viability (%V) in any of the dose levels, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Based on the %RSG and / or RTG values observed, it was considered that optimum levels of

toxicity had been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 625 μg/ml in the absence of metabolic activation, and at and above 1875 μg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 468.75 μg/ml in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis.

Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating

satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test item was observed at all of

the dose levels. The precipitate observations varied slightly from those of the preliminary toxicity test. However, the purpose and integrity of the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13 (see attached background material).

As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 9 and 12). There was evidence of significant dose

related reductions in viability (%V) in the presence of metabolic activation, therefore indicating that residual toxicity had occurred. However, it should be noted that these reductions were only observed at dose levels that had been excluded from the statistical

analysis due to excessive levels of toxicity. Based on the %RSG and / or RTG values observed, it was considered that optimum levels of toxicity had once again been achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at and above 400 μg/ml in the absence of metabolic activation, and at 1875 μg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 937.5 and 1250 μg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure group without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating

satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test item was observed at and

above 25 μg/ml in the absence of metabolic activation, and at and above 78.13 μg/ml in the presence of metabolic activation. The precipitate observations once again varied slightly from those of the preliminary toxicity test although the observations in the

presence of metabolic activation were the same as those of Experiment 1. However, the purpose and integrity of the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

Introduction.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and was 39.06 to 1250 μg/ml in the absence of metabolic activation, and 78.13 to 2500 μg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the

dose range was 25 to 600 μg/ml in the absence of metabolic activation, and 19.53 to 1875 μg/ml in the presence of metabolic activation.

Results.

The maximum dose level used in Experiment 1 was the maximum achievable dose level of 2500 μg/ml in the presence of metabolic activation, and limited by toxicity in the absence of metabolic activation. In Experiment 2 the maximum dose level was limited by toxicity for both of the exposure groups. Precipitate of test item was observed at and above 25 μg/ml in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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:
26 July 2005 to 12 August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ beta-naphthalflavone induced rat liver, S9 mix
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ beta-naphthalflavone induced rat liver, S9 mix
Test concentrations with justification for top dose:
Dose range-finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Main study: 3, 10, 33, 100 and 333 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol

Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
5 µg/plate for TA 1535 (-S9 mix)
Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
60 µg/plate for TA 1537 (-S9 mix)
Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
10 µg/plate fpr TA 98 (-S9 mix)
Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
650 µg/plate for TA100 (-S9 mix)
Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 µg/plate with E coli (-S9 mix)
Negative controls:
yes
Remarks:
spontaneous mutation rate
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Used for all tester strains (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: triplicate plating

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for the reduction of the bacterial background lawn and number of revertant colonies.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) the negative response should be reproducible in at least one independently repeated experiment.

a test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not biologically relevant.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the start and the end of the incubation period at concentrations of 333 µg/plate and upwards.


RANGE-FINDING/SCREENING STUDIES:
The dose range finding test is reported as a part of the first experiment of the mutation test (see Table 1 in attached background material).
Precipitation:
Precipitation was observed at the start of the incubation period at concentrations of 100 µg/plate and upwards. At the end of the incubation period precipitation was observed at concentrations of 333 µg/plate and upwards.
Toxicity:
No reduction of the background lawn and no decrease in the number of revertants were observed.
Mutagenicity:
In the dose range finding test, no increase in the number of revertants was observed upon treatment with EA-3098 under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Mutation assay

Based on the results of the dose range finding test, EA-3098 was tested up to concentrations of 333µg/platein the absence and presence of 59-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown in Table 1 and 2 (see attached background material).

Precipitate: Precipitation was observed at the start and end of the incubation period at the concentration of 333 µg/plate.

Toxicity: In both mutation assays there was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9 -mix.

Mutagenicity: In both mutation assays, no increase in the number of revertants was observed upon treatment with EA-3098 under all conditions tested.

Discussion

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

 

The negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that EA-3098 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Conclusions:
Interpretation of results (migrated information):
negative

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
Based on the results of this study it is concluded that EA-3098 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The method was designed to conform to the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation. The test was performed in two independent experiments in the presence and absence of S9 -mix.

In the dose range-finding test, EA-3098 was tested up to concentrations of 5000 µg per plate in the absence and presence of 5% (v/v) S9 -mix in the strains TA100 and WP2uvra. EA-3098 precipitated on the plates at dose levels of 333 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

Based on the results of the dose range-finding test, EA-3098 was tested in the first mutation assay at a concentration range of 3 to 33 µg/plate in the absence and presence of 5% (v/v) S9 -mix in tester strains TA 1535, TA 1537 and TA98. In an independent repeat of the assay with additional parameters, EA-3098 was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9 -mix in tester strains TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. EA-3098 precipitated on the plates at the top dose of 333 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

EA-3098 did not induce a dose-related, two-fold increase in the number of reverants (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

In the study, the negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that EA-3098 is not mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli:

The method was designed to conform to the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation. The test was performed in two independent experiments in the presence and absence of S9 -mix.

In the dose range-finding test, EA-3098 was tested up to concentrations of 5000 µg per plate in the absence and presence of 5% (v/v) S9 -mix in the strains TA100 and WP2uvra. EA-3098 precipitated on the plates at dose levels of 333 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

Based on the results of the dose range-finding test, EA-3098 was tested in the first mutation assay at a concentration range of 3 to 33 µg/plate in the absence and presence of 5% (v/v) S9 -mix in tester strains TA 1535, TA 1537 and TA98. In an independent repeat of the assay with additional parameters, EA-3098 was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9 -mix in tester strains TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. EA-3098 precipitated on the plates at the top dose of 333 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

EA-3098 did not induce a dose-related, two-fold increase in the number of reverants (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

In the study, the negative and strain-specific positive control values were within laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that EA-3098 is not mutagenic.

Chromosome aberration test in human lymphocytes:

The ability of EA-3098 to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted to the following guidelines: OECD Guideline 473 and EU Method B.10. The possible clastogenicity was tested in two independent experiments.

In the first cytogenetic assay, EA-3098 was tested up to 33 µg/ml for a 3 h exposure time with a 24 hour fixation time in the absence and presence of 1.8 % (v/v) S-9 fraction. EA-3098 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, EA-3098 was tested up to 100 µg/ml for a 24 -h and 48 -h continuous exposure time with a 24 -h and 48 -h fixation time in the absence of S9 -mix. In the presence of S9 -mix EA-3098 was tested up to 33 µg/ml for a 3 -h exposure time with a 48 -h fixation time. EA-3098 precipitated in the culture medium at these dose levels.

Positive control chemicals, mytomycin-C and cyclophosphamide, both produced statistically significant increases in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

EA-3098 did not induce a statistically significant or biologically relvenat increase in the number of cells with chromosomeome aberrations in the absence or presence of S9 -mix, in two independently repeated experiments.

No effects of EA-3098 on the number of polyploid cells and cells with andoreplicated chromosomes were observed both in the absence and presence of S9 -mix. Therefore it can be concluded that EA-3098 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditins described in this report.

It is concluded that this test is valid and that EA-3098 is not clastogenic to human lymphocytes under the experimental conditions in the study.

Mouse lymphoma assay:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at up to ten dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test, and was 39.06 to 1250 μg/ml in the absence of metabolic activation, and 78.13 to 2500 μg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the

dose range was 25 to 600 μg/ml in the absence of metabolic activation, and 19.53 to 1875 μg/ml in the presence of metabolic activation.

The maximum dose level used in Experiment 1 was the maximum achievable dose level of 2500 μg/ml in the presence of metabolic activation, and limited by toxicity in the absence of metabolic activation. In Experiment 2 the maximum dose level was limited by toxicity for both of the exposure groups. Precipitate of test item was observed at and above 25 μg/ml in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for selection of genetic toxicity endpoint

Three separate in vitro genetic toxicity studies have been conducted on the test item as follows:

OECD Guideline 471 Bacterial Reverse Mutation Test "Ames Test"

OECD Guideline 473 Chromosome Aberration Test

OECD 476 In Vitro Mammalian Cell Gene Mutation Tests (Mouse Lymphoma Assay)

All 3 studies have been conducted according to OECD Guidelines and GLP and are adequately reported. All studies have been assigned a reliability 1.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in the three following in-vitro studies, in substance is not classified for mutagencity.

 

- Reverse mutation assay 'Ames Test' using S. typhimurium and E. coli:

The test item was considered to be non-mutagenic under the conditions of this test.

- Chromosome aberration test in human lymphocytes:

The test item is considered to be non-clastogenic in this chromosome aberration test.

- Mouse lymphoma assay:

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.