1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, Ames, Salmonella typhimurium, plate incorporation and preincubation, ±S9: not mutagenic in bacteria
OECD 473, Chromosomal Aberration, V79 cells, ±S9: not clastogenic in mammalian cells in vitro
OECD 476, HPRT, V79 cells; ±S9: not mutagenic in mammalian cells in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT-locus (hypoxanthine-guanine phosphoribosyl transferase locus)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: PPA Ready Mix, commercially available by PAA, Paching, Austria, consisting of Eagle's minimal essential medium (MEM, Earle) with 10% FCS and supplements. During treatment with AE 1170437 technical, PAA Ready Mix with 2% FCS was used. For selection of mutants, culture medium containing 10 µg/mL of 6-thioguanine (6-TG) was used.
- Properly maintained: yes, V79 cell stocks are stored in liquid nitrogen. Laboratory cultures were maintained in plastic tissue culture vessels at 37 °C in a humidified atmosphere containing approximately 5% CO2. Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in order to subculture, an adequate dilution (ranging between 1:2.5 and 1:5) in phosphate buffered saline (PBS) of a commercially available PBS solution consisting of 0.5% trypsin and 0.2% EDTA (ethylenediamine- N,N,N',N'-tetraacetic acid) has been employed.
- Periodically checked for Mycoplasma contamination: yes, using a DNA-Staining Kit (Biochrom) according to method provided by supplier
- Periodically checked for karyotype stability: yes, utilising a modified protocol of the method by Moorhead et al. (1960)
- Periodically "cleansed" against high spontaneous background: yes, by subcloning due to plating of about 1000 cells per culture vessel at least every 2 weeks. If necessary, the spontaneous frequency of HPRT-mutants was additionally reduced by supplementing the culture medium with thymidine (9 µg/mL), hypoxanthine (10 µg/mL), glycine (22.5 µg/mL) and methotrexate (0.3 µg/mL). A 6-TG sensitive subclone was then used for the HPRT-test.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix

Test concentrations with justification for top dose:

Pretest: 2.5, 5, 10, 20, 40, 80, 160, 320 µg/mL (± S9)
Main test: 15, 30, 60, 90, 120, 150 µg/mL (-S9); 10, 20, 40, 80, 160, 320 µg/mL (+S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (1% v/v)
- Justification for choice of solvent/vehicle: solubility of test substance in this vehicle up to 250 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): normally 6 days
- Selection time (if incubation with a selection agent): 6-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): on day 12 to 14, depending on selection period

SELECTION AGENT (mutation assays): 10 µg/mL 6-thioguanine (6-TG)
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2 x 8 plates per concentration per trial, independently repeated

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth

OTHER:
Pretest: Determination of cytotoxicity after 5 hours exposure. Cells were plated out to Petri dishes (3 dishes, 200 cells each), and the colonies formed were counted. Colonies in treated cultures were compared to those in vehicle control cultures (relative cloning efficiency). Based on these findings concentrations were chosen for the main test.

Main test:
The method is based on the publication of Myhr and DiPaolo (1978). Exponentially growing V79 cells (4x10e6) were plated in duplicate flasks and exposed to various test substance concentrations after attachment (16-24 hours) for 5 hours in 20 mL culture medium. The cell monolayers were washed, trypsinised, and 1.5x10e6 cells were replated in new flasks. Additionally, 3 Petri dishes per culture were plated with 200 cells each for determination of colony development and cytotoxicity associated with test substance directly after treatment. The dishes were incubated for 6 days.

The cells in flasks were subcultured after 3 days by reseeding 1.5x10e6 cells into new flasks which were cultured for another 3 days (6 days expression period in total). At the end of the expression period cultures were reseeded in 8 Petri dishes per culture at 3x10e5 cells per dish in selection medium containing 10 µg/mL 6-TG. In addition, 200 cells per dish were seeded into 3 Petri dishes per culture in culture medium without 6-TG to determine absolute cloning efficiency for each concentration. After incubation for 6-8 days colonies were fixed, stained with Giemsa, and 6-TG resistant colonies were counted. At the same time the number of colonies in the cloning efficiency dishes was counted.

The activation assay was performed independently. The procedure was identical to the non-activation assay except for the replacement of 1 mL of culture medium in the flask by 1 mL of S9-mix during the exposure period.

Two trials were performed each with and without activation.

Evaluation criteria:

- The average cloning efficiency of the negative and vehicle controls should be at least 50%.
- The average of mutant frequency of the vehicle controls should not exceed 25x10e-6 cells.
- The mutant frequency of the two cultures of the vehicle and/or the negative control should differ only to an acceptable extent. As a rule of thumb, the difference of mutant frequencies should not be greater than 5x10e-6.
- The positive control should induce an average mutant frequency of at least three times that of the vehicle control.
- If not limited by the solubility of the test substance in the vehicle the highest concentration should induce cytotoxicity of about 80 to 90% or should be a concentration where precipitation occurs in the medium. The survival at the lowest concentration should be in the range of the negative control.
- For the calculation of an acceptable mutant frequency at least 5 dishes per culture should be available and relative survival to treatment, relative population growth and absolute cloning efficiency should be 10% or greater.

Statistics:

Mutant frequencies are submitted to a weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights (Hsie et al., 1981; Arlett et al., 1989).
According to the acceptance criteria mutant frequencies based on less than 5 plates and/or on a relative survival to treatment and/or a relative population growth and/or an absolute cloning efficiency below 10% are not included in the statistical analysis.
The two mutant frequency values obtained per group are considered as independent measurements thus increasing the power of the statistical tests applied. Since the protocol of the HPRT assay requires at least two independent trials, the overall analysis without respectively with activation is the most important one for classifying substances into mutagens and non-mutagens. However, separate analyses were run for each trial in order to examine the consistency of the results.
All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the vehicle control on a nominal significance level of α = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, negative and vehicle controls. If there is a significant concentration related increase of the mutant frequency (α = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 320 µg/ml AE 1170437 technical did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 320 µg/mL AE 1170437 technical.
- Water solubility: The test substance was not soluble in water, therefore, the respective amounts were dissolved in DMSO which did not exceed a final concentration of 1% (v/v) in the medium.
- Precipitation: In the absence of S9-mix substance precipitation occurred in the medium at the concentration 150 µg/mL. With S9-mix substance precipitation was noted at 160 µg/mL and above.


RANGE-FINDING/SCREENING STUDIES:
Precipitation of AE 1170437 technical in the culture medium started at 160 µg/mL. Without S9 mix cytotoxic effects of AE 1170437 technical were observed at 80 µg/mL and above. With S9 mix cytotoxicity was evident at 160 µg/mL and above. Due to these findings, AE 1170437 technical was tested in the respective first mutation experiment in concentrations ranging from 15 µg/mL to 150 µg/mL without metabolic activation and from 10 µg/mL to 320 µg/mL with metabolic activation. The same concentrations were used for the independent repeats.

COMPARISON WITH HISTORICAL CONTROL DATA
The results of the control groups were well comparable with the historic control data provided in the report.

The means of the absolute cloning efficiency for the vehicle controls in the mutation experiments were 69.6% and 98.7% in the experiments without activation. In experiments with metabolic activation 64.2% and 65.2% were observed. These results demonstrate good cloning conditions for the experiments.

Two trials were performed with and without metabolic activation. The mutant frequencies of the negative controls and of the vehicle controls were all within the normal range. The positive controls EMS and DMBA induced clear mutagenic and statistically significant effects in all trials with and without metabolic activation, respectively. All controls were well comparable to the historical data provided in the report.

Without S9-mix clear cytotoxic effects were induced; concentration-related decreases in relative survival to treatment and/or in relative population growth were observed for test substance-treated cultures in the absence of metabolic activation, whereas

no such decreases were found in the presence of metabolic activation. However, precipitation of the test substance in the medium was observed at 150 µg/mL without S9-mix and at 160 and 320 µg/mL with S9-mix. The test substance induced no relevant increases in mutant frequencies. In addition, the overall statistical analysis reveals no statistically significant increases, as well.

V79/HPRT Mutagenesis assay – examples for results (trials with highest mutation frequencies):

Concentration (µg/mL)	Survival to treatment		Relative population growth (% vehicle control)	Total mutant colonies (8 plates)	Absolute cloning efficiency (%) ± SD	Mutant frequency x10e-6
	Mean colony number ± SD	% Vehicle control
Without activation
negative	192.0±25.2	106.3	62.6	0	70.8±4.7	0.0
negative	237.0±9.5	97.9	207.4	3	131.3±21.2	1.0
vehicle	180.7±11.2	100.0	100.0	2	118.0±11.5	0.7
vehicle	242.0±14.7	100.0	100.0	4	79.3±20.7	2.1
EMS900 µg/mL	165.3±6.7	91.5	53.4	192	104.3±8.8	76.7
EMS900 µg/mL	156.7±8.5	64.7	82.2	208	52.7±7.3	164.6
15	231.3±23.7	128.0	80.2	1	64.8±12.1	0.6
15	195.3±4.0	80.7	155.4	6	82.0±9.6	3.0
30	223.7±7.5	123.8	48.0	2	65.8±19.8	1.3
30	190.0±12.5	78.5	108.4	0	83.2±10.0	0.0
60	210.7±20.6	116.6	51.5	4	82.7±5.3	2.0
60	189.3±8.7	78.2	151.5	12	88.7±5.5	5.6
90	206.0±13.0	114.0	64.3	4	108.8±16.6	1.5
90	255.0±16.5	105.4	82.2	3	78.8±5.0	1.6
120	176.0±11.5	97.4	95.8	2	114.7±10.4	0.7
120	212.3±5.1	87.7	40.7	0	80.2±4.3	0.0
150 (P)	228.0±20.7	126.2	42.0	2	112.0±7.1	0.7
150 (P)	197.3±21.1	81.5	45.5	0	81.5±6.9	0.0
With activation
negative	148.3±20.5	89.2	111.2	10	78.3±7.9	5.3
negative	198.7±8.6	134.2	106.6	6	56.3±0.8	4.4
vehicle	166.3±21.6	100.0	100.0	6	58.7±9.5	4.3
vehicle	148.0±12.2	100.0	100.0	5	69.7±3.0	3.0
DMBA 20 µg/mL	82.0±15.4	49.3	14.7	58	69.8±6.0	34.6
DMBA 20 µg/mL	64.3±6.1	43.5	17.6	57	55.3±9.5	42.9
10	168.3±4.0	101.2	143.1	14	52.8±0.8	11.0
10	172.7±13.8	116.7	76.1	5	61.0±4.9	3.4
20	171.0±2.6	102.8	176.5	10	59.0±6.3	7.1
20	175.0±14.1	118.2	87.9	13	70.3±4.6	7.7
40	141.7±9.5	85.2	152.2	6	66.3±4.0	3.8
40	110.0±1.7	74.3	85.2	1	53.3±8.0	0.8
80	153.0±15.5	92.0	109.0	12	67.5±2.0	7.4
80	199.0±19.1	134.5	54.8	12	55.8±3.3	9.0
160 (P)	159.7±11.9	96.0	108.5	10	50.5±10.8	8.3
160 (P)	166.3±8.1	112.4	39.2	13	66.5±3.5	8.1
320 (P)	158.0±18.0	95.0	69.1	4	70.2±3.8	2.4
320 (P)	149.7±16.6	101.1	57.5	9	58.2±1.2	6.4

 P: precipitation

CONCLUSION:

The test substance was evaluated as non-mutagenic with and without metabolic activation under the conditions chosen.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: PPA Ready Mix, commercially available by PAA, Paching, Austria, consisting of Eagle's minimal essential medium (MEM, Earle) with 10% FCS and supplements. During treatment with AE 1170437 technical, PAA Ready Mix with 2% FCS was used.
- Properly maintained: yes, V79 cell stocks are stored in liquid nitrogen. Laboratory cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2 (incubator). Cells were passaged on the day prior to treatment. For detachment during maintenance culture and after exposure cells were trypsinised.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9-mix

Test concentrations with justification for top dose:

Pre-test:
0, 1, 5, 10, 25, 50, 100, 200, 400 µg/ml (±S9, 4 hour exposure, harvest after 24 hours); 0, 1, 5, 10, 20, 30, 60, 120 µg/mL (-S9, 18 hours exposure, harvest after 18 hours)

Main test:
0, 15, 30, 60, 90, 120 µg/mL (-S9, 4 hour exposure, harvest after 18 hours); 0, 50, 100, 160, 200, 240 µg/mL (+S9, 4 hour exposure, harvest after 18 hours); 0, 60, 90, 120 µg/mL (-S9, 4 hour exposure, harvest after 30 hours); 0, 160, 200, 240 µg/mL (+S9, 4 hour exposure, harvest after 30 hours); 0, 4, 8, 16, 20, 24 µg/mL (-S9, 18 hour exposure, harvest after 18 hours)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of AE 1170437 technical in DMSO up to 250 mg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 hours, respectively (additionally, 8 hours total incubation period in main-test for determination of cytotoxicity only)
- Expression time (cells in growth medium): 14 and 26 hours, respectively (4 hour exposure, harvest after 18 and 30 hours, respectively), and 0 hours (18 hour exposure, harvest after 18 hours)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 30 hours, respectively (main test); 18 and 24 hours, respectively (pre-test)

SPINDLE INHIBITOR (cytogenetic assays): 0.2 mL colcemid (40 µg/mL)
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: duplicate cultures, at least two slides generated per culture

NUMBER OF CELLS EVALUATED: 200 metaphases (1000 cells for mitotic index)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: cell survival (visual inspection in improved Neubauer hemocytometer)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
- Other: chromosome disintegration as indication of a cytotoxic effect (recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase)

OTHER:
The general protocol of the test system was similar to published procedures (e.g. Dean and Danford, 1984). Chinese hamster V79 cells were passaged on the day prior to treatment. Approximately 1 x 10e6 cells were seeded in 20 mL of medium per 75 cm² flasks and incubated at 37 °C, 5% CO2 in medium with 10% FCS. For exposure the normal culture medium was exchanged with medium containing only 2% FCS. After the exposure period medium with 10% FCS was used again. Two hours before harvest colcemid was added to the cultures for metaphase arrest.

Evaluation criteria:

An increased incidence of gaps of both types without concomitant increase of other aberration types was not considered as indication of a clastogenic effect.
A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
A test was considered negative, if there was no such increase at any time interval.
A test was also considered negative, if there were statistically significant values, which were, however, within the range of historical negative controls.
A test was considered equivocal, if there was an increase above the range of historical negative controls which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.

An assay was considered acceptable, if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results from the laboratory and from published studies.

Statistics:

The statistical analysis was performed by pair-wise comparison of treated and positive control groups to the respective solvent control group. The mitotic index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chi²-test.
The numbers of metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges were compared (provided that these data superceded the respective solvent control). The statistical analysis followed the recommendations outlined by Richardson et al. (1989). The one-sided chi²-test was used for the statistical evaluation.
A difference was considered to be significant, if the probability of error was below 5%.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 30 µg/ml and 16 µg/mL without S9 (4 and 18 h exposure, respectively), and at 100 µg/mL with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Concentrations of up to 2500 µg/mL AE 1170437 technical did not change the pH in the medium in the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 400 µg/mL AE 1170437 technical.
- Evaporation from medium: not relevant, incubations performed in an incubator
- Water solubility: up to 250 mg/mL
- Precipitation: Precipitation of AE 1170437 technical was observed in the culture medium of the pre-test at 100 µg/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The selection of the concentrations used for the main study was based on the results of pre-tests in which cells were exposed with and without S9 mix for 4 hours to various concentrations of the test substance. In addition, cells were exposed without S9 mix for 18 hours to various concentrations of the test substance. Duplicate cultures were tested for every concentration and control.
As indicators of cytotoxic effects, mitotic indices and numbers of surviving cells (survival index) were used. The mitotic index was determined by counting a total of 1000 cells per culture. The results of the solvent controls were set 100% and compared to the AE 1170437 technical treated cultures. Non-attached cells were observed without S9-mix at 100 µg/mL and above and alteration in cell morphology was observed without S9-mix (18 hours treatment) at 60 µg/mL and above. With S9-mix non-attached cells were observed at 100 µg/ml and above and alteration in cell morphology was observed at 400 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The amount of metaphases with aberrations was well comparable to that reported in the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assessed in the pre-test as well as in the main-study. In the main study, cultures with a total incubation period of 8 hours were additionally and exclusively used to determine the cytotoxicity of AE 1170437 technical. At the end of the respective incubation period cells of all cultures of the respective period were trypsinised, and an appropriate dilution was counted using a hemocytometer (improved Neubauer) to determine cell survival. The mitotic index was determined for all cultures. The number of mitotic cells among a total of 1000 cells per culture was determined. All cells which were not in interphase were defined as mitotic.

Precipitation was observed starting at 120 µg/ml without and 160 µg/ml with activation. Altered morphology and non-attached cells were found at 60 µg/mL in cultures without S9. In cultures with metabolic activation altered cell morphology was observed starting at 160 µg/mL, and non-attached cells were observed starting at 100 µg/mL.

Mitotic indices were found to be relevantly reduced beginning at 60 and 16 µg/mL (4- and 18-hour exposure, respectively) without metabolic activation and beginning at 100 µg/mL with activation. Both positive controls mitomycin C and cyclophosphamide reduced the mitotic index, as well.

Survival indices were also relevantly reduced, starting at 30 and 16 µg/mL (4- and 18-hour exposure, respectively) without activation and 100 µg/mL with activation.

Based on the results of the survival index and the mitotic index, the following concentrations were selected for reading:

4-hour exposure, 18-hour harvest: 15, 30, 60 µg/mL (-S9); 50, 100, 160 µg/mL (+S9)

4-hour exposure, 30-hour harvest: 60 µg/mL (-S9); 160 µg/mL (+S9)

18-hour exposure, 18-hour harvest: 8, 16, 20 µg/mL (-S9 only)

Chromosomal aberrations in V79 cells (200 cells scored for aberrations, 2000 for mitotic index):

			Gaps		Metaphases with aberrations (%)
Conc. test substance (µg/mL)	Mitotic index (%)	Survival index (%)	gap	isogap	incl. gaps	excl. gaps	exchanges
4 hours treatment, 18 hours harvest, -S9
DMSO	100.0	100.0	0	0	1.0	1.0	0.0
15	103.7	89.5	0	0	1.5	1.5	0.0
30	110.5	54.7#	0	0	3.0	3.0	1.0
60	54.7**	60.0#	0	0	2.5	2.5	1.0
90	21.1**	53.7#	n.r.	n.r.	n.r.	n.r.	n.r.
120	26.8**	47.4#	n.r.	n.r.	n.r.	n.r.	n.r.
MMC 0.1	60.0**	45.3#	5	5	60.5**	59.5**	37.5**
4 hours treatment, 18 hours harvest, +S9
DMSO	100.0	100.0	1	0	3.0	2.5	1.0
50	113.1	91.7	0	0	4.5	4.5	0.5
100	64.6**	66.7#	0	1	2.0	1.5	0.0
160	50.8**	59.7#	2	0	4.5	3.5	0.0
200	47.7**	43.1#	n.r.	n.r.	n.r.	n.r.	n.r.
240	53.1**	72.2#	n.r.	n.r.	n.r.	n.r.	n.r.
CP 2.0	60.8**	69.4#	10	7	63.5**	61.5**	34.0**
4 hours treatment, 30 hours harvest, -S9
DMSO	100.0	100.0	1	0	5.0	4.5	0.0
60	88.0	63.0#	1	0	5.0	5.0	1.5
90	72.6**	34.1#	n.r.	n.r.	n.r.	n.r.	n.r.
120	53.0**	24.6#	n.r.	n.r.	n.r.	n.r.	n.r.
4 hours treatment, 30 hours harvest, +S9
DMSO	100.0	100.0	0	0	3.0	3.0	0.0
160	75.3**	65.8#	2	0	7.5^a	7.0	4.5^b
200	43.4**	56.1#	n.r.	n.r.	n.r.	n.r.	n.r.
240	53.9**	43.0#	n.r.	n.r.	n.r.	n.r.	n.r.
18 hours treatment, 18 hours harvest, -S9
DMSO	100.0	100.0	0	0	3.0	3.0	0.5
4	131.9	100.0	n.r.	n.r.	n.r.	n.r.	n.r.
8	123.1	100.0	1	0	2.5	2.0	0.0
16	75.8*	64.2#	2	0	3.0	2.0	0.0
20	65.9**	79.2#	1	0	3.0	2.5	1.0
24	35.2**	54.7#	n.r.	n.r.	n.r.	n.r.	n.r.
MMC 0.03	81.3**	45.3#	3	3	40.0**	37.0**	16.5**

n.r. not selected for reading

*p<0.05

**p<0.01

# relevant reduction of survival index

^anot considered relevant

^bnot considered relevant

Without metabolic activation:

No biologically relevant and statistically significant increases in numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 or 30 hours. The same was true for a treatment period and total culture time of 18 hours. The positive control mitomycin C resulted in a clear and statistically significant increase in metaphases with aberrations and demonstrated the sensitivity of the test system.

With metabolic activation:

No biologically relevant and statistically significant increases in numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 18 hours, and no biologically relevant increases of numbers of metaphases with aberrations were detected after 4 hours treatment and total culture times of 30 hours. The positive control cyclophosphamide induced statistically significant and biologically relevant increases in metaphases with aberrations and demonstrated the sensitivity of the test system and the activity of the used S9 mix.

CONCLUSION

The test substance AE 1170437 technical does not induce chromosomal aberrations in V79 cells in the absence and presence of metabolic activation under the conditions of the study. Based on the results of this test, the test substance is considered not to be clastogenic for mammalian cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His-operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

16, 50, 158, 500, 1581, 5000 µg/plate (plate incorporation assay); 100, 200, 400, 800, 1600, 3200 µg/plate (preincubation assay)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test compound

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

mitomycin C

other: nitrofurantoin (0.2 µg/plate, -S9, TA 100); 4-nitro-1-,2-phenylene diamine (10 µg/plate, -S9, TA 1537; 0.5 µg/plate, -S9, TA 98); cumene hdydroperoxide (50 µg/plate, -S9, TA 102 in pre-incubation assay); 2-aminoanthracene (3 µg/plate, +S9, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (served as independent repeat)

DURATION
- Preincubation period: 20 minutes (only preincubation assay)
- Exposure duration: 48 hours (both plate incorporation and preincubation)

SELECTION CRITERIA (mutation assays): reverse mutation to histidine prototrophy

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in background growth; reduction in mutant count per plate; titer

Evaluation criteria:

Acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Even if the criteria for points 1 and 2 were not met, a trial was accepted if it showed mutagenic activity of the test compound.

Criteria for a positive result:
A reproducible and dose-related increase in mutant counts of at least one strain (For TA1535, TA100 and TA98 about twice that of negative controls, for TA1537 at least threefold; for TA102 an increase of about 100 mutants should be reached). Otherwise the result is evaluated as negative.

Statistics:

Means and standard deviation of triplicate plates were calculated.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain-specific bacteriotoxic effect, starting at 158 µg/plate in TA98 and 500 µg/plate in the other strains in the plate incorporation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate in the plate incorporation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The plate incorporation assay (1st trial) served as range finder for the determination of the test concentrations and was considered as pre-test for toxicity. As more than three doses could be used for assessment, the first trial was included in the assessment. The doses of the repeats were chosen on the basis of the results obtained in the first experiment. The independent repeat was performed as preincubation assay. Due to the substance's toxicity and precipitation, doses ranging from 100 µg to 3200 µg per tube were chosen for the repeat test. As the preincubation assay confirmed the results of the first trial, no additional repeat was performed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The experimental data is well comparable with the provided historical control data.

Maximum number of revertants (Mean ± SD):

Plate incorporation assay
	-S9			+S9
Strain	control	test compound (µg/plate)	pos. control	control	test compound (µg/plate)	pos. control
TA1535	12 ± 4	10 ± 2 (158)	495 ± 25	9 ± 1	12 ± 3 (16)	135 ± 6
TA 100	147 ± 15	139 ± 17 (16)	316 ± 35	144 ± 25	159 ± 22 (158)	1256 ± 117
TA 1537	7 ± 1	8 ± 2 (158)	122 ± 14	11 ± 6	12 ± 4 (16)	124 ± 36
TA 98	24 ± 5	23 ± 7 (16)	173 ± 15	26 ± 3	28 ± 8 (158)	1128 ± 81
TA102	187 ± 11	218 ± 15 (158)	650 ± 62	197 ± 11	276 ± 30 (500)	548 ± 71
Preincubation assay
	-S9			+S9
Strain	control	test compound (µg/plate)	pos. control	control	test compound (µg/plate)	pos. control
TA1535	9 ± 3	16 ± 3 (200)	409 ± 30	7 ± 1	9 ± 5 (800)	93 ± 9
TA 100	108 ± 13	96 ± 6 (100)	489 ± 47	139 ± 19	120 ± 6 (100)	934 ± 183
TA 1537	11 ± 1	8 ± 1 (100)	119 ± 6	7 ± 1	12 ± 4 (800)	105 ± 14
TA 98	22 ± 8	18 ± 7 (100)	143 ± 18	32 ± 10	36 ± 10 (100)	795 ± 87
TA102	255 ± 19	215 ± 44 (100)	427 ± 11	224 ± 47	252 ± 25 (800)	640 ± 86

Precipitation occurred at concentrations of 3200 µg/plate and above in all assays.

No indications of a mutagenic effect of the test substance occurred at assessable doses up to 1600 µg/plate in any of the strains tested.

Conclusion:

Under the test conditions chosen in this study the test substance has to be regarded as non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

OECD 474, Micronucleus test, mouse: not clastogenic in mammalian cells in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: 6-12 weeks
- Weight at study initiation: 37-45 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: individually in type II cages; bedding of soft wood granules, type BK8/15 (J. Rettenmaier & Soehne, Fuellstoff-Fabriken, 73494 Ellwangen-Holzmuehle) was used
- Diet (e.g. ad libitum): fixed-formula feed 3883 (10 mm cubes), produced according to specification by Provimi Kliba SA, CH-4303 Kaiseraugst, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22.5
- Humidity (%): 45-54
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% aqueous Cremophor emulsion, resulting suspension was set to pH 6 to 8 using 0.02 N aqueous NaOH. Cyclophosphamide was dissolved in physiological saline solution.
- Justification for choice of solvent/vehicle: suitability for intraperitoneal application
- Concentration of test material in vehicle: 1, 2, 4 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg intraperitoneal
- Lot/batch no. (if required): Fluka, batch 444193/1 52304077

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in 0.5% aqueous Cremophor emulsion, using a microdismembrator for 3 minutes, and formed white turbid suspensions. The suspensions were set to pH 6 to 8 using 0.02 N aqueous NaOH. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally. Cyclophosphamide was dissolved in physiological saline solution and administered in the same way.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

2 injections 24 hours apart

Post exposure period:

24 hours

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in the form of Endoxan 100 mg injection vials of dry substance (Baxter Oncology GmbH), batch 4N120
- Justification for choice of positive control(s): proven cytostatic agent and known clastogen with bifunctional alkylation action.
- Route of administration: intraperitoneal (only 1 injection)
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Bone marrow cells (erythrocytes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the range-finder study with doses of 20, 40, 100 and 1000 mg/kg, each applied to groups of 3 males and 3 females; 40 mg/kg AE 1170437 technical were chosen as MTD for males.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals received 2 intraperitoneal injections 24 hours apart, 24 hours after the last application the animals were sacrificed and the bone marrow cells were isolated.

DETAILS OF SLIDE PREPARATION:
Bone marrow smears were prepared according to the method of Schmid (Mutat Res 31: 9-15; 1975. DFG; Kommission fuer Mutagenitaetsfragen; Mitteilung III, 53-61). At least one intact femur was prepared from each sacrificed animal. The bone marrow cells were flushed out from the femur with fetal calf serum. After centrifugation the supernatant was discarded and a homogeneous cell suspension prepared from the pellet. One drop of this suspension was then placed on a well-cleaned slide and spread out with a suitable object. The slides were dried overnight and stained automatically on the following day with an Ames Hema-Tek Slide Stainer from the Miles Company. Unspecific background staining was subsequently removed with methanol, then the slides were rinsed with deionised water and left to dry. Thereafter the slides were fixed with xylene, embedded in covering agent and covered with a covering glass. Slides were not evaluated until the covering agent had dried.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus. Normally, 2000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.

Evaluation criteria:

A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible. In both cases, normally a second test will be performed.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

Statistics:

The AE 1170437 technical group(s) with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (1s ranges) were calculated for all the means.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing for up to 3 hours after the second treatment

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The pilot test was performed in the laboratory which conducted the main study using animals of the same source, strain and age. Groups consisting each of three males and three females received two intraperitoneal injections (animals of the 1000 mg/kg group died prior to second treatment) separated by 24 hours.

- Dose range: 20, 40, 100, 200, 1000 mg/kg bw AE 1170437 technical

- Solubility: AE 1170437 technical was suspended in 0.5% aqueous Cremophor (Fluka, batch 444193/1 52304077) emulsion, using a microdismembrator for 3 minutes, and formed white turbid suspensions. The suspension was set to pH 6 to 8 using 0.02 N aqueous NaOH. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.

- Clinical signs of toxicity in test animals: In males the following symptoms were recorded up to their death or for up to at least 3 hours after the second application, starting at 20 mg/kg: apathy, running in circles, roughened fur, lateral and sternal recumbency, spasm, palmospasm, twitching, difficulty in breathing, and slitted eyes. In addition, 3 of 3 males died in the 1000 mg/kg group, 2 of 3 males died in the 200 mg/kg group and 1 of 3 males died in the 100 mg/kg group. In females, except twitching, the same symptoms plus periodically stretching of body were recorded. In addition, 3 of 3 females died in the 1000 mg/kg group and 2 of 3 females died in the 200 mg/kg group as well as in the 100 mg/kg group.
- Rationale for exposure: Based on these findings, 40 mg/kg AE 1170437 technical were chosen as MTD for males. Due to the results of the dose range finder it is concluded, that there are no substantial differences between sexes in toxicity. Therefore, no females were used. Intraperitoneal application is one of the common routes recommended by the guideline.
- Harvest times: 24 hours after last treatment
- High dose with and without activation: activation not required, in vivo study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations existed for males between the negative control and the groups treated intraperitoneally with AE 1170437 technical, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.4/2000 (1s=1.1) in the negative control, and 3.0/2000 (1s=1.9), 3.0/2000
(1s=2.2) and 3.0/2000 (1s=2.6) in the AE 1170437 technical groups (10, 20 and 40 mg/kg bw, respectively).

- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic to normochromatic erythrocytes in males was not altered by the treatment with AE 1170437 technical, being 2000: 3247 (1s=362) in the negative control, 2000: 3741 (1s=976) in the 10 mg/kg group, 2000: 3465 (1s=436) in the 20 mg/kg group and 2000: 4219 (1s=1050) in the 40 mg/kg group. No relevant variations were thus noted for males.

- Appropriateness of dose levels and route: After two intraperitoneal administrations of 10, 20 and 40 mg/kg AE 1170437 technical, treated males showed the following compound-related symptoms for up to 3 hours after the second treatment: apathy, roughened fur, spasm, periodically stretching of body and difficulty in breathing. These symptoms demonstrate relevant systemic exposure of males to AE 1170437 technical. Thereafter, their external appearance and physical activity remained unaffected. There was no substance-induced mortality. Except for loss of weight in one male, no symptoms were recorded for the control groups. No animals died in these groups.

- Statistical evaluation: There was no biologically significant variation between the negative control and AE 1170437 technical groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The results with AE 1170437 gave no indications of clastogenic effects for male mice after two intraperitoneal treatments with doses up to and including 40 mg/kg bw.

Summary of Results of the Micronucleus Test with AE 1170437 technical:

Experimental groups	Number of evaluated PCE	Number of NCE/2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
negative	10000	3247 ± 362	1.2 ± 0.6	2.4 ± 1.1
2 x 10 mg/kg	10000	3741 ± 976	1.4 ± 0.9	3.0 ± 1.9
2 x 20 mg/kg	10000	3465 ± 436	1.2 ± 0.4	3.0 ± 2.2
2 x 40 mg/kg	10000	4219 ± 1050	0.7 ± 0.7	3.0 ± 2.6
Cyclophosphamide 20 mg/kg	10000	4036 ± 714	1.1 ± 0.8	29.0 ± 9.6*

*P<0.01 in non-parametric Wilcoxon ranking test

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MNNCE: micronucleated normochromatic erythrocytes

MNPCE: micronucleated polychromatic erythrocytes

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 29.0/2000 (1s=9.6), which represents biologically relevant increases in comparison to the negative control. A biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control was not possible since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [2000: 4036 (1s=714), as against 2000: 3247 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Conclusion:

There was no indication of a clastogenic effect of intraperitoneally administered AE 1170437 technical in the micronucleus test on the male mouse, i.e. in a somatic test system in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are reliable tests available investigating genetic toxicity in bacteria and mammalian cells in vitro and in mammalian cells in vivo.

Mutagenicity in bacteria was assessed by two GLP-studies performed according to OECD guideline 471, following the method of Ames. In both studies the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102 were tested. In both studies a plate incorporation assay served as range-finder for the determination of the test concentrations and was considered as a pre-test for toxicity; in both studies the bacteria were exposed to concentrations of 16, 50, 158, 500, 1581 and 5000 µg/plate in the plate incorporation assay in the absence and presence of metabolic activation by rat liver S9-mix. In the newer study from 2007 a strain-specific bacteriotoxic effect, starting at 158 µg/plate in TA 98 and 500 µg/plate in the other strains, was observed, therefore, in the second trial concentrations of 100, 200, 400, 800, 1600 and 3200 µg/plate were tested in the preincubation assay. Although a comparable effect starting at 500 µg/plate in all strains was also observed in the older study from 2006, the same concentrations as in the pre-test were used in the preincubation assay. Precipitation was observed at concentrations of 3200 µg/plate and above in the newer study and at 5000 µg/plate in the old study. Therefore, the newer study was chosen as key study. However, the results of both studies corresponded well to each other and to the historical control data; in both studies the maximum numbers of revertants observed in the test substance-treated plates were comparable to those in the negative controls with and without metabolic activation in all strains tested.

The clastogenic potential of the test substance in vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD guideline 473 under GLP-conditions. In a pre-test Chinese hamster V79 cells were exposed to test substance concentrations of 1, 5, 10, 25, 50, 100, 200 and 400 µg/ml for 4 hours in the absence and presence of metabolic activation by rat liver S9 and were harvested after 24 hours. Additionally, cells were exposed for 18 hours to concentrations of 1, 5, 10, 20, 30, 60 and 120 µg/ml without metabolic activation and harvested after 18 hours. Based on the findings of precipitation, non-attached cells and changes in cell morphology, the following concentrations were chosen for the main test: The V79 cells were exposed for 4 hours to 15, 30, 60, 90 and 120 µg/ml without activation and to 50, 100, 160, 200 and 240 µg/ml with activation and harvested after 18 hours. Additionally, cells exposed for 4 hours to 60, 90 and 120 µg/ml without and to 160, 200 and 240 µg/ml with S9-mix were harvested after 30 hours. At last, cells exposed for 18 hours to concentrations of 4, 8, 16, 20 and 24 µg/ml without S9-mix were harvested after 18 hours. Precipitation was observed starting at 120 µg/ml without and 160 µg/ml with activation. Altered morphology and non-attached cells were found at 60 µg/ml without S9 and starting at 160 and 100 µg/ml, respectively, with S9. Cytotoxicity was observed at 30 and 16 µg/ml (survival indices; 4- and 18-hour exposure, respectively) without metabolic activation and beginning at 100 µg/ml with activation. There were no biologically and statistically significant increases in numbers of metaphases with aberrations either with or without metabolic activation at any exposure duration and at any total culture time. In contrast, the positive controls resulted in clear and statistically significant increases in metaphases with aberrations and demonstrated the functionality of the test system.

The mutagenic potential of the test substance in mammalian cells in vitro was assessed by a HPRT-assay according to OECD guideline 476 under GLP-conditions. In the pre-test Chinese hamster V79 cells were exposed for 5 hours to test substance concentrations of 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/ml in the absence and presence of metabolic activation by rat liver S9-mix. Precipitation of the test substance in the culture medium started at 160 µg/ml, cytotoxicity was evident at 160 µg/ml and above with S9 and at 80 µg/ml and above without. Based on these results the test substance was tested at concentrations of 15, 30, 60, 90, 120 and 150 µg/ml without activation and 10, 20, 40, 80, 160 and 320 µg/ml with activation in the main test, two trials were performed. Cells were exposed for 5 hours, as well. Without metabolic activation clear cytotoxic effects were induced. Precipitation occurred at 160 µg/ml with S9 and at 150 µg/ml without. However, the test substance did not induce any statistically significant increases in mutant frequencies at any concentration tested. Therefore, the test substance was not mutagenic in mammalian cells in vitro.

The genetic toxicity of the test substance in vivo was addressed in a Mammalian Erythrocyte Micronucleus test in the mouse, performed according to OECD guideline 474 under GLP conditions. Based on a range-finder study a dose of 40 mg/kg bw of the test substance was determined as MTD for males. Five males per dose received 2 intraperitoneal injections 24 hours apart of 10, 20 and 40 mg/kg bw of the test substance in an appropriate vehicle; 24 hours after the last application the animals were sacrificed, and bone marrow smears were prepared. The slides were analysed microscopically, and the number of micronucleated polychromatic erythrocytes was determined. Clinical signs of toxicity were observed up to 3 hours after the last injection, demonstrating relevant systemic exposure of the animals to the test substance in the absence of mortality. There was no biologically important or statistically significant variation in the number of micronucleated polychromatic erythrocytes noted for the test substance-treated groups. The incidence of these micronucleated cells was 3.0 per 2000 polychromatic erythrocytes (PCE) in all treatment groups compared to 2.4 per 2000 PCE in the negative control group. These results gave no indication of a clastogenic effect of the test substance in male mice in vivo after two injections of up to and including 40 mg/kg bw.

In conclusion, the test substance did not induce gene mutations in bacteria and mammalian cells in vitro or clastogenicity in mammalian cells both in vitro and in vivo.

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008 the test substance does not have to be classified for genetic toxicity.