2-Butenoic acid, 1-ethyl-2-methylpropyl ester, (2E)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance, TM 12-209, was assessed according to internationally recognized guidelines for its genotoxicity: 1. AMES 2. In vitro mammalian chromosome aberration assay No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation according to OECD Test Guideline 471 in the Ames test. The chromosome aberration potential of the test item was negative both with and without metabolic activation according to OECD Test Guideline 473 using an in vitro mammalian chromosome aberration method.

Link to relevant study records

Reference

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:

The study was conducted between 15 January 2013 and 13 January 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1as it has been conducted according to OECD Test Guideline 473 using an in vitro chromosome aberration test method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared in-house from the livers of male rats.

Test concentrations with justification for top dose:

Experiment 1
There were 2 exposure conditions conducted for Experiment 1:
i) 4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 13.28, 26.56, 53.13, 106.25, 212.5, 425, 850 and 1700 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 13.28, 26.56, 53.13, 106.25, 212.5, 425, 850 and 1700 µg/ml.

Experiment 2
There were 2 exposure conditions conducted for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 75, 100 and 200 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 100, 150 and 200 µg/ml.

Vehicle / solvent:

Eagle's minimal essential medium with HEPES buffer (MEM), supplemented in-house with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Absence of S9: MMC at 0.4 and 0.2 µg/ml for cultures in Experiment 1 and 2 respectively. Presence of S9: CP at 5 µg/ml in both experiments.

Details on test system and experimental conditions:

Cell culture
Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Preparation of Test Item and Control Items
The test item was immiscible in aqueous media at 17 mg/ml but soluble in dimethyl sulphoxide at 170 mg/ml in solubility checks performed in house. Dimethyl sulphoxide was, therefore, selected as the vehicle.
The test item was accurately weighed, formulated in dimethyl sulphoxide and serial dilutions prepared. The molecular weight of the test item was provided by the Sponsor as 170.13, therefore the maximum dose level was 1700 µg/ml, which was calculated to be equivalent to 10 mM the maximum recommended dose level. The purity of the test item was 99% and, therefore, was not accounted for in the formulations.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated (Scott et al, 1991).
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.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 ml MEM, 10% (FBS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With metabolic activation (S9) treatment
After approximately 48 hours incubation at approximately 37ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 ml of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 ml of 10% S9-mix (i.e. 1% final concentration of S9 in standard co factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.
After 4 hours at approximately 37ºC, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re incubated for a further 20 hours at approximately 37ºC in 5% CO2 in humidified air.

Without metabolic (S9) treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 1.0 ml of the appropriate vehicle control, test item solution or 0.1 ml of positive control solution. The total volume for each culture was a nominal 10 ml.
After 4 hours at approximately 37ºC, 5% CO2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.0 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 ml of vehicle control, test item dose solution or 0.1 ml of positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at approximately 37ºC, 5% CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary toxicity test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 6.64, 13.28, 26.56, 53.13, 106.25, 212.5, 425, 850 and 1700 µg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Additionally, one slide from the 24-hour exposure (106.25 µg/ml) was selected for metaphase analysis. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
There were 2 exposure conditions conducted for Experiment 1:
i) 4-hour exposure to the test item without S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 13.28, 26.56, 53.13, 106.25, 212.5, 425, 850 and 1700 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 13.28, 26.56, 53.13, 106.25, 212.5, 425, 850 and 1700 µg/ml.

Experiment 2
There were 2 exposure conditions conducted for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 75, 100 and 200 µg/ml.
ii) 4-hour exposure to the test item with S9-mix followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 12.5, 25, 50, 100, 150 and 200 µg/ml.

Cell harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC to ensure complete fixation.

Preparation of metaphase spreads
The lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative slide assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerised random number generator.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 6.64 to 1700 µg/ml. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure. In the 4(20)-hour exposure groups, greasy/oily precipitate was observed at and above 425 µg/ml, and at and above 212.5 µg/ml in the 24-hour continuous exposure group. Haemolysis was also observed at and above 26.56 µg/ml in all three exposure groups. Haemolysis is the toxic effect on the erythrocytes in the culture and does not normally represent toxicity to the lymphocytes but in this study there was evidence that toxicity was observed with the lymphocytes also.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 850 µg/ml in the 4(20)-hour without S9 exposure group, at 1700 µg/ml in the 4(20)-hour with S9 exposure group and up to 106.25 µg/ml in the 24-hour continuous exposure group. The mitotic index data are presented. The test item induced clear evidence of toxicity in the absence of S9.
Additionally, and at the request of the Sponsor, slides from this experiment were assessed for chromosome aberrations. Therefore, in the 24-hour exposure in the absence of S9, the negative (0 µg/ml) and 106.25 µg/ml slides were assessed for chromosome damage because the Mitotic Inhibition was 57%, close enough to the OECD 473 Test Guideline recommendation of 50%. No increase in the frequency of cells with chromosome aberrations was noted at this dose level.
The selection of the maximum dose level was therefore based on toxicity for all exposure groups in both experiments.

Chromosome aberration test - Experiment 1
The qualitative assessment of the slides determined that the toxicity was more excessive than that observed in the Preliminary Toxicity Test. There were metaphases suitable for scoring present up to 106.25 µg/ml in both the absence and presence of metabolic activation (S9). Above this concentration, the test item was too toxic to the metaphases. A greasy/oily precipitate of the test item was observed at the end of exposure, at and above 425 µg/ml in the absence of S9 and at and above 850 µg/ml in the presence of S9. Haemolysis was observed at and above 26.56 µg/ml in the absence of S9, and at and above 53.13 µg/ml in the presence of S9.

Results confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 65% mitotic inhibition and 37% mitotic inhibition were achieved at 106.25 µg/ml in the absence and presence of S9 respectively. Although the Mitotic Inhibition was in excess of 50% in the absence of S9, this dose level was assessed for chromosome damage in an effort to assess the clastogenic potential of the test item beyond that recommended by the OECD 473 Guideline due to the steep toxicity response. Additionally, no suitable metaphases for scoring were observed at and above 212.5 µg/ml in either exposure group.

The maximum dose level selected for metaphase analysis was the maximum surviving dose level of 106.25 µg/ml in both exposure groups.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation, even though a dose level in excess of 50% Mitotic Inhibition was selected in the absence of S9.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome aberration test - Experiment 2
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to the test item dose level of 100 µg/ml in the absence and presence of S9. Above this level, there was excessive toxicity excluding the higher dose levels from metaphase analysis.
No precipitate of the test item was observed at the end of exposure, at any dose level. Haemolysis was observed at the end of the exposure period at and above 50 µg/ml in both the absence and presence of S9.
In the absence of S9, there was 4%, 25% and 7% mitotic inhibition achieved at 50, 75 and 100 µg/ml. In the absence of S9, the data from the preliminary toxicity test and Experiment 1 suggested that the test item would be toxic above 106.25 µg/ml. So intermediate dose levels were included below 100 µg/ml in Experiment 2 but not between 100 and 200 µg/ml. Unexpectedly the test item exhibited very little toxicity in dose levels below 100 µg/ml. In the presence of S9, the response was not as marked with a modest 26% mitotic inhibition being observed at 50 µg/ml. However, in the presence of S9, a modest decrease in Mitotic Inhibition (37%) was observed at 106.25 µg/ml in Experiment 1 allowing the addition of two intermediate dose levels at 150 and 200 µg/ml in an effort to reach 50% Mitotic Inhibition. Unfortunately, the test item exhibited excessive toxicity above the 100 µg/ml dose level in the presence of S9 with 79% toxicity being observed at 150 µg/ml.
The maximum dose level selected for metaphase analysis was limited by toxicity to 100 µg/ml in both the absence and presence of S9.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.
Additionally, and at the request of the Sponsor, slides from the preliminary toxicity test 24-hour exposure group were assessed for chromosome aberrations. The negative control (0 µg/ml) and 106.25 µg/ml were assessed for chromosome damage because the Mitotic Inhibition observed in the 106.25 µg/ml dose level was 57%, close enough to the OECD 473 Guideline recommendation of 50%. No increase in the frequency of cells with chromosome aberrations was noted at this dose level. This gave weight to the argument that the test item had been adequately tested.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Discussion

This test item exhibited very varied toxicity over all experiments performed which was ably demonstrated by the fact that Experiment 1 was repeated. The first attempt had a dose range of 53.13, 106.25, 212.5, 425, 637.5 and 850 µg/ml in the absence of S9 and 106.25, 212.5, 425, 850, 1275 and 1700 µg/ml, in the presence of S9. These dose ranges were based on the Preliminary Toxicity Test but, unfortunately, the test item was excessively toxic at and above 212.5 µg/ml. This extensive toxicity meant there were not enough dose levels to adequately assess the test item. Therefore, the experiment was repeated using the expanded dose range detailed in the table for Experiment 1. The results of this experiment confirmed the steep toxicity of the test item where there were no analysable dose levels above 106.25 µg/ml. The steep toxicity of the test item made dose selection for Experiment 2 a challenge. In the absence of S9, the data from previous experiments suggested that the test item was toxic above 106.25 µg/ml so intermediate dose levels were included below 100 µg/ml in Experiment 2. However, in the presence of S9, a modest decrease in Mitotic Inhibition (37%) was observed at 106.25 µg/ml allowing the addition of two intermediate dose levels at 150 and 200 µg/ml in an effort to reach 50% Mitotic Inhibition. Unfortunately, the test item exhibited insufficient toxicity below 100 µg/ml in the absence of S9, and excessive toxicity above this dose level in the presence of S9. Despite this, it was considered that the test item had been adequately tested over the course of the study as we scored a dose level with excessive toxicity in both the Preliminary Toxicity Test (106.25 µg/ml in 24hr without S9, with mitotic index of 43%) and Experiment 1 (106.25 µg/ml in 4hr treatment without S9, with mitotic index of 35%). In Experiment 2, although the analysable dose levels exhibited no or only modest decreases in Mitotic Inhibition, the test item was still adequately tested because the toxicity and haemolysis observed in all the experiments confirms the cells were exposed to the test item.

Results

Mitotic Index

Mitotic Index - Experiment 1 DOSE LEVEL µg/ml 4 HOURS TREATMENT WITHOUT S9 4 HOURS TREATMENT WITH S9 A B MEAN % OF CONTROL A B MEAN % OF CONTROL 0 3.50 3.05 3.28 100 5.40 4.90 5.15 100 13.28 2.80 3.75 3.28 100 4.10 6.20 5.15 100 26.56 3.30 H 4.50 H 3.90 119 5.30 5.00 5.15 100 53.13 2.55 H 2.75 H 2.65 81 4.00 H 4.80 H 4.40 85 106.25 0.70 H 1.60 H 1.15 35 2.60 H 3.90 H 3.25 63 212.5 NM H NM H - - NM H NM H - - 425 NM H GP NM H GP - - NM H NM H - - 850 NM H GP NM H GP - - NM H GP NM H GP - - 1700 NM H GP NM H GP - - NM H GP NM H GP - - MMC 0.4 0.65 0.70 0.68 21 NA NA NA NA CP 5 NA NA NA NA 0.70 0.85 0.78 15 MMC = Mitomycin C CP = Cyclophosphamide H = Haemolysis NA  = Not applicable -         = Not assessed for mitotic index NM     = No metaphases suitable for scoring GP     = Greasy/oily precipitate observed at end of exposure period   Mitotic Index - Experiment 2 DOSE LEVEL µg/ml 24 HOURS TREATMENT WITHOUT S9 4 HOURS TREATMENT WITH S9 A B MEAN % OF CONTROL A B MEAN % OF CONTROL 0 4.35 5.20 4.78 100 3.90 3.65 3.78 100 12.5 - - - - - - - - 25 - - - - 4.20 5.10 4.65 123 50 4.30 H 4.90 H 4.60 96 3.15 H 2.45 H 2.80 74 75 3.50 H 3.70 H 3.60 75 NA NA NA NA 100 5.10 H 3.75 H 4.43 93 3.15 H 4.40 H 3.78 100 150 NA NA NA NA 0.40 H 1.20 H 0.80 21 200 NM H NM H - - NM H NM H - - MMC 0.2 1.55 1.05 1.30 27 NA NA NA NA CP 5 NA NA NA NA 0.85 0.55 0.70 19

CP      = Cyclophosphamide

MMC  = Mitomycin C

H        = Haemolysis

NA      = Not applicable

-         = Not assessed for mitotic index

NM    = Too few or no metaphases suitable for scoring

Results of Chromosome Aberration Test – Preliminary Toxicity Test 24-Hour, Without Metabolic Activation (S9)

Treatment group	Replicate	Mitotic index (%)	Number of cells scored	Number of aberrations						Total no. of aberrations		Frequency of aberrant cells (%)
				Gaps	Chromatid		Chromosome		Others	+ Gaps	- Gaps	+ Gaps	- Gaps
					Breaks	Exchanges	Breaks	Exchanges
Vehicle control (DMSO)		5.50	100	0	0	0	0	0	0	0	0	0	0
	Total	(100)	100	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)
106.25µg/mL		2.35	100	0	0	3	0	0	0	3	3	3	3
	Total	(43)	100	0	0	3	0	0	0	3	3	3 (3.0)	3 (3.0)

Results of chromosome Aberration Test- Experiment 1 Without Metabolic Activation (S9) (4 h treatment period)

Concentration (µg/mL)		Number and percentage of cells showing structural chromosome aberrations (%)							g	Cell growth index	Number and percentage of cells showing numerical aberrations (%)
		Observed	Ctb	Cte	Csb	Cse	Others	Total		Mitotic index (%)	Observed	Polyploids	Others	Total
Negative control (DMSO) 0	A	100	2	0	0	0	0	2	0	3.50	100	0	0	0
	B	100	0	0	0	0	0	0	0	3.05	100	0	0	0
	Total (%)	200 (100)	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (1.0)	0 (0.0)	(100)	200	0	0	0 (0.0)
13.28	A	100	0	0	0	0	0	0	0	2.80	100	0	0	0
	B	100	0	0	0	0	0	0	1	3.75	100	0	0	0
	Total (%)	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	(100)	200	0	0	0 (0.0)
26.56	A	100	0	0	0	0	0	0	0	3.30	100	0	0	0
	B	100	0	0	0	0	0	0	0	4.50	100	0	0	0
	Total (%)	200 (100)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	(119)	200	0	0	0 (0.0)
53.13	A	100	0	0	0	0	0	0	0	2.55	100	0	0	0
	B	100	1	0	0	0	0	1	0	2.75	100	0	0	0
	Total (%)	200 (100)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	(81)	200	0	0	0 (0.0)
106.25	A	100	1	0	0	0	0	1	0	0.70	100	0	0	0
	B	100	1	0	0	0	0	1	0	1.60	100	0	0	0
	Total (%)	200 (100)	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	(35)	200	0	0	0 (0.0)
Positive Control (MMC) 0.4	A	50^a	9	12	3	0	0	19	3	0.65	50	0	0	0
	B	50^a	9	16	0	0	1	21	0	0.70	50	0	0	0
	Total (%)	100 (100)	18 (18.0)	28 (28.0)	3 (3.0)	0 (0.0)	1 (1.0)	40*** (40.0)	3 (3.0)	(21)	100	0	0	0 (0.0)

 

 

MMC = Mitomycin C

a = slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

*** = P<0.001

Conclusions:

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

The test item was toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that was limited by test item induced toxicity. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The chromosome aberration potential of the test item, TM 12-209, was negative both with and without metabolic activation according to OECD Test Guideline 473 using an in vitro mammalian chromosome aberration method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Based on the negative result in AMES , the test item is considered non-mutagenic.

In the chromosome aberration test, the test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system, and was therefore considered non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint
The study was conducted on the target substance using an appropriate in vitro method according to internationally recognised test guidelines.

Justification for classification or non-classification

Based on the negative results in the AMES and in vitro mammalian chromosome aberration assay, classification of the test substance TM 09 -217 for genotoxicity is not warranted in accordance with EU Directive 67/548 and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.