Ethyl 2-chloroacetoacetate

Genetic toxicity: in vitro

Administrative data

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:

2010-03-01 till 2010-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline 473

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-chloroacetoacetate

EC Number:

210-180-4

EC Name:

Ethyl 2-chloroacetoacetate

Cas Number:

609-15-4

Molecular formula:

C6H9ClO3

IUPAC Name:

ethyl 2-chloro-3-oxobutanoate

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment IA: 11.4, 20.0, 34.9, 61.1, 106.9, 187.1, 327.5, 573.1, 1002.9, 1755.0 µg/mL


Without metabolic activation:
Experiment IA: 1.1, 2.0, 3.5, 6.1, 10.7, 18.7, 32.7, 57.1, 100.0, 175.0 µg/mL
Experiment IB: 5.0, 10.0, 15.0, 17.5, 20.0, 22.5, 25.0, 27.5, 30.0, 40.0, 50.0, 75.0, 100.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment IA the exposure period was 4 hours with and without metabolic activation. In Experiment IB the exposure period was 4 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: 250 per culture

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in at least 250 metaphase cells (% polyploid metaphases) was scored.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item EACA, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
In Experiment IA the exposure period was 4 hours with and without S9 mix. Experiment IA was repeated because the highest evaluated concentration did not achieve a reduction in mitotic index below 50% of the control. In Experiment IB the exposure period was 4 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. IA & IB) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations and at least 250 metaphases per culture were analysed for numerical aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 1755.0 µg/mL (approx. 10 mM) was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the molecular weight and the purity (93.3 %) of the test item.
No visible precipitation of the test item in the culture medium was observed at the end of treatment (up to and including the highest concentration 1755 µg/mL). No relevant influence in the osmolarity or pH value was observed (Exp. IA: solvent control: 399 mOsm, pH 7.4 versus 406 mOsm and pH 7.2 at 175.0 µg/mL; Exp. IB: solvent control: 406 mOsm, pH 7.4 versus 410 mOsm and pH 7.3 at 100.0 µg/mL).
In Experiment IA in the absence and presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage due to missing and / or low number of metaphases and / or bad metaphase quality. The test item showed a steep dose-cytotoxicity course making it difficult to comply with the criterion to have evaluable concentration with a reduced mitotic index below 50% of the control. In Experiment IB in the absence of S9 mix, the mitotic index was markedly reduced to 59.2 % of control at the highest evaluated concentration. Concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.
In Experiment IA in the absence of S9 mix no relevant increase in the number of aberrant cells was observed. The aberration rate of the cells after treatment with the test item (1.0 - 2.0 % aberrant cells, excluding gaps) was slightly above the solvent control value (1.0 % aberrant cells, excluding gaps) and in the range of the laboratorys historical solvent control data (0.0 -3.5 % aberrant cells, excluding gaps). In Experiment IA in the presence of S9 mix clastogenicity was observed after treatment with 20.0 and 34.9 µg/mL of the test item (4.0 and 21.5 % aberrant cells, excluding gaps, respectively). A dose-dependent increase was observed. In addition, the number of cells carrying exchanges increased in a dose-dependent manner (0, 1 and 5 %).
In Experiment IB in the absence of S9 mix statistically significant increases were observed after treatment with 27.5 and 40.0 µg/mL of the test item (8.5 and 6.0 % aberrant cells, excluding gaps, respectively). All values clearly exceeded the range of the laboratorys historical solvent control data (without S9 mix: 0.0 - 3.5 % aberrant cells, excluding gaps). Additionally, slight increases in cells carrying exchanges were observed in Experiment IB.
In both experiments in the absence of S9 mix, single values for polyploidies were slightly above the historical control range (0.0 - 0.4 %).
In both experiments, either EMS (825.0 µg/mL) or CPA (7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. The solvent controls were within the historical control range.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the chromosomal aberration study with EACA

Exp.	Preparation	Test item	Polyploid	Mitotic indices	Aberrant cells
	interval	concentration	cells	in %	in %
		in µg/mL	in %	of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
IA	22 hrs	Solvent control^1	0.2	100.0	1.5	1.0	0.0
		Positive control^2	0.4	126.2	18.0	16.0^S	1.0
		6.1	0.2	97.3	1.0	1.0	0.0
		10.7	0.0	88.9	1.5	1.5	0.0
		18.7	0.7**	69.3	2.0	2.0	0.0
IB	22 hrs	Solvent control^1	0.0	100.0	1.5	1.5	0.0
		Positive control^2	0.0	66.9	9.0	8.5^S	0.5
		25.0	0.6**	70.8	2.5	2.5	0.5
		27.5^#	0.4	78.9	8.5	8.5^S	1.0
		30.0^#	0.5**	82.0	3.8	3.8	0.3
		40.0^#	0.0	59.2	6.0	6.0^S	0.3
Exposure period 4 hrs with S9 mix
IA	22 hrs	Solvent control^1	0.0	100.0	1.0	1.0	0.0
		Positive control^3	0.0	64.9	17.0	16.5^S	4.0
		11.4	0.2	76.4	1.0	0.5	0.0
		20.0	0.4	79.0	4.5	4.0^S	1.0
		34.9	0.2	85.6	21.5	21.5^S	5.0

*  Including cells carrying exchanges

** Evaluation of 500 metaphases per culture

^#   Evaluation of 200 metaphases per culture

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Ethanol 0.5 % (v/v)

²     EMS 825.0 µg/mL

³   CPA     7.5 µg/mL

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced chromosomal aberrations in human lymphocytes in vitro.
Therefore, EACA is considered to be clastogenic in this chromosome aberration test, when tested up to the highest evaluable concentrations.

Executive summary:

The test item EACA, dissolved in ethanol, was assessed for its potential to induce structural and numerical chromosomal aberrations in human lymphocytes in vitro. The following study design was performed:

	Without S9 mix	With S9 mix
	Exp.IA & IB	Exp. IA
Exposure period	4 hrs	4 hrs
Recovery	18 hrs	18 hrs
Preparation interval	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were scored for structural chromosomal aberrations and at least 250 metaphases were scored for numerical aberrations.

The highest applied concentration in the pre-test on toxicity (1755.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (93.9 %) of thetest item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473.

In in the absence and presence of S9 mix, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. The test item showed a steep dose-cytotoxicity course. In Experiment IB in the absence of S9 mix, the mitotic index was markedly reduced below 60 % of control at the highest evaluated concentration. Concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In the absence of S9 mix no clastogenicity was observed. In the presence of S9 mix clastogenicity was observed after treatment with 20.0 and 34.9 µg/mL of the test item (4.0 and 21.5 % aberrant cells, excluding gaps, respectively). A dose-dependent increase was observed. In addition, the number of cells carrying exchanges increased in a dose-dependent manner (0, 1 and 5 %).

In Experiment IB in the absence of S9 mix statistically significant increases in chromosomal aberrations were observed after treatment with 27.5 and 40.0 µg/mL of the test item (8.5 and 6.0 % aberrant cells, excluding gaps, respectively). All values clearly exceeded the range of the laboratorys historical solvent control data (without S9 mix: 0.0  3.5 % aberrant cells, excluding gaps). Additionally, slight increases in cells carrying exchanges were observed in Experiment IB.

In both experiments in the absence of S9 mix, single values for polyploidies were slightly above the historical control range (0.0 0.4 %).

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. The negative control values were well within the historical control ranges.