2-[[(butylamino)carbonyl]oxy]ethyl acrylate

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:

2 Dec 2014 - 13 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Gyógyszerészeti és Egészségügyi Minőség- és Szervezetfejlesztési Intézet (National Institute for Quality- and Organizational Development in Healthcare and Medicines), Budapest, Hungary

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

EC Number:

264-036-0

EC Name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Cas Number:

63225-53-6

Molecular formula:

C10H17NO4

IUPAC Name:

2-[[(butylamino)carbonyl]oxy]ethyl acrylate

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Cell proliferation: doubling time 12 - 14 h
- Type and identity of media:
DME medium supplemented with:
- L-glutamine (2mM)
- 1% of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B)
- heat-inactivated bovine serum (final concentration 10 %)
During the 3 and 20 h treatments the serum content was reduced to 5%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding study:
3 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix
3 h treatment: 5, 10, 15, 30, 60, 120 and 240 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4, 8, 16 and 32 µg/mL without S9 mix

Experiment 1:
3 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix

Experiment 2:
3 h treatment: 10, 20, 40, 80 and 90 µg/mL with S9 mix
20 h treatment: 0.5, 1, 2, 4 and 4.5 µg/mL without S9 mix

4.5 µg/mL was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 20 h and 28 h; 20 h treatment: 20 h and 28 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine, 0.2 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Validity criteria for the test:
The chromosome aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data.
- the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Evaluation of test results:
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed.

A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data).
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same).
- the increase is statistically significant.
Both, biological and statistical significance should be considered together.

Statistics:

For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were as follows: number of aberration and number of cells with aberration (with and without gaps).

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 80 µg/mL and higher following the 3 h treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 4 µg/mL following the 3 h and 20 h treatment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant differences between test item treatment and control groups were observed.
- Effects of osmolality: No significant differences between test item treatment and control groups were observed.
- Precipitation: A clear solution was obtained up to a concentration of 10 mg/mL. There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection was performed. The cells were treated using increasing concentrations of test item in the absence or presence of S9 mix and were incubated at 37 °C for 3 h. Cell counts were performed after 20 h. Additional groups of cells were treated for 20 h without metabolic and for 3 h with metabolic activation, with cell counts conducted after 20 h (without S9 mix only) and 28 h (without and with S9 mix).

COMPARISON WITH HISTORICAL CONTROL DATA: In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, confirming the suitability of the cell line used.

Any other information on results incl. tables

Table 1. Results of chromosomal aberration test.

Test item	Concentration	RICC	Aberrant cells per 200 metaphase chromosome spreads
	in µg/mL	in %	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (- gaps)
Exposure period 3 h, sampling time 20 h, without S9 mix
DMSO	0.45 µL	100	4	2
EMS	1.0 µL	-	26**	21**
Test substance	0.5	97	6	2
	1	86	5	2
	2	77	5	2
	4	50	7	2
Exposure period 3 h, sampling time 20 h, with S9 mix
DMSO	9 µL	100	6	3
CP	5.0	-	35**	30**
Test substance	10	99	6	3
	20	86	7	4
	40	80	10	4
	80	50	9	6
	90	44	11	7
Exposure period 20 h, sampling time 20 h, without S9 mix
DMSO	0.45 µL	100	4	2
EMS	0.4 µL		32**	26**
Test substance	0.5	94	5	3
	1	82	4	2
	2	67	4	2
	4	49	6	3
Exposure period 20 h, sampling time 28 h, without S9 mix
DMSO	0.45 µL	100	4	2
EMS	0.4 µL	-	33**	27**
Test substance	0.5	98	4	2
	1	81	4	2
	2	74	4	2
	4	50	5	2
Exposure period 3 h, sampling time 28 h, with S9 mix
DMSO	9 µL	100	3	1
CP	5.0	-	34**	29**
Test substance	10	97	4	2
	20	92	4	2
	40	79	5	3
	80	49	7	4
	90	43	11*	7*

* (p ≤ 0.05); ** (p ≤ 0.01)

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulphonate

CP: Cyclophosphamide

RICC: Relative Increase in Cell Counts

In Experiment 1, the test item caused a moderate increase in the number of cells with structural chromosome aberrations in the presence of metabolic activation, up to and including cytotoxic concentrations. This increase was dose associated and biologically important.

In Experiment 2, the test item caused an increase in the number of cells with structural chromosome aberrations without gaps in the presence of S9 mix at concentration of 90 µg/mL following 3 h treatment. This increase was biologically and statistically significant.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.