Potassium (Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Jul 2009 - 19 Feb 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-N-methyl-N-(1-oxo-9-octadecenyl)glycine

EC Number:

203-749-3

EC Name:

(Z)-N-methyl-N-(1-oxo-9-octadecenyl)glycine

Cas Number:

110-25-8

Molecular formula:

C21H39NO3

IUPAC Name:

N-methyl-N-oleoylglycine

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: in whole blood treated with heparin

Details on mammalian cell type (if applicable):

RPMI 1640 medium supplemented with
- 15% (v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (10000 U /10000 µg/mL)
- 4.8 µg/mL Phytohaemagglutinin solution in H2O
During exposure to the test substance, RPMI medium was used without FCS supplementation.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Pre-Experiment I:
4h treatment: 55.3, 110.6, 221.3, 442.5, 885, 1770 and 3540 µg/mL, with and without metabolic activation.
Pre-Experiment II:
4h treatment: 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 µg/mL, without metabolic activation.

Experiment I:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with and without metabolic activation.
Experiment II:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with metabolic activation.
22h treatment: 0.5, 1, 5, 10, 15, 20, 25, 30, 40 an 50 µg/mL, without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 35 µg/mL in 0.9% NaCl, +S9; methanesulphonate, 600 and 350 µg/mL (experiment I and II, respectively) in nutrient medium, –S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.1 pg/mL
STAIN (for cytogenetic assays): Giemsa 10% (v/v) in Soerensen buffer
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test item is classified as non-mutagenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is below 5.0 % aberrant cells, excluding gaps. No significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if: the number of induced structural chromosome aberrations is above 5.0 % aberrant cells, excluding gaps and either a concentration-related or a significant increase in the number of cells with structural chromosome aberrations is observed.

Statistics:

Statistical significance was calculated by means of the Fisher's exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: in whole blood treated with heparin

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 30 µg/mL without and from 40 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed from 221.3 µg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The applied concentrations ranged from 55.3 µg/mL to 3540 µg/mL with and without metabolic activation (exposure time 4 h, pre-experiment I). In pre-experiment II concentrations from 0.78 to 50 µg/mL were used.

ADDITIONAL INFORMATION ON CYTOTOXICITY: All concentraitons of the pre-experiment I showed cytotoxicity.

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results of cytogenicity experiments.

Test item		Concentration	Mitotic Index	Aberrant cells in %
		in µg/mL	in %	with gaps	without gaps		with exchanges
	Exposure period 4h, fixation time 22h, without S9 mix
DMSO		0.5% (v/v)	100.0	5	1		0
EMS		600	48.4	23	21.5*		2.5
Test substance		10	94.0	2.5	1.5		0
		20	70.2	3	2		0.5
		30	50.4	0.5	0		0
	Exposure period 4h, fixation time 22h, with S9 mix
DMSO		0.5% (v/v)	100	2	2		0
CPA		35	77.2	21.5	16.5*		0.5
Test substance		15	82.6	2.5	2		0
		30	39.1	2.5	1.5		0.5
		40	18.8	3	2		0
	Exposure period 22h, fixation time 22h, without S9 mix
DMSO		0.5% (v/v)	100.0	0	0		0
EMS		350	44.8	39.5	33.5*		4.5
Test substance		50	97.1	5.5	2		0
		100	77.6	3.5	2		0
		125	36.8	2	1		0
	Exposure period 4h, fixation time 22h, with S9 mix
DMSO		0.5% (v/v)	100.0	1.5	1.5		0
CPA		35	70.1	33	22*		1
Test substance		25	94.8	3.5	2		0
		35	69.7	3.5	3		0
		40	29.5	1	1		0

*:Aberration frequency statistically significant higher than corresponding control values

CPA: Cyclophosphamide

EMS: Ethyl methanesulphonate

Applicant's summary and conclusion

Conclusions:

Intgerpretation of results: negative