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Genetic toxicity: in vitro

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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 guidelineopen allclose all
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
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

Constituent 1
Chemical structure
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

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