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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
09 Apr - 21 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance CAS 111-82-0. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl laurate
- Physical state: clear colourless liquid
- Analytical purity: 99.33%
- Lot/batch No.: SME02/1301/K
- Expiration date of the lot/batch: 1 February 2011
- Storage condition of test material: stable at room temperature in the dark under nitrogen

Method

Target gene:
not applicable
Species / strain
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation)
- Other: blood was collected from healthy adult, non-smoking, male volunteers
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:
3h treatment: 33, 100 and 200 µg/mL
24h treatment: 100, 120 and 140 µg/mL
48h treatment: 30, 120 and 140 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix

Migrated to IUCLID6: 10 µg/mL
Negative controls:
yes
Solvent controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix

Migrated to IUCLID6: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h treatment: 24 and 48 h; 24h treatment: 24 h; 48 h treatment: 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

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 substance is considered positive (clastogenic) in the chromosome aberration test if: a) it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations b) and a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance is considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test, one-sided, p < 0.05

Results and discussion

Test results
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: as specified above
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 200 µg/mL methyl laurate precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES
The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, i.e., inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

COMPARISON WITH HISTORICAL CONTROL DATA
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

Any other information on results incl. tables

Table 1: Cytotoxic and genotoxic observations

Test item

Concentration

Mitotic Index

Aberrant cells in % (200 cells were scored)

 

in µg/mL

in %

with gaps

without gaps

Exposure period 3h, fixation time 24h, without S9 mix

Ethanol

1.0% (v/v)

100

0.5

0.5

MMC

0.5

64

29*

29*

Test substance

33

90

0

0

100

75

2

1.5

200

66

2

1.5

Exposure period 3h, fixation time 24h, with S9 mix

Ethanol

1.0% (v/v)

100

0.5

0.5

CP

10

54

21*

21*

Test substance

33

100

0.5

0

100

89

1

1

200

66

0.5

0

Exposure period 24h, fixation time 24h, without S9 mix

Ethanol

1.0% (v/v)

100

2

1.5

MMC

0.2

54

47.3*c)

46*c)

Test substance

100

99

0

0

120

80

2.5

2

140

42

2.5

2

Exposure period 48h, fixation time 48h, without S9 mix

Ethanol

1.0% (v/v)

100

1

0.5

MMC

0.1

102

60*b)

59*b)

Test substance

30

93

0

0

120

59

0.5

0.5

140

44

1

0.5

Exposure period 3h, fixation time 48h, with S9 mix

Ethanol

1.0% (v/v)

100

1

1

CP

10

-b)

52*b)

51*b)

Test substance

30

103

1

0.5

100

101

1

0.5

200

106

0

0

MMC: Mitomycin C

CP: Cyclophosphamide

a): CP was fixed after 24 hours. The mitotic index could not be calculated as percentage of control

b): 100 cells were scored

c): 150 cells were scored

*: Significantly different from control group (Chi-square test), P < 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative