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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09 Apr - 15 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-guideline study, tested with the source substance isopropyl laurate (CAS 10233-13-3). According to the ECHA guidance document 'Practical guide 6: How to report read-across and categories' (ECHA, 2012), 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
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(adopted July 21, 1997).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopropyl laurate
EC Number:
233-560-1
EC Name:
Isopropyl laurate
Cas Number:
10233-13-3
Molecular formula:
C15H30O2
IUPAC Name:
isopropyl laurate
Details on test material:
- Name of test material (as cited in study report): Isopropyl Laurate
- Physical state: clear colorless liquid
- Analytical purity: 99.4±0.2%
- Lot/batch No.: 7000876+7000948 combined
- Expiration date of the lot/batch: 4 February 2012
- Storage condition of test material: stable at RT in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
3 h exposure time: 10, 33 and 100 µg/mL (with and without S9 mix)
24 h exposure time: 66, 150 and 250 µg/mL (without S9 mix)
48 h exposure time: 3, 125 and 150 µg/mL (without S9 mix)
(At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (10 µg/mL, with S9); mitomycin C (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 without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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

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 was considered positive (clastogenic) in the chromosome aberration test; it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations; 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 was 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 / choice of top concentrations:
cytotoxicity
Remarks:
at 150 µg/mL (exposure period 24 h, fixation time 24 h, -S9 mix) and at 125 µg/mL (exposure period 48 h, fixation time 48 h, -S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium
- Polyploidy did also not occur in a significant way and was also observed in control group.

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, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

COMPARISON WITH HISTORICAL CONTROL DATA: Negative controls were in range of the historical control data from experiments performed between January 2007 and December 2009.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Cytotoxic and Genotoxic observations

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

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

Ethanol

1.0% (v/v)

100

6

4

MMC

0.5

97

56

56

Test substance

10

97

6

1

33

92

4

2

100

81

2

2

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

Ethanol

1.0% (v/v)

100

7

3

CP

0.5

59

52

50

Test substance

10

101

7

7

33

96

2

2

100

91

5

3

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

Ethanol

1.0% (v/v)

100

3

3

MMC

0.2

86

45

45

Test substance

66

96

1

1

150

55

4

4

250

41

2

2

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

Ethanol

1.0% (v/v)

100

3

3

MMC

0.1

89

56

56

Test substance

3

94

6

6

125

70

3

3

150

46

5

5

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

Ethanol

1.0% (v/v)

100

2

2

CP

10.0

--

42

42

Test substance

10

99

0

0

33

96

0

0

100

95

1

1

MMC: Mitomycin          

CP: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative