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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
08 Mar - 15 Apr 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study tested with the source substance (Z)-octadec-9-enyl oleate (CAS 3687-45-4). 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:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted April 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
3687-45-4 (purity: 55-70%)
IUPAC Name:
3687-45-4 (purity: 55-70%)
Details on test material:
- Name of test material (as cited in study report): oleyl oleate
- Batch No.: 00034039
- Physical state: yellow liquid
- Molecular weight: approx. 510-535
- Purity: approx. 55 - 70%
- Impurities (identity and concentrations): other wax esters
- Stability: the pure substance is stable for months
- Storage: room temperature, light protected
- Expiration date: 09 Aug 1994

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
Test concentrations with justification for top dose:
10, 30, 60 and 100 µg/mL, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells.

Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate (600 µg/mL in MEM without FCS, -S9) and 7,12-dimethylbenz(a)anthracene (3.85 µg/mL in DMSO, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)

Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)

NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time)
Evaluation criteria:
The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.

In a case-by-case evaluation the decision depends on the level of the corresponding negative control data.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at limit of water solubility at 100 µg/mL observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.

No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: mutagenicity data, experiment 1

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.2

54

9.4

 100.0

Vehicle control

-

-

1.0

66

3.9

 100.0

EMS

600

-

89.6

50

416.4

 73.8

Test substance

10

-

1.0

72

3.3

 91.8

Test substance

30

-

2.0

72

6.8

 72.0

Test substance

60

-

0.2

81

0.6

 68.1

Test substance

100

-

2.8

65

10.2

 64.2

Negative control

-

+

2.0

69

7.3

 100.0

Vehicle control

-

+

3.2

68

11.4

 100.0

DMBA

3.85

+

112.0

57

496.2

 53.1

Test substance

10

+

5.4

78

16.1

 92.4

Test substance

30

+

0.2

65

0.8

 95.6

Test substance

60

+

1.4

67

5.4

 97.3

Test substance

100

+

0.2

74

0.7

 109.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

 

Table 2: mutagenicity data, experiment 2

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.0

69

6.6

 100.0

Vehicle control

-

-

1.8

64

6.4

 100.0

EMS

600

-

145.8

60

496.9

 61.5

Test substance

10

-

4.2

62

18.4

 93.6

Test substance

30

-

1.0

63

4.1

 107.8

Test substance

60

-

3.2

60

13.5

 56.4

Test substance

100

-

0.8

59

3.5

 61.4

Negative control

-

+

2.4

60

10.3

 100.0

Vehicle control

-

+

2.2

61

9.0

 100.0

DMBA

3.85

+

141.6

64

550.4

 50.3

Test substance

10

+

1.4

61

5.7

 104.8

Test substance

30

+

2.2

72

7.5

 106.1

Test substance

60

+

0.6

69

2.1

 102.8

Test substance

100

+

1.4

79

4.4

 107.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative