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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:
key study
Study period:
March 4th, 1994 - June 20th, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Oleyloleate . In accordance 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:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Fatty acids, C18-unsatd., C16 and C18-unsatd. alkyl esters
EC Number:
297-617-2
EC Name:
Fatty acids, C18-unsatd., C16 and C18-unsatd. alkyl esters
Cas Number:
93685-70-2
IUPAC Name:
93685-70-2
Details on test material:
- Name of test material (as cited in study report): Oleyloleate
- Batch No.: 00034039
- Physical state: yellow liquid
- Molecular weight: approx. 510-535
- Purity: approx. 55 - 70 %
- Impurities (identity and concentrations): other wax esters
- Stability: pure: stable for months; in solvent: not indicated by the sponsor
- Storage: room temperature, light protected
- expiration date: August 9th, 1994

Method

Target gene:
The test was used to detect the potential of a test material to induce structural chromosomal aberrations in the Chinese hamster cell line V79.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
- doubling time of clone V79/T5 in stock cultures: 12 h as determined on April 29th, 1993.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver microsomal fraction); 50 µL /mL cell culture was used.
Test concentrations with justification for top dose:
With and without S9 mix:
18 h: 1, 3, 10, 30, 60, 100 µg/mL
28 h: 10, 30, 60, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
On the day of the experiment (immediately before treatment), the test substance was dissolved in ethanol (E. Merck, Darmstadt, Germany, purity > 99.8 %). The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate (final conc.: 600 µg/mL, without metab. act.) and cyclophosphamide (final conc.: 0.47µg/mL, with metab. act.)
Remarks:
purity of control substances were > 98 %
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4h with metabolic activation, 18 and 28 h without metabolic activation
- Expression time (cells in growth medium): Preparation interval was 18 and 28 hours for all experiments. Therefore, for the cells exposed for 4 h with metabolic activation, the medium was changed to complete medium after the exposure for the remaining time up to preparation. The cells treated without metabolic activation were kept for the whole exposure duration of 18 or 28 hours in medium containing the test substance.
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 h after adding colcemid (at 18 and 28 h)

SPINDLE INHIBITOR (cytogenetic assays): 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: at least 100 well spreach metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
up to 100 µg/mL no substantial reduction of mitotic indices was found.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, the number of polyploid cells was scored (% polyploid metaphases)
- Determination of endoreplication: no data
Evaluation criteria:
Significant increases of the number of cells with structural chromosomal aberrations (outside the historical range of 0.00 - 4.00 %).
The test material is considered as mutagenic, if it includes either a concentration-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
The test material neither producing a concentration-related increase in the number of structural chromosomal aberrations nor a significant and positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
chi-square test at five per cent level (p < 0.05)

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 occured at concentrations > 100 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: limited
Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES/ADDITIONAL INFORMATION ON CYTOTOXICITY:
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.
No toxic effects were observed, however a slight precipitation occured a this concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
Range of historical control data: 0.00 - 4.00 %

In the absence and presence of S9-mix, the mitotic indices were not substantially reduced after treatment with the highest evaluated concentration at each fixation interval.
In the cytogenetic experiment, at both fixation intervals in the absence and presence of S9-mix, the test material did not increase the frequency of cells with aberrations to a biologically and statistically relevant extent. No biological relevant increase of polyploid metaphases as compared to the rates of the controls was found after treatment with the test material.
EMS and CPA were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

Any other information on results incl. tables

Table 1: Number of Polyploid Cells and Mitotic Index

 

conc.

per mL

Exposure

duration

(hours)

S9

mix

fixation

interval

mean number

of polyploid cells*

mitotic

index**

(%)

neg. control

 

4

-

18

1.5

100.0

vehicle control

1.0 %

4

-

18

1.5

100.0

EMS

600 μg

4

-

18

3.0

68.9

test substance

10 μg

4

-

18

1.0

83.4

test substance

60 μg

4

-

18

2.5

100.0

test substance

100 μg

4

-

18

2.0

89.7

neg. control

 

18

+

18

2.0

100.0

vehicle control

1.0 %

18

+

18

3.0

100.0

CPA

0.47 μg

18

+

18

3.0

63.0

test substance

10 μg

18

+

18

1.5

127.1

test substance

60 μg

18

+

18

2.0

113.7

test substance

100 μg

18

+

18

2.0

104.3

vehicle control

1.0 %

4

-

28

4.0

100.0

test substance

100 μg

4

-

28

5.0

119.0

vehicle control

1.0 %

28

+

28

2.5

100.0

test substance

100 μg

28

+

28

2.5

91.5

* The number of polyploid cells was determined in 100 cells per culture of each test group.

** The mitotic index was determined in 1000 cells per culture of each test group.

 

Table 2: Structural chromosomal aberrations

 

conc.

per mL

S9

mix

fixation

interval

cells scored

aberrant cells

(%mean)

exchanges

incl.

gaps

excl.

gaps

neg. control

 

-

18

200

0.0

0.0

0.0

vehicle control

1.0 %

-

18

200

1.5

0.5

0.0

EMS

600 μg

-

18

200

23.5

22.0

16.5

test substance

10 μg

-

18

200

1.5

0.5

0.0

test substance

60 μg

-

18

200

1.0

1.0

1.0

test substance

100 μg

-

18

200

5.0

3.0

0.0

neg. control

 

+

18

200

1.0

1.0

0.0

vehicle control

1.0 %

+

18

200

4.0

3.5

0.5

CPA

0.47 μg

+

18

200

23.0

22.0

11.0

test substance

10 μg

+

18

200

3.0

1.0

0.0

test substance

60 μg

+

18

200

2.0

2.0

1.5

test substance

100 μg

+

18

200

3.0

1.5

0.0

vehicle control

1.0 %

-

28

200

4.0

2.0

0.0

test substance

100 μg

-

28

200

3.5

2.0

0.0

vehicle control

1.0 %

+

28

200

4.0

2.5

0.5

test substance

100 μg

+

28

200

4.0

2.0

1.0

EMS = Ethylmethanesulphonate

CPA = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Under the given conditions the test substance did not induce significant increases of chromosomal aberrations in V79 chinese hamster cells in vitro.

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