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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 4, 1983 - August 24, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted similar to OECD guideline 476, and in compliance with GLP Standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
2 hours treatment was performed with 3 concentrations of the test compound.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 4-methoxycinnamate
EC Number:
226-775-7
EC Name:
2-ethylhexyl 4-methoxycinnamate
Cas Number:
83834-59-7
Molecular formula:
C18H26O3
IUPAC Name:
2-​Propenoic acid, 3-​(4-​methoxyphenyl)​-​, 2-​ethylhexyl ester, (2E)​-
Details on test material:
- Name of test material (as cited in study report): 2-Ethylhexyl 4-methoxycinnamate
- Physical state: liquid

Method

Target gene:
HPRT gene
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles minimal essential medium (EMEM) enriched with amino acids and vitamins but free of added thymidine and hypoxanthine, i.e. Dulbeccos medium.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: the V79 cells have a stable karyotype with a modal chromosome number of 22 ± 1
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 (from Aroclor 1254 treated male Sprague Dawley rats)
Test concentrations with justification for top dose:
5, 10 and 20 µg/mL
Vehicle / solvent:
- Solvent used: Methanol
- Justification for choice of solvent: The solvent was chosen because it proved to be relatively non-toxic.
Controls
Untreated negative controls:
yes
Remarks:
untreated cells (± S9 mix)
Negative solvent / vehicle controls:
yes
Remarks:
Methanol (± S9 mix), DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
N-dimethylnitrosamine
ethylmethanesulphonate
Remarks:
(EMS: 2.0 mg/mL (- S9 mix)), N-dimethylnitrosamine (DMN: 11.1 mg/mL (+ S9 mix)), 7,12-dimethylbenzanthracene (DMBA: 60 µM (+ S9 mix), used in parallel to DMN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): 9 days

SELECTION AGENT (mutation assays): 6-thio-guanine (6-TG)
STAIN (for cytogenetic assays): 10 % methylene blue

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 10*6

DETERMINATION OF CYTOTOXICITY
- Method: concentration related plating efficiency (PE, absolute and PE relative) (% survival)
Evaluation criteria:
A result is considered negative if the number of mutant colonies in the exposed groups to the test compound is in the range of the corresponding negative controls.
Statistics:
Mean and standard deviations; mutant frequency

Results and discussion

Test results
Key result
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:
20 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: The treatment of the cells with DMN did not induce sufficiently enhanced mutations for this positive control with metabolic activation. Therefore, DMBA was included as additional positive control. With this compound the mutation rate clearly was enhanced.
DMN also gave very variable toxicity tests response. Also because of this DMBA was taken in parallel in these experiments.

RANGE-FINDING/SCREENING STUDIES: The solubility and toxicity of the test compound was determined in a pre-experiment by establishing the concentration related plating efficiency (PE). According to these data the concentration range was chosen

ADDITIONAL INFORMATION ON CYTOTOXICITY: In a pre-experiment at concentrations higher than 20 µg/mL the test compound showed a tendency to precipitate in the medium. Therefore, 20 µg/mL was chosen as highest concentration.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
The results do not indicate a mutagenic activity of the test compound 2-Ethylhexyl 4-methoxycinnamate in the HGPRT-test with the concentrations applied: 5, 10, 20 µg/mL. Higher concentrations could not be tested because of beginning precipitation of the test compound in the culture medium. Therefore, the substance does not need to be classified as genotoxic according to the criteria outlined in Annex I of 1272/2008/EC.
Executive summary:

The sunscreen 2-Ethylhexyl 4-methoxycinnamate was evaluated for induction of mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V79, derived from Chinese hamster lung cells. Treatment of cells with 5, 10 and 20 µg 2-Ethylhexyl 4-methoxycinnamate per mL for two hours with and without a rat liver activation system did not induce mutations to 6-thio-guanine resistance. 20 µg 2-Ethylhexyl 4-methoxycinnamate per mL were not cytotoxic. No higher concentrations could be used because of precipitation of the compound. Sensitivity of the test system and activity of S-9 mix were shown by using the known mutagens ethyl methanesulfonate (EMS) and 7,12-dimethylbenzo(a)anthracene (DMBA) as reference substances. It is concluded that under the experimental conditions described in the report neither 2-Ethylhexyl 4-methoxycinnamate per se nor one of its metabolites formed by rat liver enzymes induced mutations at the HGPRT locus in Chinese hamster cells in vitro. Therefore, the substance does not need to be classified as genotoxic according to the criteria outlined in Annex I of 1272/2008/EC.

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