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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro transformation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 7, 1984- March 29, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test was conducted similar to IARC/NCI/EPA Working Group (1985) Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for the prediction of carcinogenic chemicals: overview and recommended protocols, Cancer Res., 45, 2395-2399., and under GLP Standards, and QA.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.21 (In Vitro Mammalian Cell Transformation Test)
Version / remarks:
1988
Deviations:
yes
Remarks:
Balb/c 3T3 cells used and in absence of metabolic activation system
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-Ethylhexyl 4-methoxycinnamate
- Physical state: liquid
- Analytical purity: no data
- Storage condition of test material: 4°C

Method

Target gene:
Not relevant
Species / strain
Species / strain / cell type:
mammalian cell line, other: Balb/c 3T3 clone A31-11
Details on mammalian cell type (if applicable):
- Type and identity of media:
Growth media: EMEM-10: Minimal Essential Medium with Earle salts (EMEM) (Gibco). 10 % serum HJ, L-glutamine, NaHCO3
EMEM-5: 5 % serum HJ, L-glutamine, NaHCO3
Treatment medium: EMEM-5
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Metabolic activation system:
Not applicable (Balb/c 3T3 clone A31-11 have intrinsic metabolic activation)
Test concentrations with justification for top dose:
1.25, 2.5, 5.0, 7.5, 10.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 20-Methylcholanthrene (MC)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: about 48 hrs.
- Exposure duration: 3 days
- Expression time (cells in growth medium): about 4 weeks
- Fixation time (start of exposure up to fixation or harvest of cells): 4 to 5 weeks

STAIN (for cytogenetic assays): 10 % Giemsa, 50 % May-Grünwald

NUMBER OF REPLICATIONS: 8 or 15 petri dishes per dose

DETERMINATION OF CYTOTOXICITY
- Method: other: cell survival for each transformation assay

OTHER EXAMINATIONS:
- Other: Determination of transformed foci: Only Type III foci were taken into consideration
Evaluation criteria:
Petri dishes containing at least one Type III focus were taken as positive dishes.
Statistics:
In vitro cell transformation assay: No. of Petri Dishes Cell containing Type III Foci
Cell survival: Mean

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: Balb/c 3T3 clone A31-11
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
10.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: In this test system 2-Ethylhexyl 4-methoxycinnamate was cytotoxic. A 3-day exposure period with 10.0 µg/mL reduced survival of Balb/c 3T3 cells to 63 and 45 percent (first batch) and 41 and 53 percent (second batch).
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
It is concluded that under the experimental conditions described in the report 2-Ethylhexyl 4-methoxycinnamate did not induce mammalian cell transformation in vitro, for both batches tested. 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 UVB sunscreen 2-Ethylhexyl 4-methoxycinnamate was evaluated for its ability to induce mammalian cell transformation in vitro. Sensitivity of the test system was shown by using the well-known carcinogen 20-methylcholanthrene (MC) known to induce transformed mammalian cells in vitro. Treatment of Balb/c 3T3 cells with 1.3 µg to 10 µg 2-Ethylhexyl 4-methoxycinnamate per mL for 3 days did not induce cell transformation in vitro, for both batches tested. 10 µg/mL were taken as highest dose tested throughout the experiments because at this concentration survival of Balb/c 3T3 cells was reduced to 50 percent related to the concurrent control cultures. It is concluded that under the experimental conditions described in the report, 2-Ethylhexyl 4-methoxycinnamate did not induce cell transformation in mammalian 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.