2-Ethylhexyl trans-4-methoxycinnamate

Genetic toxicity: in vitro

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.