Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames): not mutagenic (OECD 471).
- In vitro mammalian cell transformation assay: not genotoxic (IARC/NCI/EPA Working Group (1985).
- In vitro DNA damage and repair assay (unscheduled DNA synthesis (UDS) in mammalian cells): not mutagenic (OECD 482).
- In vivo cytogenicity test (Mammalian Erythrocyte Micronucleus Test): not mutagenic, not clastogenic (OECD 474).

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May 16, 1983 - August 30, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted similar to OECD Test Guideline No. 473, under GLP Standards
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Treatment with S-9: 2 hrs.; without S-9 24 hrs.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S-9 liver homogenate fraction from Füllinsdorf albino rats
Test concentrations with justification for top dose:
Without S-9 metabolizing system: 2, 10 and 20 µg/mL for 24 hours.
With S-9 metabolizing system: 5, 25 and 50 µg/mL for 2 hours.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Remarks:
+S9: 2 cultures undosed
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S-9 metabolizing system: 50 µg/mL (2 hrs)
Untreated negative controls:
yes
Remarks:
- S9: 3 cultures undosed
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
direct-acting mutagen
Positive control substance:
other: Bleomycin
Remarks:
Without S-9 metabolizing system: 10 µg/mL (24 hrs)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Incubation in medium

DURATION:
Without S-9 metabolizing system (- S9):
- Preincubation period:
- Exposure duration: 24 hrs. treatment
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.
With S-9 metabolizing system (+ S9):
- Preincubation period:
- Exposure duration: 2 hrs. treatment
- Expression time (cells in growth medium): 22 hrs. recovery
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.15 µg/mL., last 3 hrs. of incubation
STAIN (for cytogenetic assays): Giemsa solution 10 min, 5 %

NUMBER OF REPLICATIONS:
- S9: 3 cultures at each test substance concentration, 3 for negative (undosed), 3 for solvent (DMSO), and 3 for positive (Bleomycin) controls
+ S9: 3 cultures at each test substance concentration, 2 for negative (undosed), 2 for solvent (DMSO), 2 for S9-mix control, and 3 for positive (Cyclophosphamide) control

NUMBER OF CELLS EVALUATED: 200 metaphases at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: other: Preliminary experiments: viability of cells
Evaluation criteria:
The test article is considered negative if cultures treated with 2-Ethylhexyl 4-methoxycinnamate exhibited no increase in the incidence of chromosomal damage when compared with the concurrent control groups (lymphocyte control, solvent control or S-9 mix control), either in the presence or absence of metabolic activation.
Statistics:
Statistical evaluation of the results was performed according to Kastenbaum M.A. and Bowman K.O. (1970): Tables for determining the statistical significance of mutation frequencies. Mutation Research 9, 527 - 549.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Top concentration: - S9: 20.0 µg/mL; + S9: 50.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The concentration range to be evaluated was selected on the basis of cytotoxicity (based on cell viability).
Top concentration were 20.0 µg/mL (- S9) and 50.0 µg/mL (+ S9).

Remarks on result:
other: all strains/cell types tested
Conclusions:
2-Ethylhexyl 4-methoxycinnamate did not induce chromosomal aberrations in human lymphocytes in vitro at concentrations of 2, 10 and 20 µg/mL in the absence of metabolic activation, nor in the presence of metabolic activation at concentrations of 5, 25 and 50 µg/mL. 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 a potential induction of chromosomal aberrations in vitro in human peripheral blood lymphocytes in the presence and in the absence of metabolic activation. For metaphase analysis without metabolic activation, the human lymphocyte cultures were treated with 2, 10 and 20 µg 2-Ethylhexyl 4-methoxycinnamate per mL medium for 24 hours. For metaphase analysis with metabolic activation by an aroclor 1254-induced S-9 liver homogenate fraction from Füllinsdorf albino rats, the human lymphocytes were treated with 5, 25 and 50 µg 2-Ethylhexyl 4-methoxycinnamate per mL medium for 2 hours. Cultures treated with 2-Ethylhexyl 4-methoxycinnamate exhibited no increase in the incidence of chromosomal damage when compared with the concurrent control cultures (lymphocyte control, solvent control or S-9 mix control), either in the presence or absence of metabolic activation. Higher concentrations of the compound could not be tested due to its cytotoxicity. Sensitivity of the test system could be demonstrated with the positive control compounds bleomycin as a direct acting mutagen and cyclophosphamide as an indirect acting mutagen. It is therefore concluded that under the experimental conditions described in this report, 2-Ethylhexyl 4-methoxycinnamate did not show any evidence of mutagenic activity when tested with cultured human lymphocytes either in the presence or absence of metabolic activation. Therefore, the substance does not need to be classified as genotoxic according to the criteria outlined in Annex I of 1272/2008/EC.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting 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
Qualifier:
equivalent or similar to
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
Target gene:
HPRT gene
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.
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
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
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.

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.
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
Target gene:
Not relevant
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
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
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
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.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 10, 1985 - October 7, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted similar to OECD guideline 482, and in compliance with GLP Standards
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Version / remarks:
1986
Deviations:
no
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Species / strain / cell type:
hepatocytes: rat-freshly prepared cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Williams Medium E (WME)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Metabolic activation system:
intrinsic metabolic activity
Test concentrations with justification for top dose:
2.5, 5.0, 7.5, 10.0, 15.0, 20.0 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Two experiments with different durations were conducted:
- Preincubation period:
Experiment U31: 22 hrs.
Experiment 90M85: 3 hrs.
- Exposure duration:
Experiment U31: 5 hrs.
Experiment 90M85: 18 hrs.
- Expression time (cells in growth medium): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment U31: 5 hrs. + 15 min.
Experiment 90M85: 18 hrs. + 15 min.

STAIN (for cytogenetic assays): Autoradiography: 2 % orcein

NUMBER OF CELLS EVALUATED:
Experiment U31: 50/dose
Experiment 90M85: 100/dose
All cells being in the S-phase (scheduled DNA synthesis) were excluded from evaluation.

DETERMINATION OF CYTOTOXICITY
- Method: Viability of the cells after treatment was determined by in situ trypan blue exclusion.

OTHER: The experiments were carried out avoiding sun light.
Evaluation criteria:
A positive result is a statistically significant dose-related increase in radiolabel incorporation, expressed as grains per nucleus (Net Nuclear Grain Counts).
Statistics:
Mean and medians. The nuclear grain counts, the cytoplasmic grain counts and the net nuclear grain counts are evaluated by the following significant tests:
Step I: Jonckheere-test (Jonckheere. A.R. (1954): A distribution free k-sample test against ordered alternatives. Biometrika 41. 133-145).
Step II: Kruskal-Wallis test: Test against all alternatives: (Siegel. S. (1956): Nonparametric statistics. McGraw-Hill, New York).
Mann-Whitney U-Test.
Key result
Species / strain:
hepatocytes: rat-freshly prepared cells
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
20 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: 2-Ethylhexyl 4-methoxycinnamate was slightly cytotoxic in this test system. 5 and 18 hours treatment with 20 µg/mL reduced cell viability to 71 and 86 percent (viability of the concurrent solvent control cultures was taken as 100 percent).
Remarks on result:
other: all strains/cell types tested
Conclusions:
Under the experimental conditions described, 2-Ethylhexyl 4-methoxycinnamate did not induce DNA damage resulting in unscheduled DNA synthesis in cultured rat hepatocytes. 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 UV-B sunscreen 2-Ethylhexyl 4-methoxycinnamate was tested for its ability to induce DNA damage as measured by autoradiographic evaluation of the 3H-thymidine incorporation into the nuclear DNA.

Neither 5 nor 18 hours treatment of cultured rat hepatocytes with 2.5 µg to 20 µg 2-Ethylhexyl 4-methoxycinnamate per ml induced significant changes in the nuclear labeling of the cells.

Sensitivity of the test system and responsiveness of the primary cultures were demonstrated by treating the hepatocytes with 2-acetyl aminofluorene as reference substance. The known genotoxic agent requiring metabolic activation significantly induced unscheduled DNA synthesis.

20 µg/mL were taken as highest dose tested because at this concentration survival of hepatocytes was reduced.

It is concluded that under the experimental conditions described, 2-Ethylhexyl 4-methoxycinnamate did not induce DNA damage resulting in unscheduled DNA synthesis in freshly prepared rat hepatocytes. Therefore, the substance does not need to be classified as genotoxic according to the criteria outlined in Annex I of 1272/2008/EC.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 27 - May 21, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study conducted under GLP conditions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Different loci of the bacterial histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
supernatatnt of rat liver homogenates (S9) from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: commonly used solvent
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide; 2-nitrofluorene; 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF EXPERIMENTS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
According to OECD TG 471
Statistics:
X²-test
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Experiment AM026991: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

without metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates ± standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

34 ± 3

145 ± 8

359 ± 22

16 ± 3

9 ± 4

Solvent control

0

25 ± 4

137 ± 13

337 ± 4

24 ± 3

12 ± 4

 

HR 99/9040020/1

50

29 ± 4

127 ± 7

303 ± 28

22 ± 6

8 ± 3

HR 99/9040020/1

150

28 ± 6

128 ± 12

301 ± 17

18 ± 3

12 ± 5

HR 99/9040020/1

500

29±3

130±15

312±24

21±3

11±3

HR 99/9040020/1

1500 - P

29 ± 5

141 ± 1

315 ± 23

19 ± 5

12 ± 4

HR 99/9040020/1

5000 - P

30 ± 5

129 ± 12

306 ± 12

19 ± 4

9 ± 3

 

Sodium azide

0.5

 

336 ± 15

 

 

 

Sodium azide

0.7

 

 

 

453 ± 41

 

2-nitrofluorene

2.5

331 ± 12

 

 

 

 

9-aminoacridine

50

 

 

 

 

273 ± 64

Mitomycin C

0.15

 

 

1061 ± 38

 

 

 

Experiment AM026991: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

with metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates ± standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

30 ± 2

119 ± 8

339 ± 2

24 ± 3

13 ± 5

Solvent control

0

33 ± 3

105 ± 10

315 ± 44

20 ± 5

10 ± 3

 

HR 99/9040020/1

50

37 ± 7

124 ± 4

344 ± 7

26 ± 7

9 ± 4

HR 99/9040020/1

150

47 ± 6

132 ± 10

352 ± 10

25 ± 5

10 ± 1

HR 99/9040020/1

500

31 ± 10

104 ± 2

366 ± 22

17 ± 3

12 ± 5

HR 99/9040020/1

1500 - P

41 ± 1

146 ± 22

348 ± 11

19 ± 4

12 ± 3

HR 99/9040020/1

5000 - P

33 ± 5

124 ± 10

322 ± 17

16 ± 8

15 ± 4

 

2-amino-anthracene

0.7

777 ± 27

763 ± 26

 

 

 

2-amino-anthracene

2.0

 

 

1192 ± 96

631 ± 52

414 ± 34

 

Experiment AM026992: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

without metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates ± standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

29 ± 7

114 ± 9

240 ± 9

23 ± 2

5 ± 2

Solvent control

0

23 ± 6

115 ± 13

251 ± 15

24 ± 7

9 ± 4

 

HR 99/9040020/1

50

23 ± 4

118 ± 8

241 ± 19

25 ± 3

10 ± 6

HR 99/9040020/1

150

21 ± 5

115 ± 5

233 ± 12

24 ± 5

9 ± 3

HR 99/9040020/1

500

21 ± 5

103 ± 8

238 ± 5

24 ± 3

10 ± 3

HR 99/9040020/1

1500 - P

28 ± 4

114 ± 9

233 ± 18

27 ± 3

11 ± 6

HR 99/9040020/1

5000 - P

22 ± 1

105 ± 9

234 ± 9

28 ± 4

10 ± 2

 

Sodium azide

0.5

 

468 ± 37

 

 

 

Sodium azide

0.7

 

 

 

542 ± 44

 

2-nitrofluorene

2.5

495 ± 47

 

 

 

 

9-aminoacridine

50

 

 

 

 

238 ± 32

Mitomycin C

0.15

 

 

671 ± 18

 

 

 

Experiment AM026992: Induction of revertants by HR 99/9040020/1 (2-Ethylhexyl 4-methoxycinnamate);

with metabolic activation

 

Substance

Concentration

(µg/plate)

Number of revertants/plate

(mean of 3 replicates ± standard deviation)

TA98

TA100

TA102

TA1535

TA1537

Control

0

44 ± 9

96 ± 6

273 ± 16

19 ± 4

14 ± 4

Solvent control

0

35 ± 4

86 ± 4

276 ± 14

15 ± 3

13 ± 2

 

HR 99/9040020/1

50

46 ± 7

98 ± 6

288 ± 50

14 ± 6

16 ± 5

HR 99/9040020/1

150

40 ± 7

94 ± 10

279 ± 36

13 ± 3

22 ± 3

HR 99/9040020/1

500

35 ± 5

101 ± 7

292 ± 7

15 ± 3

16 ± 4

HR 99/9040020/1

1500 - P

46 ± 7

100 ± 10

320 ± 10

20 ± 4

13 ± 4

HR 99/9040020/1

5000 - P

35 ± 9

103 ± 12

273 ± 9

16 ± 6

13 ± 4

 

2-amino-anthracene

0.5

928 ± 40

637 ± 19

 

 

 

2-amino-anthracene

0.7

 

 

1301 ± 85

635 ± 34

222 ± 11

 

Conclusions:
2-Ethylhexyl 4-methoxycinnamate was negative in a valid bacterial cell mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 of S. typhimurium  were exposed to 2-Ethylhexyl 4-methoxycinnamate (HR 99/9040020/) dissolved in DMSO, at concentrations of 0, 50, 150, 500, 1500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation, i.e. supernatant of rat liver homogenates (S9) from Aroclor 1254-induced male Sprague-Dawley rats. 2-Ethylhexyl 4-methoxycinnamate was tested up to insoluble concentration. Precipitation occurred at 1500 and 5000 µg/plate without signs of bacteriotoxicity at any tested dose level. HR 99/9040020/1 did not increase the number of revertants in any tester strain in a dose-related manner, with or without metabolic activation.The negative controls performed as expected.The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background with the test substance. This study is classified as acceptable because it was conducted equivalent to the OECD Test Guideline No. 471 and under GLP conditions, and because the documentation is sufficient for assessment. This study therefore satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation).

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 23, 1983 - April 11, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted similar to OECD guideline 474, and in compliance with GLP Standards
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Treatment Schedule
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: Füllinsdorf Albino SPF, outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Biological and Medical Research, CH-4414 Füllinsdorf
- Age at study initiation: approx. 6 - 8 weeks
- Weight at study initiation: female: 29.3 g, male: 31.8 g
- Assigned to test groups randomly: no
- Housing: Maximum number of animals per cage: 6
- Diet: Nafag 850-cubes ad libitum
- Water: Basel tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): conventional air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Solvent used: rape oil
- Concentration of test material in vehicle: different concentrations at standard dose volume of 0.1 mL/kg bw applied
Duration of treatment / exposure:
30 hours
Frequency of treatment:
Twofold dosing (over two cell cycles/30 hours)
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Basis: actual ingested
Dose / conc.:
2 500 mg/kg bw/day
Remarks:
Basis: actual ingested
Dose / conc.:
5 000 mg/kg bw/day
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent no treatment
Positive control(s):
Procarbazine hydrochloride (3 male animals used)
- Justification for choice of positive control(s): known to induce micronuclei in erythrocytes of mice
- Route of administration: orally
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
bone-marrow; polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose of 5000 mg/kg is the highest applicable one as determined by preliminary experiments.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): subacute repeated dosing over two cell cycles (approximately 30 hours in the mouse); twofold oral application of 1000, 2500 and 5000 mg 2-Ethylhexyl 4-methoxycinnamate per kg bodyweight 30 and 6 hours prior to sacrifice of the mice.

DETAILS OF SLIDE PREPARATION: Four bone-marrow smears per animal were prepared according to the method of Schmid (Schmid W., Mut. Res. 31, 1975, 9). The slides were coded and 24 hours after the preparation they were fixed in absolute methanol and stained with May-Grünwald-Giemsa.

METHOD OF ANALYSIS: For each animal 2000 polychromatic erythrocytes were examined in a double blind fashion. Only cells with clearly identifiable anomalies were recorded as cells containing micronuclei. Micronuclei contained in mature erythrocytes were recorded separately, but not taken into the statistical evaluation.
Evaluation criteria:
A positive result: a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of treated mice.
Statistics:
The results were evaluated by means of the Jonckheere-test (A.R. Jonckheere, Biometrica 41, 1954, 133) and the U-test (F. Wilcoxon et al., Biometrics 19, 1963, 58).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Remarks:
The dose of 5000 mg/kg is the highest applicable one as determined by preliminary experiments.
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Other: The dose of 5000 mg/kg is the highest applicable one as determined by preliminary experiments.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No compound-related increase of micronuclei observed

Absence of proportion of immature erythrocytes (PCE) among total erythrocytes.

Absence of data on Preliminary test.

No result data on solvent control (rape oil).

Conclusions:
After twofold oral application of 1000, 2500 and 5000 mg/kg bodyweight, 2-Ethylhexyl 4-methoxycinnamate induces neither chromosome breaks nor mitotic non-disjunctions in bone marrow cells of mice. 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:

2-Ethylhexyl 4-methoxycinnamate, a UV-B sunscreen, was evaluated for a potential induction of chromosome breaks and/or mitotic non-disjunctions in vivo by means of the micronucleus test.

After twofold oral application of 1000, 2500 and 5000 mg 2-Ethylhexyl 4-methoxycinnamate per kg bodyweight 30 and 6 hours prior to sacrifice of the mice, no compound related increase of micronuclei could be observed. The dose of 5000 mg/kg is the highest applicable one as determined by preliminary experiments.

It is therefore concluded that under the experimental conditions described in this report, 2-Ethylhexyl 4-methoxycinnamate induces neither chromosome breaks nor mitotic non-disjunctions in mouse bone-marrow cells. Therefore, the substance does not need to be classified as genotoxic according to the criteria outlined in Annex I of 1272/2008/EC.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

For this endpoint 5 tests, performed according/similar to test guidelines and under GLP conditions were selected as key studies:

- One Bacterial Reverse Mutation Assay, performed according to OECD 471 (King and Harnasch, 1999). Ethylhexyl Methoxycinnamate was negative in these valid bacterial cell mutation assay.  

- An in vitro mammalian cell transformation assay using Balb/c 3T3 cells (Strobel, 1985, according 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). Ethylhexyl Methoxycinnamate did not induce cell transformation in mammalian cells in vitro.

- A DNA damage and repair assay, unscheduled DNA synthesis (UDS) in mammalian cells (i.c. freshly prepared rat hepatocytes) in vitro (Strobel, 1985, similar to OECD Guideline 482). Ethylhexyl Methoxycinnamate did not induce DNA damage resulting in unscheduled DNA synthesis in cultured rat hepatocytes.

- A Mammalian Erythrocyte Micronucleus Test in vivo using a Füllinsdorf Albino SPF, outbred strain of mice (Hugentobler, 1983, similar to OECD Guideline 474). After twofold oral application of 1000, 2500 and 5000 mg/kg bw, Ethylhexyl Methoxycinnamate induces neither chromosome breaks nor mitotic non-disjunctions in bone marrow cells of mice.

The following tests are considered supporting studies, performed similar to test guidelines, and under GLP conditions:

1) In vitro Mammalian Chromosome Aberration Test (Hugentobler, 1984, similar to OECD Guideline 473), using human peripheral blood lymphocytes. In absence as well as in presence of a metabolic activation system the results were negative, being not clastogenic.

2) In vitro Mammalian Cell Gene Mutation Test (Strobel, 1983, similar to OECD Guideline 476, 1997). In this test evaluation was performed for induction of mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in the established cell line V79, derived from Chinese hamster lung cells. In absence as well as in presence of a metabolic activation system Ethylhexyl Methoxycinnamate did not induce mutations.

Justification for classification or non-classification

Provided key and supporting studies with 2-Ethylhexyl 4-Methoxycinnamate did not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic and therefore does not need to be classified for mutagenicity according to the criteria outlined in 1272/2008/EC (CLP/EU-GHS).