Isopentyl p-methoxycinnamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-26 to 2010-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The certificate was signed at 30. Mach 2009 by "Hessisches Ministerium für Umwelt, Ländlichen Raum und Verbraucherschutz".

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isopentyl p-methoxycinnamate

EC Number:

275-702-5

EC Name:

Isopentyl p-methoxycinnamate

Cas Number:

71617-10-2

Molecular formula:

C15H20O3

IUPAC Name:

isopentyl p-methoxycinnamate

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank´s salts, Neomycin (5 µg/mL) and Amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 (98.5% air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes; The cells have a stable karyotype with a modal chromosome number of 22.
- Periodically "cleansed" against high spontaneous background: yes; the level of spontaneous mutants was depressed by treatment with HAT-medium

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-test: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0 and 2500.0 µg/mL
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected.
Experiment I:
- without S9 mix, 4 h treatment: 0.6, 1.3, 2.5, 5.0, 10.0, 20.0 and 40.0 µg/mL
- with S9 mix, 4 h treatment: 18.8, 37.5, 75.0, 150.0, 300.0 and 2500.0 µg/mL
Experiment II:
- without S9 mix, 24 h treatment: 1.3, 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 µg/mL
- with S9 mix, 4 h treatment: 18.8, 37.5, 75.0, 150.0, 300.0 and 2500.0 µg/mL
For more details see below ("Any other information on materials and methods incl. tables").

Vehicle / solvent:

- Vehicle(s)/solvent(s): On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (purity 99.9%).
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of DMSO in the culture medium did not exceed 0.5% v/v.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent control (DMSO) were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation Migrated to IUCLID6: 0.15 mg/mL (Exp.I) and 0.075 mg/mL (Exp.II); dissolved in nutrient medium

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent control (DMSO) were performed.

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation Migrated to IUCLID6: 1.1 µg/mL; dissolved in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Exponentially growing stock cultures (more than 50% confluent) were trypsinised at 37 °C for 5 min. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The cell suspension was seeded into plastic culture flasks. Approximately 1.5x10^6 (single culture) and 5x10^2 cells were seeded in MEM with 10% FBS (complete medium) for the determination of mutation rate and toxicity, respectively.

DURATION
- Exposure duration: In the first experiment the treatment period was 4 h with and without metabolic activation. The second experiment was performed with a treatment time of 4 h with and 24 h without metabolic activation.
- Expression time (cells in growth medium): Following the expression time of 7 d cells were cultured in selection medium.
- Fixation time (start of exposure up to fixation or harvest of cells): About 15 d after treatment cultures were evaluated.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
STAIN: The colonies were stained with 10% methylene blue in 0.01% KOH solution.

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentra-tion level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case-by-case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 10^6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value is <0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No relevant and reproducible increase in mutant colony numbers/10^6 cells induced by the test item was observed in the main experiments up to the maximum concentration

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

in experiment II without S9 mix the highest concentration tested was determind by cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The osmolarity and pH-value were determined in the solvent control and the maximum concentration of the pre-experiment without metabolic activation:
- Effects of pH: solvent control = 7.21 and test item = 7.22
- Effects of osmolality: solvent control = 396 mOsm and test item = 358 mOsm
- Precipitation: Precipitation of the test item was observed following 4 h treatment without metabolic activation at 40 µg/mL. After metabolic activation test item precipitation was observed at 300 µg/mL and above in both experiments.

RANGE-FINDING/SCREENING STUDIES: The maximum evaluated concentration in the absence of metabolic activation was based on relevant test item induced cytotoxic effects. Relative cloning efficiency I was reduced to 44.9 and 30.8 % after 4 hours treatment at 5.0 µg/mL and to 33.1 and 27.3% after 24 h treatment at 40.0 µg/mL. The corresponding cell densities at the first subcultivation after treatment were 2 to 20% of the solvent control. After 4 h treatment at a test item concentration of 10 µg/mL the relative cloning efficiency I was still above 10% und fulfilled the evaluation criteria, but the cell numbers were not sufficient during the first subcultivation of the mass cultures.
The absolute value of cloning efficiency II was in between 65 and 88% in all experimental parts and therefore fulfilled the acceptability criteria of at least 50% .

COMPARISON WITH HISTORICAL CONTROL DATA: Results were compared with historical control data. In the main experiments the mutant frequencies without metabolic activation of some cultures exceeded the historical control data ranges. But these findings were not reproducible in the parallel culture and the induction factor was below 3.0. Thus this observation was not considered biologically relevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: main experiments I and II

Any other information on results incl. tables

All relevant information on results is provided in sections "Test results" and "Additional information on results".

Applicant's summary and conclusion

Conclusions:

The study was performed to investigate the potential of isopentyl p-methoxycinnamate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments with isopentyl p-methoxycinnamate.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, isopentyl p-methoxycinnamate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The potential genotoxicity of isopentyl p-methoxycinnamate was investigated using the HPRT assay in V79 cells of the Chinese hamster, an in vitro assay system for the induction of mutations in mammalian cells. The assay was performed according to OECD guideline no. 476 and EU Method B.17 and consisted of two independent experiments. In the first experiment, the cells were exposed to the test item for 4 h with and without metabolic activation. In the second experiment, cells were treated with the test item for 24 h in the absence of metabolic activation and for 4 h in the presence of metabolic activation.

In the first experiment, cells were exposed to the test item at concentrations ranging from 18.8 to 2,500 µg/mL, and from 0.6 to 40 µg/mL, for tests with and without metabolic activation, respectively. In the second experiment, the test without metabolic activation was conducted with concentrations of the test item ranging from 1.3 to 80 µg/mL, while in the test with metabolic activation, a concentration range of 18.8 to 2,500 µg/mL was used.

Accordingly, the highest concentration used in this study (2,500 µg/mL) corresponded to the highest test concentration of 10 µM that is recommended in OECD guideline no. 476.

The dose range used in the two experiments was limited by precipitation of the test item (in the first experiment, at 40 µg/mL without metabolic activation, and in both experiments at 300 µg/mL with metabolic activation) and cytotoxicity.

No substantial and reproducible, dose-dependent increase of the mutation frequency was observed in both experiments with isopentyl p-methoxycinnamate.

7,12-Dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS) were used as positive controls for experiments with and without metabolic activation, respectively. The positive control compounds induced a distinct increase in mutant colonies and thus, demonstrated the sensitivity of the test and the activity of the metabolic activation system.

 In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, isopentyl p-methoxycinnamate is considered to be non-mutagenic in this HPRT assay.