4-allyl-2-methoxyphenyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2001 to 03 June 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD Guideline 482, however a repeat experiment was not performed

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

1986

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

other: in vitro UDS assay

Test material

Test material information

Constituent 1

Reference substance name:

4-allyl-2-methoxyphenyl acetate

EC Number:

202-235-6

EC Name:

4-allyl-2-methoxyphenyl acetate

Cas Number:

93-28-7

Molecular formula:

C12H14O3

IUPAC Name:

4-allyl-2-methoxyphenyl acetate

Test material form:

liquid

Details on test material:

- Appearance: Clear colorless liquid
- Storage condition of test material: Stored at room temperature, protected from exposure to light and moisture

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

hepatocytes: rat

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Preliminary toxicity test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/mL
Main test: 1, 2.5, 5, 10, 15, 20, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on solubility or miscibility of the test item and compatibility with the target cells. The test item was soluble or miscible in DMSO at ca. 500 mg/mL, the maximum concentration prepared.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Details on test system and experimental conditions:

TEST SYSTEM:
- Source of cells and cell culture: Adult male Sprague-Dawley rats obtained from Harlan Sprague-Dawley, Inc. (Frederick, MD). The procedure used for obtaining rat hepatocyte cultures was essentially that of Williams (1977 and 1979). The rats was anesthetised with metofane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis (β-aminoethyl ether)N, N, N’, N’-tetraacetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete WME (William’s Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum) and approximately 5 x 10^5 cells were seeded into 35 mm tissue culture dishes containing complete WME. The cultures were incubated at 37 ± 1 °C in a humidified 5 ± 1% CO2 incubator for 90 to 180 minutes, washed with complete medium, refed with serum-free medium and used in the test. The dishes designated for autoradiography were also treated with 10 µci 3H-Thymidine/mL.

METHOD OF APPLICATION: in medium; Complete WME (William’s Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum)

DURATION
- Exposure duration: 18-20 h

ASSESSMENT OF CYTOTOXICITY
After 18-20 h of exposure, a portion of the medium from two of each of the three culture dishes per treatment group was removed for LDH determinations. Additionally, the cultures of the highest dose and the vehicle control which were seeded in dishes without coverslips were lysed with Triton X-100 to release 100% of the LDH in the cultures.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Duplicate cultures/dose for test item and vehicle control
Main test: Triplicate cultures/dose for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- After 18-20 h of exposure, the cells in the autoradiography dishes were washed in serum free WME, swelled in 1% sodium citrate and fixed in ethanol-glacial acetic acid fixative. The coverslip were air-dried, mounted cell side up on glass slides, and allowed to dry. The slides were coated with KodakNTB-2 emulsion (diluted 1:1 in deionized water) and stored in a refrigerator for 5 to 12 days in light tight boxes with desiccant. The slides were then developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer and stained in hematoxylin-eosin stain.

NUMBER OF CELLS EVALUATED:
- The slides were counted using automated colony counter interfaced with microscope.
- Where possible, nuclear grains were counted in 50 cells in random areas on each of the three coverslips for a total of 150 cells per treatment.

CRITERIA FOR IDENTIFICATION OF NUCLEI:
The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Replicate synthesis was identified by nuclei completely blackened with grains and such cells were not counted. Nuclei exhibiting toxic effects of treatment, such as dark or uneven staining, disrupted membranes, or irregular shape, were not counted.

DETERMINATION OF CYTOTOXICITY
- In the cytotoxicity assays, relative LDH activities were obtained by subtracting the LDH activity of the negative control cultures from the LDH activity of the treated cultures. Relative toxicities were obtained by comparing the relative LDH activities of the treated cultures to the relative LDH activity of the 100% lysis control cultures.

OTHER:
For each treatment slide in the UDS assay, the net nuclear counts were averaged and the mean ± SD reported. Also reported are the grand mean and SD for each dose level as well as the per cent of cells in repair (cells with ≥ 5 net nuclear grains).

Evaluation criteria:

All conclusions were based on scientific judgement; however, as a guide to interpretation of the data, the following criteria were used.
- If the mean net nuclear count was increased by at least 5 counts over the negative control, the results for that dose level were considered significant. A test item was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the negative control. In the absence of a dose response, a test item which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive.
-If a test item showed a significant increase in the mean net nuclear grain count at one dose level without a dose response, the activity of the test item was considered to be equivocal.
- The test item was considered negative if no significant increase in the mean net nuclear grain counts was observed.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes; At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL.
- Other confounding effects: None

PRELIMINARY TOXICITY TEST:
- At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL.
- Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 667 µg/mL. For cultures treated with 100 and 333 µg/mL, the relative toxicity was 53.5 and 59.7%, respectively. For cultures treated with 33 and 67 µg/mL, the relative toxicity was 28.9 and 25.8%, respectively. Relative toxicities of the remaining cultures were < 9.3%.
- A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 100 µg/mL, moderate toxicity in cultures treated with 33 and 67 µg/mL, and low toxicity in cultures treated with 10 µg/mL. All other cultures appeared morphologically normal.
- Based on the results of the toxicity assay, the highest concentration of the test item selected for UDS assay was 100 µg/mL.

UDS ASSAY
- At the initiation and at the termination of the treatment, precipitation was not observed in any of the test item treated cultures.
- Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 50 µg/mL. For cultures treated with 20 and 25 µg/mL, the relative toxicity was 73.3 and 84.5%, respectively. For cultures treated with 15 µg/mL, the relative toxicity was 33.7%. For cultures treated with 5 and 10 µg/mL, the relative toxicity was 14.1 and 10.7%, respectively. All other relative toxicities were ≤ 7.5%.
- A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 50 µg/mL, moderate toxicity in cultures treated with 20 and 25 µg/mL, and low toxicity in cultures treated with 10 and 15 µg/mL. All other cultures appeared morphologically normal.
- Test item did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) at any dose level in isolated rat hepatocytes.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test item did not induce a significant increase in the mean number of net nuclear grain counts at any dose level in isolated rat hepatocytes. Therefore, the test item is considered to be negative in the UDS assay.

Executive summary:

In an in vitro UDS (unscheduled DNA synthesis) assay performed according to the OECD test guideline No. 482 and in compliance with GLP, primary cultures of rat hepatocytes were exposed to test item for 18-20 h at the following concentrations:

Preliminary toxicity test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/mL

Main test: 1, 2.5, 5, 10, 15, 20, 25, 50, 75 and 100 µg/mL

Vehicle (DMSO) and positive control groups were also included in the test.

Preliminary toxicity test: At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL. Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 667 µg/mL. For cultures treated with 100 and 333 µg/mL, the relative toxicity was 53.5 and 59.7%, respectively. For cultures treated with 33 and 67 µg/mL, the relative toxicity was 28.9 and 25.8%, respectively. Relative toxicities of the remaining cultures were < 9.3%. A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 100 µg/mL, moderate toxicity in cultures treated with 33 and 67 µg/mL, and low toxicity in cultures treated with 10 µg/mL. All other cultures appeared morphologically normal. Based on the results of the toxicity assay, the highest concentration of the test item selected for UDS assay was 100 µg/mL.

Main test: At the initiation and at the termination of the treatment, precipitation was not observed in any of the test item treated cultures. Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 50 µg/mL. For cultures treated with 20 and 25 µg/mL, the relative toxicity was 73.3 and 84.5%, respectively. For cultures treated with 15 µg/mL, the relative toxicity was 33.7%. For cultures treated with 5 and 10 µg/mL, the relative toxicity was 14.1 and 10.7%, respectively. All other relative toxicities were ≤ 7.5%. A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 50 µg/mL, moderate toxicity in cultures treated with 20 and 25 µg/mL, and low toxicity in cultures treated with 10 and 15 µg/mL. All other cultures appeared morphologically normal.

Under the test conditions, the test item did not induce a significant increase in the mean number of net nuclear grain counts at any dose level in isolated rat hepatocytes. Therefore, the test item is considered to be negative in the UDS assay.