p-anisylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Chromosome aberration study for the test chemical

Author:

Galloway et al

Year:

1987

Bibliographic source:

Environmental and Molecular Mutagenesis

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Sister Chromatid Exchange test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

3-Methoxyaniline

Cas Number:

536-90-3

Molecular formula:

123.1541

IUPAC Name:

3-Methoxyaniline

Details on test material:

- Name of test material: m-Anisidine
- IUPAC name: 3-Methoxyaniline
- Molecular formula: C7H9NO
- Molecular weight: 123.1541 g/mol
- Substance type: Organic
- Physical state: No data available
- Purity: No data available
- Impurities (identity and concentrations): No data available

Method

Target gene:

No data

Species / strain

Species / strain / cell type:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consisted of 15 µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

160-1600 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: The chemical was dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.
- Justification for choice of solvent/vehicle: No data available

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9.
- Expression time (cells in growth medium): 25-26 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): After staining for 10 min in “concentrated” Hoechst 33258 (5 pg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 50 cells per dose were scored from the three highest doses

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Cells were collected by mitotic shake off for evaluation

Rationale for test conditions:

No data

Evaluation criteria:

An increase in sister chromatid exchange was noted

Statistics:

Linear regression test (trend test) of SCEs per chromosome vs the log of the dose. For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Results and discussion

Test results

Test results 1

Species / strain:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. The aim was to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis. Observations on cell growth and cell cycle kinetics were used from the SCE test to select the doses and fixation times for the chromosome aberration tests. In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ml or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

The test compound did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence of S9 metabolic activation system but it induced sister chromatid exchanges in the absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Sister chromatid exchanges test was performed to determine the mutagenic nature of the test compound. The test chemical was studied at a dose level of 160-1600µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system.

                       

5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could bemade from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis.

 

After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd.

 

Delay was noted at the top dose with S9 (5 mg/ml), and the culture was harvested 2 hr later than the controls. There were increases in aberrations, but these were statistically significant only without S9.

 

The test compound did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence of S9 metabolic activation system but it induced sister chromatid exchanges in the absence of S9 metabolic activation system.