1,4-bis(methoxymethyl)benzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2010-07-22 to 2010-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) (migrated information)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(methoxymethyl)benzene

EC Number:

229-828-2

EC Name:

1,4-bis(methoxymethyl)benzene

Cas Number:

6770-38-3

Molecular formula:

C10H14O2

IUPAC Name:

1,4-bis(methoxymethyl)benzene

Test material form:

liquid

Specific details on test material used for the study:

Purity: 99.80 %

Method

Species / strain

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) (migrated information)

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

-Cell growth inhibition study: 6.48, 13.0, 25.9, 51.9, 104, 208, 415, 830 and 1660 μg/mL (equivalent to 10 mmol/L, the highest dose to be used in the absence of toxicity according to the guideline)
- Chromosomal aberration study:
-S9 mix (short-term treatment); 0, 718, 825, 949, 1090, 1260, 1440, 1660 μg/mL
+S9 mix (short-term treatment): 0, 51.9, 104, 208, 415, 830, 1660 μg/mL
Justification for top dose: The highest dose of the test substance was set at a dose higher than IC50 because it showed a cytotoxic potential that reduced the cell growth rate to less than 50% in the absence of S9 mix when the cell growth inhibition study was conducted by the short-time treatment method. Specifically, the highest dose was set at 1,660 μg/mL followed by 6 lower doses with a common ratio of 1.15 since IC50 was found to be 1,200 μg/mL. In the presence of S9 mix, the highest dose was set at 1,660 μg/mL followed by 5 lower doses with a common ratio of 2 because the test substance did not show a cytotoxic potential that reduced the cell growth rate to less than 50%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was suspended in distilled water at 16.6 mg/mL and dissolved in DMSO and acetone at 166 mg/mL. DMSO, which is easier to handle, was selected for use as vehicle because solutions of the test substance prepared at 166 mg/mL using DMSO and acetone generated no heat or foam and showed no changes in color at room temperature for 2 hrs after preparation and DMSO is commonly used as vehicle in chromosomal aberration studies.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

Cell growth inhibition study
- Procedures:
In the study conducted by the short-time treatment method, 30 μL of solution of the test substance or vehicle mixed well with 3 mL of fresh medium was added to cells after removing pre-cultured medium in the absence of S9 mix. In the presence of S9 mix, 30 μL of solution of the test substance or vehicle added to 2.5 mL of fresh medium and mixed well with 0.5 mL of S9 mix was added to cells. In both cases, medium was removed after treatment for 6 hrs, and cells were washed 3 times with 2 mL of Ca2+- and Mg2+-free Dulbecco phosphate buffered salt solution and cultured for 18 hrs after adding 5 mL of fresh medium.
In the study conducted by the continuous treatment method, 50 μL of solution of the test substance or vehicle mixed well with 5 mL of fresh medium was added to cells after removing pre-cultured medium and treated for 24 hrs.
Regardless of whether the short-time treatment method or the continuous treatment method was used, 50 μL per culture vessel of 10 μg/mL Demecolcine solution was added 2 hrs before completing culture.
The presence or absence of precipitation of the test substance, changes in the color of medium, and corrosion of culture vessels was determined with the naked eye at the beginning and end of treatment and at the end of culture.
After completing culture, cells were exfoliated with 2 mL of 0.25 w/v% trypsin to prepare cell suspensions. Two hundred μL of cell suspension was sampled, and cells were counted using a microcell counter (CDA-500, Sysmex Corporation) after diluting with 10 mL of Cell Pack (Sysmex Corporation). The cell growth rate and 50% cell growth inhibition concentration (IC50) were calculated. IC50 was calculated using the line obtained from the cell growth rate at the lowest dose at which the cell growth rate decreases below 50% and the second lowest dose.
The remaining cell suspension was centrifuged at 1000 rpm for 5 min to recover cells and hypotonically treated for 15 min at 37C after adding 3 mL of KCl. About 0.3 mL of fixative (methanol: acetic acid = 3:1) was added to hypotonically treated cells to half-fix them. Cells were fixed using 3 mL of fixative replaced twice. Cell suspension of appropriate concentration was prepared thereafter using fixative and dropped on slides. One stained specimen was prepared per dose. After allowing them to dry spontaneously, specimens were stained for 15 min with 2 vol% Giemsa stain diluted with 1/15 mol/L phosphate buffer solution (pH 6.8).
- Observation and determination:
The presence or absence of divided cells was determined, and 50 metaphase cells were observed per dose at doses that are referred to set doses for the chromosomal aberration study to determine incidence of cells with chromosomal aberration (Structural and Numerical aberrations).

Chromosomal aberration study:
- Method: The chromosomal aberration study was conducted in the same way as the cell growth inhibition study by the short-time treatment method. Four chromosome specimens were prepared per dose (two per culture vessel). 30 μL per culture vessel of 0.01 mg/mL MMC was used as positive control in the absence of S9 mix, while 18 μL per culture vessel of 1 mg/mL CPA was used in the presence of S9 mix. Two culture vessels were used for dose.
- Doses for observation of specimens:
All specimens from the negative and positive control groups were observed. Doses of the test substance selected for observation of specimens: 1260, 1440 and 1660 μg/mL without S9 mix and 104, 208, 415, 830 and 1660 μg/mL with S9 mix
- Structural aberrations: Two hundred metaphase cells with 25±2 chromosomes (centromeres) per dose (50 per specimen) were observed. The total number of cells with structural aberrations was recorded together with the number by type of aberration. Gaps were defined as unstained areas narrower than the width of chromatid and recorded separately from structural aberrations.
- Numerical aberrations: Two hundred metaphase cells per dose (50 per specimen) were observed. The number of polyploid cells with 38 or more chromosomes was recorded.

Evaluation criteria:

The test result was considered positive if incidence of cells with structural or numerical aberrations is 10% or higher and a dose response is observed or if incidence is 5% or higher and the result is reproducible in the chromosomal aberration study and confirmatory study. Otherwise, the result was considered negative. No statistical method was employed. The D20 value (concentration at which 20% of cells show aberration) was determined for the treatment method which caused chromosomal aberrations in 5% or more cells (with the exception of treatment method that gave a negative result).

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) (migrated information)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cell growth inhibition test:
The IC50 value was found to be 1,200 μg/mL and 1,660 μg/mL, respectively, in the absence and presence of S9 mix by the short-term treatment method and 1,100 μg/mL by the 24-hour continuous treatment method.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels were not observed at any dose of the test material regardless of the treatment method at the start or completion of treatment or at the end of culture.
In the observation of specimens, incidence of cells with structural aberrations was 52.0% and 22.0%, respectively, at the maximum in the absence and presence of S9 mix when the short-term treatment method was used. Incidence of cells with numerical aberrations was below 5% at all doses in the absence of S9 mix by the short-term treatment method, but it increased up to 16.0% in the presence of S9 mix.

Chromosomal aberration test
- In the absence of S9 mix:
Cell growth rate and IC50: The cell growth rate was found to be 87.7, 85.8, 79.1, 75.1, 69.7, 51.7, and 14.0%, respectively, at 718, 825, 949, 1,090, 1,260, 1,440 and 1,660 μg/mL, and IC50 was calculated to be 1,400 μg/mL.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels: No precipitation of the test material, changes in color of the medium, or erosion of culture vessels was observed at any dose.
Incidence of cells with structural aberrations: Incidence of cells with structural aberrations was 2.5% and 75.0%, respectively, in the negative and positive control groups. In the test material groups, it was 2.5, 14.0 and 26.0%, respectively, at 1,260, 1,440 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
Incidence of cells with numerical aberrations: The test result was judged to be negative because incidence was below 5% at all doses.
D20: D20 for structural aberration was calculated to be 1.6 mg/mL.

- In the presence of S9 mix:
Cell growth rate and IC50: The cell growth rate was found to be 104.3, 98.4, 84.6, 77.6, 62.5, and 47.4%, respectively, at 51.9, 104, 208, 415, 830, and 1,660 μg/mL, and IC50 was calculated to be 1,500 μg/mL.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels: No precipitation of the test material, changes in color of the medium, or erosion of culture vessels was observed at any dose.
Incidence of cells with structural aberrations: Incidence of cells with structural aberrations was 2.0% and 24.0%, respectively, in the negative and positive control groups. In the test material groups, it was 1.0, 7.5, 7.0, 25.0, and 35.0%, respectively, at 104, 208, 415, 830 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
Incidence of cells with numerical aberrations: Incidence of cells with numerical aberrations was 0.0% and 0.5%, respectively, in the negative and positive control groups. In the test material groups, it was 1.0, 9.0, 13.0, 15.5, and 1.0%, respectively, at 104, 208, 415, 830 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
D20: D20 for structural aberration and numerical aberration was calculated to be 0.61 and 0.95 mg/mL, respectively.

Applicant's summary and conclusion

Conclusions:

The test item was thought to induce both structural and numerical aberrations.

Executive summary:

An in Vitro Chromosomal Aberration Test was conducted with Cultured Chinese Hamster CHL/IU Cells according to OECD 473.

Cell growth inhibition test was conducted at 6.48, 13.0, 25.9, 51.9, 104, 208, 415, 830 and 1660 μg/mLusingshort-time treatment methodor the continuous treatment method.

Chromosomal aberration test was conducted at 718, 825, 949, 1090, 1260 1440 and 1660μg/mL in the absence of S9 mix and 51.9, 104, 208, 415, 830 and 1660μg/mLin the presence of S9 mix using short-time treatment method.

The result of chromosomal aberration test was judged to be positive by the short-term treatment method because incidence of cells with structural aberrations increased up to 26.0% and 35.0%, respectively, exceeding 10% in the absence and presence of S9 mix, and incidence increased in a dose-dependent manner. In terms of incidence of cells with numerical aberrations, the test result was judged to be negative in the absence of S9 mix by the short-term treatment method because incidence was below 5% at all doses, but it was judged to be positive in the presence of S9 mix because incidence increased up to 15.5% exceeding 10% and increases showed dos-dependency. At 1,660 μg/mL, incidence was low (1.0%) probably due to a delay in cell cycle.

The test substance induced structural aberrations in CHL/IU cells with and without metabolic activation and induced numerical aberrations with metabolic activation.