(1R)-1-[(4R,4aR,8aS)-2,6-bis(3,4-dimethylphenyl)tetrahydro[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Commencement of test: December 1, 1994; Completion of observation: Feb 22, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Test conducted to a Japanese method which does not conform to EC/OECD requirements. The test method employed did not utilise a second experiment to confirm the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Japanese method in accordance with "The notification on Partial Revision of Testing Methods Relating to New Chemical Substances", 1986

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

413-110-2

EC Name:

-

Cas Number:

135861-56-2

Molecular formula:

C24H30O6

IUPAC Name:

1-[2,6-bis(3,4-dimethylphenyl)-hexahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 1,3:2,4-bis-O-(3,4-dimethlbenzlidene)-D-solbitol; (3,4-DMBS)
- Substance type: white powder
- Physical state: solid
- Analytical purity: ≥99.0 w/w %
- Lot/batch No.: 4073003
- Storage condition of test material: a cold and dark place

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung fibroblasts

Details on mammalian cell type (if applicable):

Modal number of chromosomes was 25 per cell. The time required for doubling of cell number was ca. 15 h. CHL cells preserved by freezing were defrosted and cultured. Tow or three days after incubation a subculture was made and the cells in logarithmic growth phase were used for each treatment. The passage number of cells used in the chromosomal aberration test were 28 for 24 h treatment and 48 h treatment by the direct method, and 29 by the metabolic activation method.

Medium:
Eagle's minimum essential medium was supplemented with newborn calf serum at a rate of 10 v/v%. The medium is referred to as 10% NCS/MEM.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and 5,6-benzoflavone induced rat liver, S9.

Test concentrations with justification for top dose:

Preliminary dose determination test: 10 to 2000 µg/ml
Main test:
Without S9 mix (direct method): 125, 250, 500 µg/ml (24 h treatment); 62.5, 125, 250, 500 µg/ml (48 h treatment)
With S9 mix (metabolic activation method): 50, 500, 5000 µg/ml

Vehicle / solvent:

0.5% aqueous solution of sodium carboxy methyl celluose (CMC)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% aqueous solution of sodium carboxy methyl celluose

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C used in direct method, cyclosphamide hydrate used in metabolic activation method.

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct incorporation method and metabolic activation method.

Exposure period (with metabolic activation): 6 hours
Exposure period (without metabolic activation): 24 hours

Fixation time:
24 hours with metabolic activation.
24 and 48 hours without metabolic activation.

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: 100 for numerical aberrations and 200 for numerical and/or strutural aberrations.

DETERMINATION OF CYTOTOXICITY:
Incidence of mitotic cells and cells with chromosomal aberration

OBSERVATION AND SCORING OF CHROMOSOMAL ABERRATIONS:
The numbers of cells with chromatid and chromosome-type structural aberrations (such as gaps, breaks, exchanges) and with numerical aberrations (polyploid, endo-reduplication) were checked by observing 100 metaphases for each dish. The incidences of cells with numerical and/or structural observations were obtained by observing 200 metaphases for each group. The incidence of structural aberration was calculated with and without gaps, respectively.
A gap was scored when a clear discontinuity (larger than a chromatid width) was evident, and when the distal part of the chromatid or chromosome showed no dislocation. Slides were all coded and blind-tested by microscopy.

Evaluation criteria:

The results obtained were assessed as follows. The incidence of cells (mean values for two dishes) with aberrations including gaps was:
less than 5%: negative (-)
5% or more and less than 10%: suspect positive (±)
10% or more and less than 20%: positive (+)
20% or more and less than 50%: positive (++)
50% or more: positive (+++)
The test substance is judged to be positive for induction of chromosomal aberration when the incidence of 10% or more was dose related or reproducible.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: as specified above

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

numerical aberrations only.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 - 300 µg/ml in preliminary testing

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Observations:
A dose-related increase in polyploidy was noted during this study in the presence and absence of metabolic activation. However, no aneuploidy or structural aberrations were observed.

All the control cultures gave values of chromosome aberrations with the expected range for normal CHL cells.

All the positive control cultures gave highly-significant increases in the frequency of aberrations, indicating the bsatisfactory performance of the test and of the activity of the metabolising system.

Remarks on result:

other: strain/cell type: as above

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Results:

The values for two dishes were not markedly different, the incidence of cells with any aberration did not exceed 5% in the negative control, the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control. There were no fluctuations in the test conditions and no contamination with microorganisms in the cultures which might have affected the results. The test results were therefore considered to be valid.

Cell Growth inhibition test and cell division inhition test

See Table 1 and Figure 1 (attached background material).

Dose levels used in cell growth inhibition test and cell division inhibition test were determined as shown in Table 1. The growth rate in the solvent control was considered to be 100%. Fifty percent growth inhibition concentrations of the test substance were about 230 µg/ml in the 24 h treatment, about 210 µg/ml in the 48 h treatment by the direct method, and about 230µg/ml by the metabolic activation method. Mitotic metaphases of chromosomes sufficient for assessing chromosomal aberration were observed at doses of 500µg/ml or less in the 24 h and 48 h treatment by the direct method, 5,000µg/ml or less by the metabolic activation method.

Cells were observed microscopically and induction of polyploid cells was observed. Based upon the results, the following 3 or 4 dose levels were employed in each method of the chromosomal aberration test.

24 h treatment by the direct method: 125, 250 and 500 µg/ml.

48 h treatment by the direct method: 62.5, 125, 250 and 500 µg/ml.

Metabolic activation method: 50, 500 and 5000 µg/ml.

Chromosomal aberration test

Direct Method (see Table 2 and figure 2, attached background material).

24 h treatment.

The incidences of cells with structural aberrations, including gaps, were 2.0% at 125 µg/ml, 0.5% at 250 µg/ml and 0.0% at 500 µg/ml.

The incidence in the solvent control was 0.5% (within the normal range). The positive control treated with MMC showed structural aberration of 62.0%.

The incidences of polyploid cells were 0.5% at 125 µg/ml, 3.5% at 250 µg/ml and 5.5% at 500 µg/ml. The incidence in the solvent control was 0.0% (within the normal range).

48 h treatment

The incidences of cells with structural aberrations, including gaps, were 1.0% at 62.5 µg/ml, 2.0% at 125 µg/ml, 2.0% at 250 µg/ml and 1.5% at 500 µg/ml.

The incidence in the solvent control was 1.5% (within the normal range). The positive control treated with MMC showed structural aberration of 75.5%.

The incidences of polyploid cells were 1.5% and 62.5 µg/ml, 6.0% at 125 µg/ml, 9.5% at 250 µg/ml and 22.5% at 500 µg/ml. The incidence in the solvent control was 0.5% (within the normal range).

Metabolic activation method (see Table 3 and figure 3, attached background material):

With S9 mix:

The incidences of cells with structural aberrations, including gaps, were 1.0% at 50 µg/ml, 2.0% at 500 µg/ml and 3.0% at 5000 µg/ml.

The incidence in the solvent control was 0.0% (within the normal range). The positive control treated with CPA showed structural aberration of 56.6%.

The incidences of polyploid cells were 3.0% at 50 µg/ml, 12.0% at 500 µg/ml and 3.0% at 5000 µg/ml.

Without S9 mix:

In the medium without S9 mix, the incidences of cells with structural aberrations, including gaps, were 1.5% at 50 µg/ml, 3.0% at 500 µg/ml and 4.0% at 5000 µg/ml.

The incidences in the solvent control and the positive control were 0.5% and 1.5% i.e. respectively within the normal range.

The incidences of polyploid cells were 1.5% at 50 µg/ml, 15.5% at 500 µg/ml and 29.0% at 5000 µg/ml.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative Polyploidy (numerical aberrations) were observed but no significant aneuploidy or structural aberrations were observed. Therefore the overall result is considered to be negative.

The chromosomal aberration test of 3,4-DMBS was carried out using CHL cells.
The test substance induced numerical aberrations dose-dependently within the dose range of 125 – 500 µg/ml in the 48 h treatment by the direct method, and 500 – 5000 µg/ml in the group without S9 mix by the metabolic activation method, respectively.
At 500 µg/ml in the group with S9 mix by the metabolic activation method and the 24 h treatment by the direct method, numerical aberrations were induced.

Executive summary:

The effect of 3,4-DMBS on the chromosomal aberration was investigated using Chinese hamster lung fibroblasts for the tests with S9 Mix (metabolic activation) and without S9 Mix (direct method), respectively.

Cell growth inhibition test and cell division inhibition tests were carried out to determine the dose levels of the test substance. From the results of these tests, chromosomal aberration tests were carried out using 125, 250 and 500 µg/ml of the test substance for 24 h treatment, 62.5, 125, 250 and 500 µg/ml for 48 h treatment by the direct method, and 50, 500 and 5000 µg/ml by the metabolic activation method.

The test substance induced numerical chromosomal aberrations dose-dependently within the dose range of 125 – 500 µg/ml in the 48 h treatment by the direct method, and 500 – 5000 µg/ml in the group without S9 mix by the metabolic activation method, respectively.

The positive controls (MMC and CPA) induced evident structural chromosomal aberrations.