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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study without deviations, performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MXDA/SM ADDUCT
- Physical state: pale yellow viscous liquid
- Analytical purity: not reported
- Impurities (identity and concentrations): not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: PMS-02A
- Expiration date of the lot/batch: not reported
- Stability under test conditions: not reported
- Storage condition of test material: room temperature in the dark, until 12 April 2002, thereafter, room temperature, in the dark, under nitrogen

The composition of test material is not reported in this study report but is reported for the same batch of the test substance in the study report "MXDA/SM ADDUCT: DETERMINATION OF GENERAL PHYSICO-CHEMICAL PROPERTIES" by O'Connor, 2002 (see e.g. IUCLID section 4.2. Melting point). Note that the components given in this study report still were based on information retrieved from old GC analytical data (for details see chapter 1.2).
Component 1: 54.9 %
Component 2: 4.7 %
Component 3: 36.4 %
Component 4: 4.0 %

Method

Species / strain
Species / strain / cell type:
other: Chinese Hamster Lung (CHL) cell line
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Experiment I, without S9: 3.75, 7.5, 15, 30, 45, 60 µg/mL
Experiment I, with S9: 15, 30, 45, 60, 90, 120 µg/mL

Experiment II, 24-hour exposure, without S9: 0.31, 0.63, 1.25, 2.5, 3.75, 5 µg/L
Experiment II, 48-hour exposure, without S9: 0.25, 0.5, 1.0, 1.5, 2.0, 2.5 µg/L
Experiment II, 6-hour exposure, with S9: 7.5, 15, 30, 45, 60, 75 µg/L
Vehicle / solvent:
Dimethyl sulfoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I, with and without S9: 6 hours
Experiment II, without S9: 24 and 48 hours
Experiment II, with S9: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
Experiment I, with and without S9: 24 hours
Experiment II, without S9: 24 and 48 hours
Experiment II, with S9: 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (2 hours prior to harvest time)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS
guidelines for mutagenicity testing. Evaluation of metaphase cells may be terminated after 50 cells if approximately 50% cells with aberrations were observed.
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells are recorded and are included in the polyploid cell total number.
If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. If the study gives a positive response then a D20 value will be calculated, which is the presumed dose level of the test substance that is required to induce aberrations in 20% of metaphases.

Results and discussion

Test results
Species / strain:
other: Chinese Hamster Lung (CHL) cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: no metaphase cells present at >=30 µg/mL (without S9) and 90 µg/mL (with S9). Experiment II: 50% growth inhibition at >=2.5 µg/mL (24-hour group) and >=0.25 µg/mL (48-hour group), 39% cell growth inhibition at 75 µg/mL (6 hours group)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the Cell Growth Inhibition test, a precipitate of the test material was seen at and above 1500 µg/mL. In the main test, precipitate of the test material was not observed at the end of the treatment period in any exposure group.

RANGE-FINDING/SCREENING STUDIES: A Cell Growth Inhibition test was performed. In all cases the test material showed evidence of cell toxicity. A precipitate of the test material was seen at and above 1500 µg/mL. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphases were present at dose levels up to 50 µg/mL in the 6(18)-hour with-S9 exposure group and at up to 15 µg/mL in the 6(18)-hour without-S9 exposure group. The maximum dose with metaphases present in the 24-hour continuous exposure was 10 µg/mL and 2.5 µg/mL in the 48-hour exposure group. The dose selection for the main experiments was based on toxicity for all exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA: The data lay within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, the test material demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test. These data show an approximate 50% growth inhibition was not achieved in the absence or presence of S9. The test material was shown to have a very steep toxicity curve, such that there were no metaphase cells present at and above 30 µg/mL in the absence of S9 and 90 µg/mLl in the presence of S9. It was considered that adequate levels of toxicity had been achieved.
In Experiment II, the test material demonstrated similar toxicity to that observed in the Cell Growth Inhibition Test. The data show that an approximate 50% growth inhibition was achieved at 2.5 µg/mL and above in the 24-hour exposure group and at 0.25 µg/mL and above in the 48-hour exposure group. In the 6(18)-hour exposure group in the presence of S9 39% cell growth inhibition was achieved at the maximum dose level tested of 75 µg/mL. In a previous experiment this same dose level had proved to be too toxic for evaluation. It was therefore considered that the test material had been adequately tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment II

The test material induced small but statistically significant increases in the frequency of cells with aberrations in the 48 hour continuous exposure group and in the 6(18)-hour exposure group. It should be noted however, that in both cases the responses were within the historical maxima for that time point, both were in comparison to very low vehicle control values and were not part of any dose-related response. Therefore, both increases were considered to be of no toxicological significance.

Polyploid cells

The test material did not induce any statistically significant increases in the number of polyploid cells at any dose level in either treatment case.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce any toxicologically significant, dose-related increases in the frequency of cells with chromosome aberrations, either in the presence or absence of a liver enzyme metabolising system, or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.
Executive summary:

This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to OECD TG 473.

Duplicate cultures of Chinese Hamster Lung (CHI+) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure, a 48-hour continuous exposure and a repeat of the 6(18)-hour exposure with metabolic activation.

The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 3.75 to 120 µg/mL for the 6(18)-hour exposure, both with and without S9, and 0.25 to 5 µg/mL for the 24 and 48-hour treatments.

The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in any of the exposure groups. The test material was shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved.

The test material was shown to be non-clastogenic to CHL cells in vitro.