Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1989-06-21 to 1989-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
inadvertently, at the first fixation time, cells were fixed 6 hours post-dosing instead of at the protocolled 4 hours post-dosing. However, this amendment was not considered to seriously affect the experimental set-up of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see above
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
m-phenylenebis(methylamine)
EC Number:
216-032-5
EC Name:
m-phenylenebis(methylamine)
Cas Number:
1477-55-0
Molecular formula:
C8H12N2
IUPAC Name:
1,3-phenylenedimethanamine
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Metaxylenediamine
- Physical state: Clear liquid
- Stability of test article: More than 6 months when under nitrogen atmosphere
- Stable for at least 4 hours in vehicles: Stable in water during 24 hours but precipitate in a carbon dioxide atmosphere. Therefore, the use of carbon dioxide during exposure time was avoided.
- Storage condition of test material: In the original container under nitrogen at room temperature in the dark
- Safety precautions: Gloves, goggles and face mask were considered sufficient to ensure personal health and safety

Method

Target gene:
No data
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cells were obtained from Dr AT Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, The Netherlands (1986) - strain CHO-K1, S1B, a subclone of CHO-K1 from Flow Laboratories. Stock cultures of the cells were stored in liquid nitrogen (- 196 degrees Centigrade). The cultures were periodically checked for mycoplasma contamination (last check: March 1989).
Metabolic activation:
with and without
Metabolic activation system:
S-9 rat liver, induced with Aroclor 1254
Test concentrations with justification for top dose:
Without S-9 mix: 75, 200, 325 and 450 µg/mL culture medium
With S-9 mix: 200, 400, 600 and 800 µg/mL culture medium
Vehicle / solvent:
The test substance was dissolved in culture medium (F10 medium buffered with 20 mM HEPES). Test substance concentrations were prepared directly prior to use.

The pH and osmolarity of the culture medium containing the highest tested concentration were recorded.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation (-S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (+S9 mix)
Details on test system and experimental conditions:
Reference substances

Negative control
The vehicle of the test article

Postive controls

Without metabolic activation (-S9-mix)
Ethylmethanesulphonate (EMS; CAS number 62-50-0; purity 98 %; Merck) was used as a direct mutagen at a final concentration of 8 mM (solvent: DMSO)

With metabolic activation (+S9-mix)
Cyclophosphamide (CP; CAS number 50-18-0, Endoxan-Astra, Asta-Werke, F.R.G.) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 5 µg/mL (solvent: HBSS).

Solvents for reference substances:
DMSO = Dimethylsulphoxide of spectroscopic quality (Merck)
HBSS = Hank's Balanced Salt Solution without calcium and magnesium

Solutions of reference substances were prepared immediately before use

Evaluation criteria:
A chromosome aberration test was considered acceptable if it met the following criteria:
a) The numbers of chromosome aberrations found in the solvent control cultures should reasonably fall within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with the chromosome aberrations.
b) A statistically significant increase (Chi-square test, P < 0.05) in the frequency of aberrations was observed in the absence of a clear dose response relationship, but the results were reproducible in an independently repeated experiment.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors were allowed to enter into the final evaluation decision.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
See Tables 3 to 8 (attached)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See Table 1 (attached)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxicity test/dosage selection

Table 1 (attached) shows the results of the toxicity test with metaxylenediamine in the presence and absence of a metabolic activation system (S9-mix). Both absolute and relative (percentage of control) cloning efficiency is tabulated. In the absence of S9-mix the cloning efficiency was reduced by 24 % at a test substance concentration of 333 µg/mL . In the presence of S9-mix a reduction of 11 % was observed at 333 µg/L. Both in the absence and presence of S9-mix (almost) total cell killing was observed at a concentration of 1000 µg/mL.

Cytogenetic test

Based on the results of the cytotoxicity test (see Table 1, attached) the following doses were selected for the cytogenetic test:
Without S9-mix: 75, 200, 325 and 459 µg/mL culture medium.
With S9-mix: 200, 400, 600 and 800 µg/mL culture medium.

The osmolarity of the culture medium containing 800 µg test substance per mL was 290 mOsm/kg (osmolarity of control medium = 281 mOsm/Kg). The pH of this solution was 8.46 (pH of control medium = 7.36).

The cultures dosed with 75µg/mL (-S9-mix) were not analysed for the presence of chromosome aberrations. These cultures would have been analysed only if the highest concentration was unscorable. In the presence of S9-mix a concentration of 400µg/mL was tested at the first fixation time, concentrations up to 600µg/mL at the second fixation time and 400 and 800 µg/mL at the third fixation time. Concentrations were selected on the basis of toxicity.

The cytogenetic test was carried out with duplicate cultures. The results of duplicate cultures are indicated by A and B. The scores for the numbers of aberrant cells (inclusive and exclusive gaps) and the numbers of the various types of chromosome aberrations at the various concentration of the test substance are presented in Tables 2 to 7 (attached). The criteria according to which the aberrations were classified are outlined in appendix 1 (attached).

Referring to Appendix 4 (attached), only in the presence of S9-mix at the second fixation time a statistically significant increase was induced at a concentration of 600 µg/mL. However, as this increase was observed only when gap-containing cells were included and as there was no evidence for a dose-response relationship, a biologically significant effect was not concluded.

The number of cells with chromosome aberrations found in the solvent control cultures (gaps excluded) fell within the laboratory historical control data range (3.1 ± 1.8 {Mean ± SD; N=58} and 3.5 ± 2.2 {N=58} aberrant cells per 100 metaphases in the absence and presence of a metabolic system, respectively). The positive control chemicals (EMS and CP) both produced statistically significant increases in the number of cells with aberrations. It was therefore conluded that the test conditions were optimal and that the metabloic activation system (S9-mix) functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
This in vitro cytogenetic test with CHO cells should be considered valid and the test substance is not clastogenic under the experimental conditions described in this report.

Categories Display