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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19.09.2016 to 26.01.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: Chromosome aberration test in mammalian cells (in vitro cytogenetic study)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254
Test concentrations with justification for top dose:
125, 250, 500, 1000, 2000 µg/mL medium of DINCD
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
yes
Remarks:
ethanol (vehicle)
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h without S9 mix; 4h with S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells):
Two hours before termination the cell division was arrested by the addition of 0.5 mL of the spindle poison Colcemid® to each culture (10 µg/mL solution). The tubes were capped and left to incubate for a further two hours.
The cells were harvested by low speed centrifugation (80 - 100 x g) and the pellets of cells collected were resuspended in hypotonic potassium chloride solution (0.56%) and fixed in freshly prepared methanol : glacial acetic acid fixative (4:1, v/v).

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid®

STAIN (for cytogenetic assays):
Giemsa stain (1:10 in WEISE's buffer12 pH 6.8)

NUMBER OF REPLICATIONS:
duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa stain (1:10 in WEISE's buffer12 pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT mountant after clearing in xylene.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
For each culture (duplicate) 150 metaphases were examined, yielding a total of 300 metaphases for each concentration analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity:To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
Rationale for test conditions:
Acceptance of the test was based on the following criteria:
o The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
o Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
o All three experimental conditions were tested unless one resulted in positive results (see paragraph 28 of the OECD 473).
o Adequate number of cells and concentrations are analysable (paragraphs 31 and 21 of the OECD 473).
o The criteria for the selection of top concentration are consistent with those described in paragraphs 22, 23 and 24 of the OECD 473.
o Both replicate cultures lead to similar results.
o Adequate numbers of cells (i.e. at least 1000 countable cells) and concentrations re-analyzable.
Evaluation criteria:
The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o The number of chromosomal aberrations is significantly (at p  0.05) increased compared with the solvent control in at least one of the test concentrations
o The increase observed is concentration-dependent
o The increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o Any of the results are outside the distribution of the historical negative control data
o A reproducible increase in the number of cells with chromosomal aberrations
o All three experimental conditions were tested unless one resulted in positive results
Statistics:
The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p  0.05) as recommended by the UKEMS guidelines (1989).

Results and discussion

Test results
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, Diisononyl 1 ,4-cyclohexanedicarboxylate (DINCD) tested up to a concentration of 2000 μg /ml medium in the absence and
presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay .