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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 17 JAN 2007 to 21 MAR 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 473) and according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
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): Dytek DCH-99
- Substance type: clear, light yellow liquid
- Physical state: fluid
- Analytical purity: 99.55 %
- Composition of test material, percentage of components:
99.55 % 1,2-diaminocyclohexane
0.24 % 2-aminomethylcyclopentylamine
0.08 % hexamethyleneimine
0.07 % 2-methyl-1,5-pentamethylenediamine
0.06 % water
- Lot/batch No.: VS52185886
- Expiration date of the lot/batch: 2007-10-02
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark under nitrogen

Method

Target gene:
none
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemented with foetal calf serum, L-glutamin, penicillin /streptomycin and heparin
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (induced with phenobarbital and beta-naphtoflavone)
Test concentrations with justification for top dose:
1. cytogenetic assay: 0, 333, 666, 1000, 1142 µg/ml culture medium
2. cytogenetic assay:
without S9 mix: 0, 33, 100, 200, 300, 400, 500, 750 µg/ml culture medium
with S9 mix: 333, 666, 1000, 1142 µg/ml culture medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI-1640 medium (test substance) or Hanks' Balanced Salt Solution without calcium and magnesium (positive controls)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
1. cytogenetic assay:
- Preincubation period of cells: 48 h
- Exposure duration: 3 h
- Expression time (cells in growth medium): 20-22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

2. cytogenetic assay:
- Preincubation period of cells: 48 h
- Exposure duration: without S9mix: 24 h (concentrations up to 400 µg/ml) or 48 h (concentraions starting from 500 mg/ml), with S9 mix: 3 h
- Expression time (cells in growth medium): 44-46 h (with S9 mix), none without S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): without S9mix: 24 h (concentrations up to 400 µg/ml) or 48 h (concentraions starting from 500 mg/ml), with S9 mix: 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: each concentration was tested in duplicate, the whole test was repeated

NUMBER OF CELLS EVALUATED:
mitotoc index: number of metaphases/ 1000 cells
chromosome aberration: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Acceptability of the assay
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations,
c) A homogeneous response between the replicate cultures is observed
d) A possible precipitate present an the slides should not interfere with the scoring of chromosome aberrations.

Data evaluation
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) lt induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test resultsopen allclose all
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic index reduced starting at concentrations of 1000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
mitotic index reduced starting at concentrations of 333 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
At the concentration of 1142 µg/ml (highest concentration tested) test substance precipiteated in the culture medium

RANGE-FINDING/SCREENING STUDIES:
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 0.01 M (3 h exposure time) or had an inhibition of the mitotic index of 50 % or greater whereas the mitotic index of the lowest dose level is approximately the same as the mitotic Index of the solvent control (24 h and 48 h continuous exposure times).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Evaluation of the results

The ability of Dytek® DCH-99 to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on toxicity (24 h and 48 h continuous exposure times), inhibition of the mitotic index of about 50% or greater or the recommended 0.01 M (3 h exposure time).

The evaluation of cells was based on the results of the cytotoxicity tests (mitotic index). Finally the chromosomal aberrations were determined in cells either

- treated with test item in concentrations of 666, 1000 and 1142 µg/ml in the presence and absence of S9 mix (3 h exposure time, 24 h fixation time; 1st cytogenetic experiment),

- or with test item in concentrations of 33, 200 and 400 µg/ml in the absence of S9 mix (24 h exposure time, 24 h fixation time, 2nd experiment),

- or with test item in concentrations of 100, 300 and 500 µg/ml in the absence of s) mix (48 h exposure time, 48 h fixation time, 2nd experiment),

- or with test item in concentrations of 333, 1000 and 1142 µg/ml in the presence of s) mix (3 h exposure time, 24 h fixation time, 2nd experiment).

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. lt was therefore concluded that the test conditions ware adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Dytek® DCH-99 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Dytek DCH-99 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Dytek® DCH-99 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the condotions of the test, the test item did not induce chromosome aberrations in human lymphocytes.
Executive summary:

In an OECD 473 guideline study induction of chromosome aberrations by the test item has been investigated in human lymphocyte cells in vitro in the presence (induced rat liver S9) and absence of metabolic activation. Based on the results of the cytotoxicity tests (mitotic index) the chromosomal aberrations were determined in cells either

- treated with test item in concentrations of 666, 1000 and 1142 µg/ml in the presence and absence of S9 mix (3 h exposure time, 24 h fixation time; 1st cytogenetic experiment),

- or with test item in concentrations of 33, 200 and 400 µg/ml in the absence of S9 mix (24 h exposure time, 24 h fixation time),

- or with test item in concentrations of 100, 300 and 500 µg/ml in the absence of s) mix (48 h exposure time, 48 h fixation time),

- or with test item in concentrations of 333, 1000 and 1142 µg/ml in the presence of s) mix (3 h exposure time, 24 h fixation time).

The test item did not induce chromosome aberrations in any of these tests performed under the described test conditions.