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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
Study period:
1999-11-10 to 1999-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Council Directive 92/69, part B
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethylene dimethacrylate
EC Number:
218-218-1
EC Name:
Tetramethylene dimethacrylate
Cas Number:
2082-81-7
Molecular formula:
C12H18O4
IUPAC Name:
butane-1,4-diyl bis(2-methylacrylate)
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-Butanediol dimethacrylate

Method

Target gene:
not applicable (chromosome aberration test)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F10 + 15% FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
- other: generation time, plating efficiency checked
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 mg/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h; second experiment: 20 h continuous exposure without S9 mix
- Expression time (cells in growth medium): 17 h (recovery period)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): 0.2 µg/mL colcemid for the last 3 h of the treatment period
STAIN (for cytogenetic assays): 3% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 from each culture, two cultures were prepared at each test point

DETERMINATION OF CYTOTOXICITY
- Method: cell count

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, recorded, but not included in the count of eligible metaphases
- Determination of endoreplication: yes, recorded, but not included in the count of eligible metaphases

Evaluation criteria:
A substance is considered clastogenic if: - any dose level shows a statistically significant increase in aberration-bearing cells - the increase is over historical controls - the increase is present in both replicates
Statistics:
Fisher's exact test with solvent control as reference point

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: opacity occurred above 313 mg/L

COMPARISON WITH HISTORICAL CONTROL DATA:
- the number of cells with chromosome aberrations were in the range of the historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in the first experiment in the absence of S9 mix cytotoxicity was observed at 625 mg/L and higher (number of viable cells reduced to 3 and 13% compared to solvent control); at 313 mg/L the number of viable cells was 61% compared to solvent control
- in the second experiment marked toxicity was observed at 313 mg/L and higher (cell counts < 22% compared to solvent control); at 156 mg/L the number of viable cells was reduced to 54% of the solvent control
- in the presence of S9 mix, no dose-related toxicity was observed
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

The test substance did not induce an increase in chromosomal aberrations at any test point selected for scoring in the absence or presence of S9 mix.

A slight, but not dose-related increase in endoreduplication, was observed in the presence of S9 mix.

Increases in gaps over the negative control value were observed in the 3 h treatment group (313 mg/L) without metabolic activation. This was seen in only one replicate and therefore not considered biologically relevant.


Significant increase in aberration-bearing cells was observed in the positive controls indicating the correct functioning of the test system.


3 h treatment, without S9 mix mg/L mean rel. Cell count % cells scored cells with aberrations (+gaps) cells with aberrations (-gaps) %CA







untreated
107 200 11 4 2.0
solvent
100 200 5 3 1.5
test substance 1250 13



625 3



313 (#) 61 200 15 5 2.5

156 (#) 93 200 8 4 2.0

78.1 (#) 79 200 12 6 3.0

39.1 87



19.5 101



9.77 94


Mitomycin C 0.3 75 100 72 68 68.0 ***







3 h treatment, with S9 mix mg/L mean rel. Cell count %


%CA







untreated
70 200 9 3 1.5
solvent
100 200 11 4 2.0
test substance 1250 (#) 89 200 16 6 3.0

625 (#) 69 200 14 8 4.0

313 (#) 103 200 19 7 3.5

156 86



78.1 68



39.1 86



19.5 107



9.77 69


Cyclophosphamide 15 88 100 62 56 56.0 ***







20 h treatment, without S9 mix mg/L mean rel. Cell count %


%CA







untreated
143 200 1 0 0.0
solvent
100 200 3 0 0.0
test substance 1250 0



625 3



313 22



156 (#) 54 200 6 2 1.0

78.1 (#) 112 200 3 1 0.5

39.1 (#) 116 200 7 1 0.5

19.5 120



9.77 94


Mitomycin C 0.3 55 150 62 62 41.3 ***


%CA = percentage of cells bearing aberrations (excl. gaps)

(#) used for scoring

* statistically significant at p< 0.05

** statistically significant at p< 0.01

*** statistically significant at p< 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In this chromsome aberration test 1,4-BDDMA did not induce chromosomal aberrations in CHO cells after in vitro treatment in the presence and absence of S9 metabolic activation.
Executive summary:

In a mammalian cell chromosome aberration assay according to OECD guideline 473, adopted 21 July 1997, CHO cell cultures were exposed to 1,4-BDDMA (93.59% a.i.) in DMSO at concentrations of 0 (control), 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 mg/L with and without metabolic activation (S9 mix). 1,4-Butanediol dimethacrylate was tested up to cytotoxic concentrations.

In the first experiment the cells were exposed to the test substance for 3 h with and without metabolic activation and in a second experiment for 20 h without metabolic activation.

The test substance did not induce an increase in chromosomal aberrations at any test point selected for scoring in the absence or presence of S9 mix.

A slight, but not dose-related increase in endoreduplication, was observed in the presence of S9 mix.

Increases in gaps over the negative control value were observed in the 3 h treatment group (313 mg/L) without metabolic activation. This was seen in only one replicate and therefore not considered biologically relevant.

Positive controls induced the appropriate response.

There was no evidence of chromosome aberrations induced over background.

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