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Genetic toxicity: in vitro

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in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 8 - March 27 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study followed OECD guidelines and was conducted under GLP assurances

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
EC Number:
EC Name:
2,2,4,4-tetramethylcyclobutane-1,3-diol, mixed isomers
Cas Number:
Molecular formula:
Details on test material:
Identity: 2,2 ,4,4-tetramethyl-1 ,3-cyclobutanediol
CAS No.: 3010-96-6
Batch No.: X29601-54-5
Aggregate state at room temperature: Solid
Colour: White
Molecular weight: 144.21 g/mol
Purity: 99.6%
Analysis: GC
Solubility in water: 5.9 g/100 gat 36.5 C
Solubility in solvent:Methanol: 47.5 g/100 gat 24 C
Storage: Room temperature
Expiration Date: December 31, 2007


Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP. Technical University Darmstadt, D-·64287 Darmstadt) were
stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
45.0, 90.0, 180.0, 360.0, 720.0, and 1440.0 ug/ml +/- S9
Vehicle / solvent:
DMSO (E. MERCK, D-64293 Darmstadt; purity 99.5 %)
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Positive control substance:
Details on test system and experimental conditions:
Thawed stock cultures were propagated at 37° C in 80 cm2 plastic flasks. About 5 x 10-5 cells per flask were seeded into 15 mL of MEM
(Minimal Essential Medium) supplemented with 10% fetal calf serum (FCS). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Phenobarbitai/beta-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old
male Wistar Hanlbm rats, (weight approx. 220 - 320 g) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and beta-Naphthoflavone p.o.
each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver
homogenate with a KCI solution (1 :3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 C. The protein concentration in the 89 preparation was 32.6 mg/mL (Lot no. 021205). An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0. 75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCI2
33 mM KCI
5 mM glucose-6-phosphate
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. The general
experimental conditions in this pre-test were the same as for the cytogenetic main experiment. The following method was used:
In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were
treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control.

The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian
cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/ml, 5ul/ml, or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. With respect to the molecular weight, 1440 ug/ml of
2,2,4,4-tetramethyl-1 ,3-cyclobutanediol was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations
between 11.3 and 1440 ug/ml (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item after 4 hrs treatment was observed.

Exponentially growing stock cultures more than 50 % confluent are treated with trypsinEDTA-solution at 37 C for approx. 5 minutes. Then the
enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all
subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution. Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution.
The Ca-Mg-free salt solution is composed as follows (per litre):
NaCI 8000 mg
KCI 200 mg
KHzP04 200 mg
NazHP04 150 mg

The cells were seeded into Quadriperm dishes which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber
1 x 10-4 to 6 x 10-4 cells were seeded with regard to the preparation time. The medium was MEM with 10% FCS (complete medium).

Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium
(for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 uL S9 mix per ml medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre):

NaCI 8000 mg
KCI 400 mg
Glucose x H20 1100 mg
Na2HP04 x 7H20 290 mg
KH2P04 150 mg
pH was adjusted to 7.2

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells. All cultures were incubated at 37 C in a humidified
atmosphere with 1.5 % C02 (98.5 % air).

Colcemid was added (0.2 IJg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the
slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 3r C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After
preparation the cells were stained with Giemsa.

For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell
number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der lndustrie, Cytogenetik") using NIKON
microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as
structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread
metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II at the 28 hrs
preparation interval with metabolic activation, where only 50 metaphase plates per culture were scored. Only metaphases with characteristic
chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index(% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Evaluation criteria:
The chromosome aberration test performed in our laboratory is considered acceptable if it
meets the following criterion:
a) The number of structural aberrations found in the solvent controls falls within the range of our historical laboratory control data: 0.0- 4.0 %
aberrant cells, exclusive gaps.

b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are
within the range of the laboratory's historical control data:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0- 4.0%
aberrant cells, exclusive gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if: the number of induced structural chromosome aberrations is not in the range of our
historical control data (0.0- 4.0 cvo aberrant cells, exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and
endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be
considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Remarks on result:
other: strain/cell type:
Migrated from field 'Test system'.

Applicant's summary and conclusion

It can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined
by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, 2,2,4,4-tetramethyl-1 ,3-cyclobutanediol is
considered to be non-clastogenic in this chromosome aberration test when tested up to the highest required concentration in the
absence and presence of metabolic activation.
Executive summary:

The test item 2,2,4,4-tetramethyl-1 ,3-cyclobutanediol, dissolved in DMSO, was assessed for

its potential to induce structural chromosome aberrations in V79 cells of the Chinese

hamster in vitro in two independent experiments. The following study design was performed:

           Without S9     With S9
   Exp I Exp II      Exp I Exp II 
 Exposure Period  4 hrs  18 hrs  28 hrs  4 hrs 4 hrs 
 Recovery  14 hrs  -  -  14 hrs  24 hrs
 Preparation Interval  18 hrs  18hrs  28 hrs  18 hrs  28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in Experiment II with metabolic activation, where only 50 metaphase plates per culture were scored. The highest applied concentration in the pre-test on toxicity (1,440 ug/ml; approx. 10 mM) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. The chosen treatment concentrations were 45.0, 90.0, 180.0, 360.0, 720.0, and 1440.0 ug/ml +/- S9. In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. In Experiment I, in the presence of S9 mix, a dose dependent increase in the number of cells carrying structural chromosome aberrations was observed, but the values were within the range of our historical control data and considered as biologically irrelevant.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases

(p < 0.05) in cells with structural chromosome aberrations.