Registration Dossier

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:
5 October 2009 - 19 November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Specific details on test material used for the study:
The test item, CTF (Cyclic Trimethylolpropane Formal), Batch No. 3977009 was received from Perstorp Holding AB on 04 September 2009. The test item was supplied with an expiry
date of 03 September 2010, which was one year from dispatch. The test item, a liquid, was stored at ambient temperature, protected from light and under Nitrogen headspace when not in
use. A Certificate of Analysis was supplied with the test item.

Method

Target gene:
Not applicable - chromosome aberration study
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cell line used was Chinese hamster ovary (CHO 10 B4) cells, obtained from the University of Leiden in 1987. The cell line is mycoplasma tested (in house) on a regular basis. The cells were grown as monolayers, and have a generation time of approximately 12 h. The modal chromosome number has been determined for these cells to be 21. The cells were incubated at ca 37°C.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Colcemid was added to all cultures at a final concentration of 0.1 μg/mL.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of Aroclor 1254 induced adult male Fischer rats
Test concentrations with justification for top dose:
For the first experiment, 9 dose levels, covering a wide concentration range, were tested. The highest dose was the limit concentration of 1462 μg/mL (10 mM) and subsequent dose levels were halving dilutions.In the second experiment, the dose levels selected were: 183, 366, 731 and 1462 μg/mL
Vehicle / solvent:
The test item was freely soluble in Ham’s F-10 medium.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9; 20-50 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; 10-40 µg/ml
Details on test system and experimental conditions:
The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10% v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.Cells were trypsinised from stock flasks at passage numbers 17 (Test 1) and 14 (Test 2), and resuspended in fresh culture medium at densities of 0.1 x 10exp6 or 0.05 x 10exp6 cells/mL. These cells, in 5 mL volumes, were dispensed into 25 cm² tissue culture flasks. The high and low cell densities were for cultures harvested at 24 or 48 h post treatment respectively. Test cultures were established from the stock flask about 20 h before testing.All experimental procedures, up to harvesting, were conducted using aseptic technique and under amber light.Tests were conducted both in the presence and absence of S9 mix. Treatments with test item or vehicle control substances were performed on du plicate cell cultures. Several concentrations of the positive controls were tested using single cultures. Cultures to be treated in the presence of S9 mix were washed before treatment with serum free medium. Exposure medium was prepared, immediately before dosing, in sterile containers. The final approximate volume per culture was 5 mL.Test 1 was conducted both with and without S9 mix, the treatment period was 0-6 h, with a recovery period of 6-22 h. Colcemid was added at 22-24 h, and the cells were harvested at 24 h.Test 2 was conducted with S9 mix in a identical manner to Test 1. Test 2 conducted without S9 mix employed either: no recovery period, colcemid was added at 22-24 h and cells harvested at 24 h; or a 22-46 h recovery period, colcemid added at 46-48 h and cells harvested at 48 h. After treatment, cells were washed twice with serum free medium, then full growth medium added, for the recovery period and colcemid treatment. The volume of medium for the recovery period was 5 mL.Harvesting of cultures: Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase; the stage of cell division at which chromosomes can be examined using light microscopy. Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation (approximately 190 g), and treated with hypotonic solution (1% trisodium citrate) for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using 4 mL of freshly prepared fixative (methanol:glacial acetic acid, 3:1). Two further changes (after sedimentation as before) of fixative were made. Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity. For both experiments, 3 slides per culture were made. All slides were marked with the study number and assigned a unique, coded number from a computer generated sequence. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with 5% Giemsa, then made permanent by mounting coverslips with DPX mountant. Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures. Slides were examined for evidence of metaphase cells and signs of cellular necrosis. Three concentration levels were selected for assessment of chromosomal aberrations. From 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture, were examined where possible. Slides were scored in order of coded number. A reduced number of metaphases were scored if a high proportion (≥40%) of metaphase cells were found to be damaged. A microscope was used for this assessment, the magnification used being x 1000 or x 1250, achieved with x 10 or x 12.5 eyepieces and x 100 objective. The number of chromosomes in each metaphase cell and all abnormalities, using the nomenclature of Gebhart (1970) was recorded. The types of structural and numerical aberrations recorded are listed in abbreviations page. The positions on the slides of any structurally aberrant cells were recorded using the Vernier scale on the microscope stage.As cultures harvested at both culture times were negative with regards to structural aberrations, a further assessment of polyploidy was made. The Study Director selected the later harvest time as being more appropriate for this analysis. This decision was based on results from the previous assessments. For this assessment, approximately 300 metaphase cells were cursorily examined at a magnification of x 400 or x 500 and deemed to be either diploid, polyploid or endoreduplicated. In this assessment no metaphase cell in a field was rejected. This assessment was considered more objective, avoiding scorer selection of either normal or polyploid cells. In addition, the larger sample size gave a more accurate frequency of such cells in the population.
Evaluation criteria:
Toxicity: A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.Clastogenicity: The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate. A test was positive if the response in at least one acceptable dose level was significant by the criterion described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No toxicity was noted in any of the cultures treated with CTF, when tested to a limit concentration of 1462 μg/mL.Cultures treated with the following dose levels of CTF were selected for assessment of chromosomal aberrations: 366, 731 and 1462 μg/mL.The vehicle control cultures had levels of structural and numerical aberrations within the 95% confidence limits of the historical negative control data. The positive control substances, cyclophosphamide in the presence and methyl methanesulphonate in the absence of S9 mix, induced positive frequencies of structural aberrations in at least one concentration level per section. These results demonstrated the sensitivity of the test system.All cultures treated with CTF had levels of structural aberrations within the 95% confidence limits for a negative response. An extra assessment of polyploidy was carried out on the cultures treated in the absence of S9 mix and harvested at 48 h. All the cultures treated with CTF had levels of polyploidy within the 95% confidence limits for a negative response.

Any other information on results incl. tables

Frequencies of aberrant cells (exluding gaps)

 

Test 1

Test 2

6h/24h

6h/24h

22h/24h

22h/48h

+S9

-S9

+S9

-S9

-S9

CTF

0 µg/mL

0.0

0.0

0.0

0.0

0.0

388 µg/mL

0.0

0.0

0.0

0.0

0.0

731 µg/mL

0.0

0.5

0.0

0.5

0.0

1462 µg/mL

0.0

0.0

0.5

0.0

0.0

CPA

20 µg/mL

 

 

12

 

 

30 µg/mL

19

 

27

 

 

40 µg/mL

40

 

 

 

 

MMS

10 µg/mL

 

 

 

1

 

20 µg/mL

 

 

 

10

3

30 µg/mL

 

6

 

 

10

40 µg/mL

 

10

 

 

 

Applicant's summary and conclusion

Conclusions:
It was concluded that CTF was not clastogenic when tested with Chinese hamster ovary cell in vitro.
Executive summary:

CTF (cyclic trimethylolpropane formal) was submitted for testing and evaluation of clastogenic potential. Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary cell cultures. This study was conducted incorporating 2 independent tests. Ham’s F-10 medium was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. Both tests were conducted in the presence and absence of a post-mitochondrial supernatant fraction obtained from the livers of adult, male rats treated with Aroclor 1254 (S9) and a NADPH-generating system. Cultures, established approximately 20 h before testing, were treated for 6 h in the presence and 6 h or 22 h in the absence of S9 mix. Cultures were harvested at 24 h (Test 1 and 2) or 48 h (Test 2) post treatment. CTF was non toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix when tested to the limit concentration of 1462 μg/mL (10 mM). There was no evidence that CTF induced structural chromosomal aberrations in either the presence or absence of S9 mix. CTF induced an increase in polyploidy in both the presence and absence of S9 mix in cultures harvested 48 h post treatment. It was concluded that CTF was not clastogenic when tested with Chinese hamster ovary cells in vitro.