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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:
02 Dec 1993 - 02 Feb 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
No version mentioned
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
No version specified
Deviations:
not specified
GLP compliance:
yes
Type of assay:
other: in vitro mammalian Chromosomal Aberration Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: volatile
Remarks:
Colourless
Details on test material:
Storage : Refrigerator (protected from light ; N2 conditions)

Method

Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
Cell line:
The V79 cell line (1, 2) being derived from the Chinese hamster has a
• high proliferation rate (doubling time of about 12 -16 hours )
• high plating efficiency (> 90% )
• stable karyotype (modal number of 22 chromosomes) .

Storage:
Stocks of the V79 cell line (1 ml portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant . Each batch used for cytogenetic experiments was checked for
• mycoplasma contamination
• karyotype stability
• plating efficiency (incl . vital staining)
Additional strain characteristics:
not specified
Cytokinesis block (if used):
Colcemid
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 fraction
Test concentrations with justification for top dose:
Doses: 250, 500 and 1000 µg/ml (2.5, 5.0 and 9.99 mM)

The doses were determined from appropriate pretests with cultures exposed to a wide dose range of the test article, i .e . 0 .1 µg/ml - 5000 µg/ml culture medium both without and with S-9 mix .
According to the findings of the pretests, 1000 µg/ml without S-9 mix and with metabolic activation were selected as top doses . This selection was based on the observed clastogenic effect, reduced cell count and on the mitotic index .
Vehicle:
- Vehicle/solvent used: DMSO
The stability of the test substance in the carrier DMSO and in aqua dest . over a period of 4 hours was verified analytically .
With the carrier DMSO a solution was obtained and therefore, it was not necessary to verify the homogeneity analytically .
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation 750 µg ethyl-methane-sulfonate (EMS)/ml culture medium added in a volume of 1 .0 ml
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation (S-9 mix) 1 µg cyclophosphamide/ml culture medium added in a volume of 1 .0 ml
Details on test system and conditions:
METHOD OF APPLICATION:
Preparation of test cultures
• Logarithmically growing cultures more than 50% confluent were trypsinized (2 .5% trypsin solution and Ca-Mg-free Hanks Balanced Salt Solution HBSS) . Prior to trypsin treatment the cells were rinsed once with 5 ml Ca-Mg-free HBSS .
• This process was stopped by adding MEM supplemented with 10% FCS.
• A single suspension was prepared and about 5 ml MEM supplemented with 10% FCS and containing about 30 000 - 50 000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture .
• The Quadriperm dishes were incubated at 37°C with 5% CO2 and > 90% humidity.

TREATMENT:
24 hours after seeding and incubating the cells the medium was replaced by fresh medium . The test article, dissolved in 50 Al DMSO, was added to the culture medium with or without 1 ml S-9 mix . Concurrent negative and positive controls (see item 3 .5 .4 .) were tested in parallel .
After incubation (37°C, 5% CO2, > 90% humidity) for 4 hours the serum-free medium was replaced by MEM supplemented with 10% FCS after rinsing twice with Hanks balanced salt solution (HBSS) . Subsequently, the Quadriperm dishes were incubated again until the cells were harvested .

CELL HARVEST AND PREPARATION OF METAPHASE SPREAD:
• 2 - 3 hours prior to harvesting the cells, 0.2 µg Colcemid/ml culture medium (= 1µg Colcemid dissolved in 0.1 ml PBS/culture) was added in each chamber in
order to arrest mitosis in the metaphase .
• After incubation at 37°C the culture medium was completely removed .
• For hypotonic treatment 5 ml of a 0 .4% KC1 solution which was at 37°C was added for about 20 minutes .
• Subsequently 5 ml of fixative (methanol : glacial acetic acid/3 : 1) which was at 4°C was added and kept for at least 15 minutes and then replaced . After about another 10 minutes fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation.
• The slides were taken out of the Quadriperm chambers, briefly dripped off and than rapidly passed through a Bunsen burner flame .
• The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 ml Giemsa, 185 ml Titrisol pH 7 .2) for 10 minutes .
• After being rinsed twice in aqua dest . and clarified in xylene, the preparations were mounted in Corbit-Balsam .

EVALUATION:
Chromosome analysis:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for the test substance, negative and carrier controls or 50 cells of each culture for the positive controls . Due to the very clear increase in the number of chromosomally damaged cells, the number of analyzed metaphases was limited to 150 (without S-9 mix) or 50 (with S-9 mix) and if cells had 20 -22 chromosomes, they were analyzed for chromosome aberrations.

Slides were coded before microscopic analysis . If only a few cells were found or if the metaphases were of low quality, a chromosome analysis was not carried out .


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A mitotic index based on 1500 cells/culture was determined for all test groups that yielded metaphase cells, for the carrier and untreated controls (= negative controls) and for the positive controls .:
- Cell counts: For determination of cytotoxicity additional cell cultures (using 25 cmz plastic flasks) were treated in the same way as in the main experiment . Growth inhibition was estimated by counting the number of cells in the dose groups in comparison to the concurrent carrier control at the end of the culture period using a counting chamber .

- Cell morphology: About 3 hours after test substance treatment cultures of all test groups were checked regarding cell morphology, which is an indication of attachment of the cells to the slides .
- Treatment conditions: pH values and osmolality were measured . The solubility of the test substance in the carrier used and in the aqueous
culture medium was checked to ensure proper culturing and to avoid extreme treatment conditions.

Evaluation criteria:
->Structural chromosome aberrations
- G' and G" chromatid gap and isochromatid gap unstained regions (so-called achromatic lesions) without dislocation of the segment which appears to be separated .
- B' and B" chromatid break and chromosome break visible discontinuity in chromatid or chromosome structure with lateral or longitudinal dislocation of
the fragment .
- F' and F" chromatid fragment and chromosome fragment acentric chromosome segments which occur singly or in pairs .
- D' and D" chromatid deletion and chromosome deletion loss of a segment on the level of chromatids or chromosomes .
- m. A . multiple aberrations metaphases with 5 or more aberrations excl . gaps .
- disintegration of chromosomal structure. The chromosomes being present as particles, a chromosomal structure cannot be detected any longer.
- Exchanges (translocations) These exchange aberrations (Ex) are divided into intrachanges and interchanges :
- Int' and Int" intrachanges on the level of chromatids and chromosomes
the joining of broken ends capable of reuniting two or several chromatid regions within a chromosome, e .g ., centric ring chromosomes,pericentric inversions .
- I' and I" interchanges on the level of chromatids and chromosomes the joining of broken ends capable of reuniting two or several chromosomes. They are classified as :- symmetric interchanges ,e .g ., reciprocal translocations between nonhomologous chromosomes, centric fusions, quadriradial
structures - asymmetric interchanges , e .g ., dicentric and polycentric chromosomes, triradial and quadriradial structures .

->Numerical chromosome aberrations (so-called heteroploidies)
- Aneuploidy metaphases with absent (hypoploid ) or additional (hyperploid) chromosomes. Only hyperploid metaphases are registered .
- Euploidy changes in the number of chromo-(= polyploidy) somes by whole chromosome sets .
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF AG) .
For each group the proportion of metaphases with aberrations was calculated .
A comparison of each dose group with the solvent control group was carried out using Fisher's exact test for the hypothesis of equal proportions . This test was Bonferoni-Holm corrected over the dose groups separately for each time point and was performed one-sided . If the results of this test are significant, labels (* p< 0 .05,** p< 0 .01) were printed in the tables .

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
clear growth inhibition was observed only with S-9 mix at 1000 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: potent chromosomedamaging (clastogenic) agent under in vitro conditions
Additional information on results:
CHROMOSOME ANALYSIS
Assay without S-9 mix ; 18 hours harvest time
- Untreated control :
8 (4 .0%) metaphases incl . gaps
3(1 .5%) metaphases excl . gaps, i .e . 1 x 2B'2Ex ; 2 x m .A . incl . Ex
1 (0 .5%) hyperploid cell
5 (2 .4%) polyploid cells
- Carrier control :
9 (4.5%) metaphases incl . gaps
7 (3 .5%) metaphases excl . gaps, i .e . 1 x B', 1 x D", 5 x Ex
4 (1 .9%) hyperploid cell s
5 (2 .4%) polyploid cells
- 1000 µg/ml :
18 (12 .0%) metaphases incl . gap s
15 (10 .0%) metaphases excl . gaps (see tables 11 - 12)
No (0 .0%) hyperploid cell s
1 (0 .7%) polyploid cell
- 750 µg EMS/ml :
With 22 (22%) aberrant cells incl . gaps and 20 (20%) aberrant mitosis excl . gaps including 1 multiple aberrant metaphase and 16 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells .

Assay with S-9 mix ; 18 hours harvest time
- Untreated control :
21 (10 .5%) metaphases incl . gaps
10 ( 5 .0%) metaphases excl . gaps, i .e . 1 x B' ; 1 x B" ; 1 x 2B" ;4xD" ; 1xF ; 2xF "
2 (1 .0%) hyperploid cells
6 (2 .9%) polyploid cells
- Carrier control :
11 (5 .5%) metaphases incl. gaps •
4 (2 .0%) metaphases excl . gaps, i .e . 1 x B' ; 1 x B" ; 2 x D "
4 (2 .0%) hyperploid cells
3 (1 .5%) polyploid cells
- 1000 µg/ml :
33 (66 .0%) metaphases incl . gap s
32 (64 .0%) metaphases excl . gaps (see tables 13 - 14)
No (0 .0%) hyperploid cell s
No (0 .0%) polyploid cells
- 1 µg cyclophosphamide/ml :
With 31 (31%) aberrant cells incl . gaps and 28 (28%) aberrant metaphases excl . gaps including 2 multiple aberrant metaphases and 19 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells .


MITOTIC INDEX
The mitotic index based on 1500 cells per culture for the different test groups without and with metabolic activation was anlyzed: a suppression of the mitotic activity was not observed .

CELL COUNTS
A clear growth inhibition was observed only with S-9 mix at 1000 µg/ml .

CELL MORPHOLOGY
At a concentration of 1000 µg/ml with and without S-9 mix some cells apeared rounded and there was slightly reduced attachment to the slides.

TREATMENT CONDITIONS
The osmolality of the untreated control was 295 and 170 with and without S-9 mix. The osmolarity of the solvent control and all treatments was from 378 to 461 mOsm
pH ranged from 7.41 to 7.79

TEST SUBSTANCE ANALYSI S
The stability of the test substance in the carrier DMSO and in aqua dest . over a period of 4 hours was verified analytically .
With the carrier DMSO a solution was obtained and therefore, it was not necessary to verify the homogeneity analytically .

Applicant's summary and conclusion

Conclusions:
According to the results of the present in vitro cytogenetic study (OECD guideline 473), the test substance Vinylpropionate (CAS 105-38-4) leads to a very clear and statistically significant increase in the number chromosomally damaged cells both incl . and excl. gaps with a high proportion of exchanges. This increase was much more pronounced after the addition of a metabolizing system when compared to the experimental part without S-9 mix.
The clastogenic activity of the test substance was already observed by scanning the slides of the pretest for quality control and now confirmed in the 1st main experiment. Therefore, a 2nd experiment for further confirmation including an additional sampling time as required by current guidelines was not carried out.
An increase in the number of cells containing numerical chromosomal aberrations was not demonstrated. Thus, under the experimental conditions chosen here Vinylpropionate is considered to be a potent chromosome damaging (clastogenic) agent under in vitro conditions using V79 cells .
Executive summary:

The substance Vinylpropionate (CAS 105-38-4) was assessed for its potential to induce structural and/or numerical chromosomal aberrations in V79 cells in vitro both in the presence and absence of a metabolizing system.

According to pretests for the determination of the highest experimental dose, 250 /µg/ml, 500 µg/ml and 1000 µg/ml culture medium both in the experiment with and without metabolic activation were selected.

Chromosomes were prepared 18 hours after test substance treatment, which lasted for about 4 hours . Duplicate cultures were used for all experimental groups.

About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells in a metaphase-like stage of mitosis (C-metaphase). After preparation of the chromosomes and staining with Giemsa, metaphases were analyzed for chromosomal aberrations.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line.

Both of the positive control chemicals led to the expected increase in the number of cells containing structural chromosomal aberrations.

According to the results of the present study, the test substance caused a clear increase in the number of structural aberrant metaphases incl. and excl. gaps with a high proportion of exchanges. This increase was much more pronounced after adding a metabolizing system in comparison to the experiment without S-9 mix. An increase in the frequency of cells containing numerical aberrations was not demonstrated.

Thus, under the experimental conditions chosen here Vinylpropionate is considered to be a potent chromosome damaging (clastogenic) agent in V79 cells in vitro.