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Genetic toxicity: in vitro

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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:
1995-05-22 to 1995-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted at a concentration of ca. 39.5 % test substance in an aqueous solution but limit dose of 10 mM was achieved.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 1983/Draft September 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
December 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Version / remarks:
May 1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride
EC Number:
947-115-0
Molecular formula:
C2H2Na2O5S2.NaCl
IUPAC Name:
Reaction mass of Disodium (sulphonatothio)acetate and sodium chloride
Test material form:
liquid
Details on test material:
Purity of Reaction mass of Disodium (sulphonatothio)acetate and Sodium chloride: ca. 39.5 %
Rest: ca. 60.5 % (water and impurities)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- ot/batch No.of test material: 80-9159

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELL LINE, STORAGE
The V79 cell line 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).

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 the cytogenetic experiments was checked for
- mycoplasma contamination
- karyotype stability
- plating efficiency (incl. vital staining).

Deep-frozen cell suspensions were thawn at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5.0 mL MEM (minimal essential medium incl. glutamine), supplemented with 10% FCS (fetal calf serum) and antibiotics. Cells were grown with 5 % CO2 at 37 °C and >= 90% humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after dispersion with 2.5 % trypsin solution (about 0.1 mL).
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 fraction
Test concentrations with justification for top dose:
1st experiment: without S-9 mix 0, 500, 1000, 2000 µg/mL (i.e. 0, 197.5, 395, 790 µg/ml of active compund); with S-9 mix 0, 3500, 5000, 6500 µg/mL (i.e. 0, 1382.5, 1975, 2567.5 µg/ml of active compund)
2nd experiment: without S-9 mix, 18 hours harvest time: 0, 500, 1000, 2000 µg/mL (i.e. 0, 197.5, 395, 790 µg/ml of active compund); with S-9 mix, 18 hours harvest time 0, 3500, 5000, 6500 µg/mL (i.e. 0, 1382.5, 1975, 2567.5 µg/ml of active compund);without S-9 mix, 28 hours harvest time 0, 2000, 3000 µg/mL; (i.e. 0, 790, 1382.5, µg/ml of active compund) with S-9 mix; 28 hours harvest time 0, 5000, 6500 µg/mL (i.e. 1975, 2567.5 µg/ml of active compund)
3rd experiment: without S-9 mix 0, 1000, 2000, 3000 µg/mL (i.e. 0, 395, 790, 1185 µg/ml of active compund); with S-9 mix 0, 6000, 7000 µg/mL (i.e. 0, 2370, 2765, µg/ml of active compund)
Vehicle / solvent:
Due to the good solubility of the test substance in water, the aqueous culture medium (MEM) was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (S-9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
with metabolic activation (S-9 mix)
Details on test system and experimental conditions:
DOSES PER EXPERIMENT
- 1st experiment
The following doses were selected for the 1st experiment using an exposure time of 4 hours with S-9 mix and of 18 hours without S-9 mix:

Doses without S-9 mix; 18 hours harvest time
250 µg/mL aqueous test substance = 98.75 µg/mL active component
500 µg/mL aqueous test substance = 197.5 µg/mL active component
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component

Doses with S-9 mix; 18 hours harvest time
2000 µg/mL aqueous test substance = 790 µg/mL active component
3500 µg/mL aqueous test substance = 1382.5 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

In general, depending on the toxicity actually found three dose levels were assessed.

- 2nd experiment
The following doses were selected for the 2nd experiment again with an exposure time of 4 hours using S-9 mix and with a continuous treatment of
18 hours without S-9 mix:

Doses without S-9 mix; 18 hours harvest time
500 µg/mL aqueous test substance = 197.5 µg/mL active component
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component

Doses without S-9 mix; 28 hours harvest time
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component

Doses with S-9 mix; 18 hours harvest time
3500 µg/mL aqueous test substance = 1382.5 µg/mL active component
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

Doses with S-98 mix; 28 hours harvest time
5000 µg/mL aqueous test substance = 1970 µg/mL active component
6500 µg/mL aqueous test substance = 2567.5 µg/mL active component

This selection was based on the findings from the 1st cytogenetic experiment. Again, three dose levels were assessed at a sampling time of 18 hours. At the additional later harvest time of 28 hours two doses were evaluated both with and without metabolic activation.

- 3rd experiment
The following doses were selected for the 3rd experiment with an exposure time of 4 hours with S-9 mix and with a continuous treatment of 18 hours
without S-9 mix:

Doses without S-9 mix; 28 hours harvest time
1000 µg/mL aqueous test substance = 395 µg/mL active component
2000 µg/mL aqueous test substance = 790 µg/mL active component
3000 µg/mL aqueous test substance = 1185 µg/mL active component
4000 µg/mL aqueous test substance = 1580 µg/mL active component

Doses with S-9 mix; 28 hours harvest time
4000 µg/mL aqueous test substance = 1580 µg/mL active component
5000 µg/mL aqueous test substance =1970 µg/mL active component
6000 µg/mL aqueous test substance = 2370 µg/mL active component
7000 µg/mL aqueous test substance = 2765 µg/mL active component

This selection was based on the findings from the 2nd experiment. Two doses with metabolic activation and 3 doses without S-9 mix were evaluated.

CELL CYCLE TIME
The cell cycle of the untreated V79 cells lasted for about 13 - 14 hours [last measurement based on the BrdU method of SPEIT, G. et al.: December 1995] under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours was within the 1 - 1.5 x the normal cell cycle time, as recommended in an "EEC Guidance Note - The practical interpretation of Ames V Test Method B 10, the in vitro mammalian cell cytogenetic test".
The later sampling time of 28 hours was chosen to cover a possible cell cycle delay.

SAMPLING TIMES
Chromosomal aberrations were generally analyzed in the first metaphase after they had formed to avoid loss during mitoses or conversion of the initial aberrations into more complex derivatives during subsequent cell cycles. Since aberrations are induced by the majority of chemical clastogens during DNA replication, the harvest time must allow cells to progress through the 5-phase after treatment to convert initial DNA damage
into chromosome alterations visible at mitosis. Since V79 cells are asynchronous and different chemicals may affect different stages of the cell
cycle, more than one sampling time is necessary. Furthermore, mitotic delay may result from clastogen exposure and thus considerably delay the first post-treatment mitosis. Thus, samples were taken at 18 hours and 28 hours after the beginning of a 4-hour treatment (with S-9 mix) or of an 18-hour treatment (without S-9 mix) covering the intervals at which maximum aberration frequencies were expected.

TREATMENT OF TEST CULTURES
About 24 - 30 hours after seeding and incubating the cells, the medium was replaced by fresh medium. The test article, dissolved in 1 mL serum-free medium (4-hour treatment) or in medium with FCS (18-hour treatment), was added to the culture medium with or without 1 mL S-9 mix. Concurrent negative and positive controls were tested in parallel. After incubation (5 % CO2, 37 °C and >= 90 % humidity) for 4 hours with S-9 mix the serum-free medium was replaced by MEM supplemented with 10 % FCS after being rinsed twice with Hanks' balanced salt solution (HBSS). Subsequently, the Quadriperm dishes were incubated again for another 14 hours or 24 hours until the cells were harvested. In the experiments without S-9 mix, cells were treated for 18 hours in culture medium supplemented with 10 % FCS.

CELL HARVEST AND PREPARATION OF METAPHASE SPREADS
The cells were prepared based on the methods described by SCHMID, W. and SPEIT, G. and S. HAUPTER.
- 2 - 3 hours prior to harvesting the cells, 0.2 µg colcemid/mL culture medium (= 1 µg colcemid dissolved in 0.1 mL PBS/culture) was added to 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 % KCl 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, the 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 then 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 purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

CHROMOSOME ANALYSIS
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations. Numerical chromosome aberrations were also recorded. lf there is a clear increase in chromosomally damaged cells the number of metaphases to be analyzed is reduced from the planned 200 mitoses/test group.

MITOTIC INDEX
A mitotic index based on 1000 cells/culture was determined for all test groups.

CELL COUNTS
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 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 with the concurrent vehicle 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 for 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 vehicle used and in the aqueous culture medium about 3 hours after treatment was checked to ensure proper culturing and to avoid extreme treatment conditions.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
- The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies.
- The aberration frequencies were within the historical control range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHIAN program System (BASF AG). The proportion of metaphases with aberrations was calculated for each group. A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
other: slight but statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after a sampling time of 28 hours in two experiments independent of each other
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Both of the positive control chemicals, i.e. EMS and cyclophosphamide, 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 slight but statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after a sampling time of 28 hours without S-9 mix in two experiments independent of each other.

Thus, under the experimental conditions of this assay, the test substance is considered to have a weakly chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells.

Any other information on results incl. tables

1st Experiment

Without S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 7 3.5 4 2 3 1.5 0 0 0 0 0 0 1 0.5
500 (198) 18 200 19 9.5* 3 1.5 2 1 0 0 0 0 0 0 3 1.5
1000 (395) 18 200 19 9.5* 4 2 0 0 1 0.5 0 0 0 0 0 0
2000 (790) 18 200 34 17.0** 9 4.5 2 1 0 0 0 0 0 0 1 0.5
EMS 350 18 100 15 15.0** 13 13.0** 8 8.0* 0 0 0 0 0 0 0 0

With S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H.  Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 13 6.5 4 2 3 1.5 0 0 0 0 1 0.5 4 2
3500 (1383) 18 200 15 7.5 10 5 6 3 0 0 0 0 0 0 2 1
5000 (1975) 18 200 11 5.5 4 2 1 0.5 1 0.5 0 0 0 0 1 0.5
6500 (2568) 18 200 9 4.5 5 2.5 3 1.5 0 0 0 0 3 1.5 1 0.5
CPP 0.5 18 100 15 15.0* 13 13.0** 9 9.0** 0 0 0 0 0 0 0 0

2nd Experiment

Without S-9 mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Metaphases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 8 4 1 0.5 1 0,5 0 0 0 0 0 0 0 0
500 (198) 18 200 9 4.5 3 1.5 2 1 1 0.5 0 0 0 0 0 0
1000 (395) 18 200 4 2 3 1.5 3 1.5 0 0 0 0 0 0 0 0
2000 (790) 18 200 18 9 6 3 4 2 0 0 0 0 0 0 0 0
EMS 350 18 100 13 13.0* 13 13.0** 10 10.0** 0 0 0 0 0 0 0 0

With S-9 Mix, 18 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 18 200 8 4 2 1 0 0 0 0 0 0 1 0.5 0 0
3500 (1383) 18 200 18 9 8 4 3 1.5 0 0 0 0 0 0 0 0
5000 (1975) 18 200 13 6.5 2 1 1 0.5 0 0 0 0 0 0 0 0
6500 (2568) 18 200 11 5.5 5 2.5 1 0.5 0 0 0 0 1 0.5 2 1
CPP 0.5 18 100 17 17.0** 15 15** 9 9.0** 0 0 0 0 0 0 0 0

Without S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 10 5 3 1.5 0 0 0 0 0 0 1 0.5 0 0
2000 (790) 28 200 24 12.0* 2 1 0 0 0 0 0 0 1 0.5 1 0.5
3000 (1185) 28 200 21 10.5* 13 6.5* 8 4.0** 0 0 0 0 0 0 1 0.5

With S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. cultures Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 6 3 3 1.5 0 0 0 0 0 0 0 0 0 0
5000 (1975) 28 200 11 5.5 5 2.5 2 1 0 0 0 0 0 0 0 0
6500 (2568) 28 200 21 10.5** 0 0 0 0 0 0 0 0 0 0 1 0.5

3rd Experiment

Without S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 11 5.5 5 2.5 2 1 0 0 1 0.5 3 1.5 0 0
1000 (395) 28 200 25 12.5* 8 4 4 2 0 0 0 0 0 0 2 1
2000 (790) 28 200 26 13.0* 16 8.0* 9 4.5 0 0 0 0 1 0.5 0 0
3000 (1185) 28 200 39 19.5** 16 8.0* 10 5 0 0 0 0 1 0.5 0 0
EMS 350 28 100 19 19.0** 16 16.00** 6 6 1 1 0 0 0 0 0 0

With S-9 Mix, 28 hours harvest time

Dose (active compound) [µg/mL] H. Meta-phases Incl. Gaps Excl. Gaps Exchanges Mul. Aber. Chr. Dis. Aneupl. Polypl.
N % N % N % N % N % N % N %
Vehicle MEM 28 200 11 5.5 6 3 0 0 0 0 0 0 0 0 0 0
6000 (2370) 28 200 19 9.5 8 4 3 1.5 0 0 0 0 2 1 1 0.5
7000 (2765) 28 200 16 8 9 4.5 4 2 0 0 0 0 0 0 1 0.5
CPP 0.5 28 100 18 18.0** 16 16.0** 6 6.0** 0 0 0 0 0 0 0 0

Fisher's Exact Test (one-sided) with Bonferroni-Holm correction: *: p <= 0.05, **: p <= 0.01

A pairwise comparison of each dose group with the solvent control group, Bonferroni-holm corrected for each time.

MEM: Minimal essential medium incl. glutamine

CPP: Cyclophosphate

Applicant's summary and conclusion