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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:
weight of evidence
Study period:
1998-09-14 to 1999-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro chromosome aberration

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
see below

Method

Target gene:
n.a.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
MEDIA USED:
MEM + 10% FCS (complete medium)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The test concentration were chosen based on the results from a pre-test.
Main study:
without S9: 0.625, 1.25, 2.5, 5.0 and 10.0 µg/mL
with S9: 3.125, 6.25, 12.5, 18.75 and 25.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionized water
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group. In each chamber 44000 cells/slide were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT:
EXPOSURE PERIOD 4 hours (with S9 mix): The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing different concentrations of the test article and 50 µL/mL S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
EXPOSURE PERIOD 18 hours (without S9 mix): The culture medium of exponentially growing cell cultures was replaced with complete medium (10 % FCS) containing different concentrations of the test article 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 4.5 % C02 (95.5 % air).

PREPARATION OF THE CULTURES:
16 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa. Additionally, two cultures V79 cells per treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the substance is given as reduction of % cells as compared to the solvent control.

ANALYSIS OF METAPHASE CELLS:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (9)) 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 metaphases per culture were scored for cytogenetic damage on coded slides. 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 was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Rationale for test conditions:
according to OECD test guideline 473
Evaluation criteria:
A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are 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 are observed.
A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
RANGE-FINDING/SCREENING STUDIES:
The highest applied concentration in the pre-test on toxicity (1600 µg/ml) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test concentrations between 12.5 and 1600 µg/ml (with and without S9 mix) were applied for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers below 50 % of the corresponding control were observed after treatment with 12.5 µg/ml (21 % of control) and above. In the presence of S9 mix after 4 h treatment reduced cell numbers were determined at 25.0 µg/ml (22 % of control) and above. Precipitation of the test article in culture medium was observed in the absence of S9 mix after treatment with 100 µg/ml and at 200 µg/ml in the presence of S9 mix. No influence of the test article on the pH value or osmolarity was observed (solvent control: 280 mOsm, pH 7.3 versus 278 mOsm and pH 7.3 at 1600 µg/ml).

MAIN EXPERIMENT:
In the main experiment the mitotic indices of the evaluated concentrations in the presence and absence of S9 were reduced to 39.1 % (2.5 µg/mL) and 26.5 % (12.5 µg/mL) of control. However, the corresponding cell numbers were not reduced. The evaluation of cultures after treatment with the next higher concentrations was not feasible, since no or not enough mitotic cells were found. Thus, only the concentrations groups 2.5 µg/mL (without S9) and 12.5 µg/mL (with S9) were chosen for further evaluation for structural chromosome aberrations. Neither a significant nor a statistically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test article (0.5 % and 1.0 %, excluding gaps) were near to the range of the solvent control values (1.0 % - 2.0 %) and within the range of our historical control data: 0.0 % - 4.0 %. EMS (600 µg/ml) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Any other information on results incl. tables

Table 1: Pre-test: Determination of Toxicity
Concentration µg/mL mean number of cells % of solvent control
without S9
solvent control 870 100
12.5 185 21
25 189 22
50 167 19
100 149 17
200 204 24
400 185 21
800 141 16
1600 172 20
with S9 mix
solvent control 554 100
12.5 385 69
25 123 22
50 176 32
100 81 15
200 227 41
400 147 27
800 196 35
1600 not evaluated not evaluated
Table 2: Main experiment: Determination of Toxicity
Concentration µg/mL mean number of cells % of solvent control
without S9
solvent control 441 100
0.625 655 149
1.25 462 105
2.5 268 61
5.0 421 96
10.0 82 19
with S9 mix
solvent control 676 100
3.125 332 49
6.25 386 57
12.5 343 51
18.75 203 30
25 321 48

Table 3: Summary of the main study results 
Exposure period Concentration of the test item in µg/mL Polyploid cells in % Mitotic index in % of control Aberrant cells in %
incl. gaps excl. gaps* exchanges
without S9
18 h negative control 4.7 100** 1.5 1.5 0
solvent control 2.7 100*** 3.5 2 0.5
positive control 3.5 94.2 18 (s) 18 (s) 9.5
2.5 3.5 39.1 0.5 0.5 0
with S9 mix
4h negative control 3.8 100** 1.5 0.5 0
solvent control 4.5 100*** 2 1 0
positive control 4 86.9 27 25.5 (s) 11
12.5 4.3 26.5 1.5 1 0

*= inclusive cells carrying exchanges

**= for the positive control groups, the relative values of the mitotic index are related to the negative controls

***= for the test item treatment groups the values are related to the solvent controls

(s)= Aberration frequency statistically significant higher than corresponding solvent control values

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
Executive summary:

In an in vitro chromosome aberration test (in accordance to OECD 473), V79 Chinese hamster lung cell cultures were exposed to the test item (90% purity), solved in deionised water, at concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10.0 µg/mL without metabolic activation and with metabolic activation of concentrations of 0, 3.125, 6.25, 12.5, 18.75 and 25.0 µg/mL. The test item was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of structural chromosome aberrations induced over background. This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.