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Toxicological information

Genetic toxicity: in vitro

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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:
2013-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21,1997
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: Japanese Guidelines: "Kanpoan No. 287 - Environmental Agency" "Eisei No. 127 - Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry".
GLP compliance:
yes (incl. certificate)
Remarks:
RCC CYTOTEST CELL RESEARCH GMBH, In den Leppsteinswiesen 19, D-64380 Rossdorf, Germany
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark and use / dissolve under minimum light
- Stability under test conditions: Stable for hours in water, saline, PEG, and CMC

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Thawed stock cultures were propagated at 37° C in 80 cm2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5x10^5 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Colbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37°C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/p-Naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I, 4h exposure, -S9: (115.6), 231.3, 462.5, 925, (1850), (3700)
Experiment I, 4h exposure, +S9: 115.6, 231.3, 462.5, (925), (1850), (3700)
Experiment II, 18h exposure, -S9: 57.8, 115.6, 231.3, (462.5), (925), (1850)
Experiment II, 28h exposure, -S9: 231.3, (462.5), (925), (1850)
Experiment II, 4h exposure, +S9: (115.6), (231.3), 462.5, 925, 1850, (3700)

all values in µg/mL
numbers in brackets were not analyzed for chromosmal aberrations.
Vehicle / solvent:
- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
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 metablic activation; 200 mg/mL (1.6 mM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; 0.7 - 1.0 mg/mL (2.5 - 3.5 mM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Experiment I:
Exposure period 4 hrs, recovery period 14 hours, without S9 mix: 115.6, 231.3, 462.5, 925, 1850, 3700 µg/mL
Exposure period 4 hrs, recovery period 14 hours, with S9 mix: 115.6, 231.3, 462.5, 925, 1850, 3700 µg/mL
Experiment II:
Exposure period 18 hrs, recovery period 0 hours, without S9 mix: 57.6, 115.6, 231.3, 462,5, 925, 1850 µg/mL
Exposure period 28 hrs, recovery period 0 hours, without S9 mix: 231.3, 462,5, 925, 1850 µg/mL
Exposure period 4 hrs, recovery period 24 hours, with S9 mix: 115,6, 231,3, 462.5, 925, 1850, 3700 µg/mL

15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2.5 hrs 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 a mixture of methanol and glacial acetic acid (3:1 parts respectively). Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF CELLS EVALUATED
In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
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). and/or
- 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 % aberrant cells exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

However, both biological and statistical significance should be considered together. 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).
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (10) (p < 0.05).

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
Remarks:
from 925 µg/mL onward
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment I, in the absence and the presence of S9 mix as well as in experiment II in the absence of S9 mix at preparation interval 18 and 28 hrs, precipitation of the test item in culture medium was observed with 462.5 µg/mL and above. In experiment II, in the presence of S9 mix precipitation occurred at preparation interval 28 hrs with 925 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In the pre-test on toxicity, precipitation of the test item was observed 4 hrs after treatment with 462.5 µg/mL and above in the absence of S9 mix and with 3700 µg/mL in the presence of S9 mix. Due to a technical error in the absence of S9 mix the cell numbers after 4 hrs treatment were strongly reduced in all test groups inclusive the solvent control. Therefore, the toxicity data after 24 hrs continuous treatment was taken for dose selection. In this pre-experimental part in the absence of S9 mix clear toxic effects were observed with 1850 µg/mL and above. In addition, in the presence of S9 mix clear toxic effects were observed after treatment with 462,5 µg/mL and above. Therefore 3700 µg/mL were chosen as top concentration for 4 hrs treatment in main experiment I with and without metabolic activation.
Dose selection of experiment II was also influenced by test item toxicity and the occurrence of precipitation. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 1850 µg/mL and above. Therefore, 1850 µg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 3700 µg/mL were chosen as top treatment concentration with respect to the results obtained in experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects indicated by reduced cell numbers and/or mitotic indices of about and below 50% of control were observed in experiment I in the absence of S9 mix after 4 hrs treatment with 925 µg/mL (37 % of control) and in experiment II, in the presence of S9 mix after 4 hrs treatment with 1850 µg/mL (53 % of control) at the 28 hrs preparation interval. In all other experimental parts no clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at evaluated test item concentrations. However, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Any other information on results incl. tables

Details on experimental results

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Two significant (p < 0.05) increases were observed in experiment I, in the presence of S9 mix at preparation interval 18 hrs after 4 hrs treatment with 115.6 and 231.3 µg/mL. Although these increases of 2.5 % and 4.0 % aberrant cells, exclusive gaps, were statistically significant compared to the low response (0.0 % aberrant cells, exclusive gaps) in the solvent control data, the responses are within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significances have to be regarded as being biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 - 2.6 %) as compared to the rates of the solvent controls (1.6-2.7%).

In both experiments, EMS (200 µg/mL) and CPA (0.7 and 1.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Aberrant cells
Exp Exposure Period Preparation Interval concentration (µg/ml) S9 Mix Polyploid cells (%) cell numbers in % of control Mitotic indices in % of control incl. Gaps excl. Gaps with exchanges
I 4 h 18 h neg. control - 2 n.t. 100 2.5 0.5 0.5
4 h 18 h solvent control1 - 2.7 100 100 1.5 1.5 0
4 h 18 h pos. control2 - 1.2 n.t. 97 14 8.5S 5
4 h 18 h 231.3 - 2.2 98 130 2.5 2.5 0.5
4 h 18 h 462.5P - 1.8 55 139 1 1 0
4 h 18 h 925P - 0.9 37 138 1 0 0
II 18 h 18 h neg. control - 2.2 n.t. 100 1 1 0
18 h 18 h  solvent control1 - 1.6 100 100 0.5 0 0
18 h 18 h  pos. control2 - 2.4 n.t. 85 16 14.5S 7
18 h 18 h 57.8 - 2.6 135 118 0 0 0
18 h 18 h 115.6 - 1.9 90 129 0 0 0
18 h 18 h 231.3 - 1.7 119 96 0 0 0
II 28 h 28 h neg. control - 2.2 n.t. 100 2.5 1.5 0
28 h 28 h  solvent control1 - 1.8 100 100 0 0 0
28 h 28 h  pos. control2 - 2.9 n.t. 85 15 13.5S 6.5
28 h 28 h 231.3 - 2 87 109 0.5 0 0
I 4 h 18 h neg. control + 0.9 n.t. 100 1 0.5 0
4 h 18 h  solvent control1 + 1.6 100 100 1 0 0
4 h 18 h pos. control3 + 0.8 n.t. 124 14.5 11.5S 4
4 h 18 h 115.6 + 1 79 92 2.5 2.5S 0
4 h 18 h 231.3 + 1.4 101 121 4 4S 0.5
4 h 18 h 462.5P + 1.4 91 85 1.5 1 0.5
II 4 h 28 h neg. control + 1.9 n.t. 100 1.5 1.5 0.5
4 h 28 h  solvent control1 + 1.8 100 100 1.5 1 0
4 h 28 h  pos. control4 + 2.2 n.t. 90 9.5 8S 4
4 h 28 h 462.5 + 2.2 89 98 2.5 2 0.5
4 h 28 h 925P + 2.2 60 101 1 0.5 0
4 h 28 h 1850P + 2.1 53 93 2 2 0.5

n.t. = not tested

P precipitation occurred

S aberration frequency statistically significant higher than corresponding control values

1 acetone 0.5 % (v/v)

2 EMS 200 mg/mL

3 CPA 0.7 mg/mL

4 CPA 1.0 mg/mL

Applicant's summary and conclusion

Conclusions:
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, the test article is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.
Executive summary:

In a chromosome aberration test performed according to OECD guideline No. 473 and in compliance with GLP, the test item dissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (exp. I and II) and 28 hrs (exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 4.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. Two significant (p < 0.05) increases were observed in experiment I, in the presence of S9 mix at preparation interval 18 hrs after 4 hrs treatment with 115.6 and 231.3 µg/mL. Although these increases of 2.5 % and 4.0 % aberrant cells, exclusive gaps, were statistically significant compared to the low response (0.0 % aberrant cells, exclusive gaps) in the solvent control data, the responses are within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significances have to be regarded as being biologically irrelevant. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).