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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03-10 to 2004-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; Seromed)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 (protein concentration 35.7 mg/mL and 28.9 mg/mL)
Test concentrations with justification for top dose:
Evaluated test concentrations:
Experiment I: without S9 mix/ 4 hrs exposure: 25.0, 50.0, 100.0 µg/mL
with S9 mix/ 4 hrs exposure: 37.5, 75.0, 300.0, 600.0 µg/mL
Experiment II: without S9 mix/ 18 hrs exposure: 6.3, 12.5, 25.0 µg/mL
without S9 mix/ 28 hrs exposure: 18.8 µg/mL
with S9 mix/4 hrs exposure:: 18.8, 37.5, 150.0, 300.0 µg/mL
Experiment III: with S9 mix/ 4 hrs exposure: 300.0, 400.0, 500.0 µg/mL
A table containing all exposure concentrations is given under "Any other informations of materials and methods"
Vehicle / solvent:
deionised water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (MEM with 10 % FCS)

DURATION
- Exposure duration:
Experiment I: with and without S9 mix 4 hrs
Experiment II: without S9 mix 18 and 28 hrs; with S9 mix 4 hrs
Experiment III: with S9 mix 4 hrs

- Expression time (cells in growth medium): 14 hrs for the 4 hrs exposure groups, no for the 18 and 28 hrs groups.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hrs respectively

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 independent experiments, per group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: at least 100 methaphase plates were scored per culture. 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.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: number of polyploidy cells in 500 metaphase cells per culture
was determined (% polyploid metaphases)
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 laboratories 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.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and
endoreduplications.
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of laboratories historical control data (0.0 – 8.5 % polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: water was chosen as solvent, test substance was suspended in deionised water immediately before treatment.
- Precipitation: was observed, corresponding concentrations were identified in the exposure concentration table under
"any other information on materials and methods".

RANGE-FINDING/SCREENING STUDIES:
In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 39.1 and 5000µg/mL were applied. Clear toxic effects were observed after 4 hrs treatment with 156.3 µg/mL and above in the absence of S9 mix and with 625µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 78.1 µg/mL and above in the absence of S9 mix induced strong toxic effects.
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 39.1 µg/mL and above in the absence and the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 288 mOsm, pH 7.2 versus 323 mOsm and pH 7.2 at 5000 µg/mL).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In this study, cytotoxicity was observed in all cytogenetic experiments. In the presence of S9 mix the cell numbers were strongly reduced after treatment with 600 µg/mL (32 % of control) at the 18 hrs preparation interval and with 500µg/mL (43 % if control) at the 28 hrs preparation interval.

MAIN TEST:
In experiment I and II, in the absence and the presence of S9 mix, no biological 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 - 4.8 % abberant cells, exclusive gaps) were close to the range of the solvent control values (0.0 – 1.5 % aberrant cells, exclusive gaps) and close to the range of our historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).
In the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). To prove this slightly increase value exceeding the upper border of our laboratory´s historical control data range an increased sample of 200 metaphase plates per culture was evaluated for cytogenetic damage.
However, the borderline value was confirmed. In addition, a dose related increase in the number of cells carrying structural chromosome aberrations (0.5 %, 2.5 % and 4.8 %) was observed after 4 hrs treatment at 28 hrs preparation interval in the presence of metabolic activation at the upper concentrations evaluated (37.5, 150.0 and 300.0 µg/mL) respectively.

A confirmatory experiment, designated experiment III, was performed to proof these observations. In the repeated experiment in the presence of S9 mix after 4 hrs treatment at prolonged 28 hrs preparation interval 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 (1.0 – 3.0 % aberrant cells exclusive gaps) were close to the value of the solvent control (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data, (0.0 – 4.0 % aberrant cells, exclusive gaps).

Beside the aberration rates were dose related increased (1.0 %, 1.5 % and 3.0 %) in the concentration range evaluated (300 to 500 mg/mL), but the values were clearly within our laboratory´s control data range (0.0 – 4.0 % aberrant cells, exclusive gaps). Finally, the observations of experiment II in the presence of S9 mix (statistical significance, dose dependency, and borderline value) were not confirmed in experiment III and therefore they have to be regarded as biologically irrelevant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 – 2.5 %) as compared to the rates of the solvent controls (1.9 – 3.0 %).

EMS and CPA were used as positive controls and showed distinct increase in cells with structural chromosome aberrations.

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. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.
Executive summary:

In a mammalian cell cytogenetics assay according to OECD Guideline 473, 1997 and EU Method B.10, 2000, V79 cell cultures were exposed to partially unsaturated TEA-Esterquat at concentration ranges of 3.1 – 200 µg/mL without metabolic activation and 4.7 – 600 µg/mL in the presence of mammalian metabolic activation. The following concentrations were selected for microscopic analysis:

Experiment I:           without S9 mix/ 4 hrs exposure:       25.0, 50.0, 100.0 µg/mL

                                  with S9 mix/ 4 hrs exposure:            37.5, 75.0, 300.0, 600.0 µg/mL

Experiment II:          without S9 mix/ 18 hrs exposure:     6.3, 12.5, 25.0 µg/mL

                                  without S9 mix/ 28 hrs exposure:     18.8 µg/mL

                                 with S9 mix/4 hrs exposure::            18.8, 37.5, 150.0, 300.0 µg/mL

Experiment III:        with S9 mix/ 4 hrs exposure:            300.0, 400.0, 500.0 µg/mL

In experiment I and II, in the absence and the presence of S9 mix, no biological 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 - 4.8 % abberant cells, exclusive gaps) were close to the range of the solvent control values (0.0 – 1.5 % aberrant cells, exclusive gaps) and close to the range of our historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).

 

In the presence of S9 mix two significant (p < 0.05) increases were observed, in experiment I at preparation interval 18 hrs after treatment with 37.5 µg/mL (4 % aberrant cells, exclusive gaps), and in experiment II at preparation interval 28 hrs with 300 µg/mL (4.8 % aberrant cells, exclusive gaps). To prove this slightly increase value exceeding the upper border of our laboratory´s historical control data range an increased sample of 200 metaphase plates per culture was evaluated for cytogenetic damage.

However, the borderline value was confirmed. In addition, a dose related increase in the number of cells carrying structural chromosome aberrations (0.5 %, 2.5 % and 4.8 %) was observed after 4 hrs treatment at 28 hrs preparation interval in the presence of metabolic activation at the upper concentrations evaluated (37.5, 150.0 and 300.0 µg/mL) respectively.

A confirmatory experiment III was performed to proof these observations. In the repeated experiment in the presence of S9 mix after 4 hrs treatment at prolonged 28 hrs preparation interval 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 (1.0 – 3.0 % aberrant cells exclusive gaps) were close to the value of the solvent control (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data, (0.0 – 4.0 % aberrant cells, exclusive gaps).

Beside the aberration rates were dose related increased (1.0 %, 1.5 % and 3.0 %) in the concentration range evaluated (300 to 500 mg/mL), but the values were clearly within our laboratory´s control data range (0.0 – 4.0 % aberrant cells, exclusive gaps). Finally, the observations of experiment II in the presence of S9 mix (statistical significance, dose dependency, and borderline value) were not confirmed in experiment III and therefore they have to be regarded as biologically irrelevant.

In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.9 – 2.5 %) as compared to the rates of the solvent controls (1.9 – 3.0 %).

Positive controls induced the appropriate response. EMS and CPA were used as positive controls and showed distinct increase in cells with structural chromosome aberrations. 

In conclusion 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. The test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic test item concentrations.

There was no evidence of chromosome aberration induced over background.