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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
01-06-2005- 05-08-2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 473, GLP. Study according to relevant guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Monomer Mix average C14.8
IUPAC Name:
Monomer Mix average C14.8
Details on test material:
- Name of test material (as cited in study report): Monomer Mix
- Supplier: Evonik Oil Additives GmbH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Dez. 31, 2005
- Stability under test conditions: Stability in solvent: 30 days at room temperature in water, saline, DMSO, ethanol, tetrahydrofurane and acetone
- Storage condition of test material: At room temperature, protected from light

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian rat liver microsomal fraction induced with phenobarbital / ß-naphthoflavone (S9 mix)
Test concentrations with justification for top dose:
Experiment I: 18 hrs prep. interval, 4 hrs exposure period: without S9 mix: 5.9, 11.7, 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p),
3000.0 (p) µg/ml
18 hrs prep. interval, 4 hrs exposure period: with S9 mix: 5.9, 11.7, 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p),
3000.0 (p) µg/ml
(p): Precipitation occurred

Experiment II: 18 hrs prep. interval, 18 hrs exposure period: without S9 mix: 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p), 3000.0 (p) µg/ml
28 hrs prep. interval, 28 hrs exposure period: without S9 mix: 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p), 1500.0 (p),
3000.0 (p) µg/ml
Experiment II: 28 hrs prep. interval, 4 hrs exposure period: with S9 mix: 23.4, 46.9, 93.8, 187.5 (p), 375.0 (p), 750.0 (p) µg/ml

(p): Precipitation occurred
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF) (E. Merck, D-64293 Darmstadt, Germany; purity: 99.5%)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells.
The final concentration of THF in the culture medium was 0.5 % (v/v).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
concurrent solvent controls (THF) were performed
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in: nutrient medium
Positive control substance:
ethylmethanesulphonate
Remarks:
Final concentration: 300 - 400 µg/ml (2.4 - 3.2 mM)

Migrated to IUCLID6: without metabolic activation
Positive controls:
yes
Remarks:
dissolved in: saline (0.9 % (w/v)
Positive control substance:
cyclophosphamide
Remarks:
Final concentration: 1.4 µg/ml (5.0 µM)

Migrated to IUCLID6: with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period:
- Exposure duration: 4 h (with S9 mix); 18 and 28 hours (without S9 mix)




SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa (Merck, D-64293 Darmstadt, Germany)


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 metaphases were scored



DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; in addition the number of polyploid cells in 500 metaphase plates per culture was scored (% polyploid metaphases)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control
data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations, which are within the range of the laboratory's historical control data:

Test group Aberrant cells in % Test group Aberrant cells in %
Final concentration (excl. gaps) Range Final concentration (excl. gaps) Range
Without S9 mix With S9 mix
EMS 200 - 400 µg/mL 7.0 - 63.0 CPA 0.7 - 1.4 µg/mL 7.0 - 49.0


EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural
chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.

A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor
a significant and reproducibly positive response at any one of the test points is considered nonmutagenic in this system.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's exact test. Evaluation was performed only for cells
carrying aberrations exclusive gaps.

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:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both cytotogenetic experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In the Chromosomal aberration test with Chinese hamster V79 cells in vitro according to OECD 473 in the presence and absence of mammalian metabolic activation structural chromosomal aberrations were not observed when tested up to cytotoxic and/or precipitationg concentrations.
Therefore, the test substance is considered to be non-mutagenic under the experimental conditions reported.
Executive summary:

The test item Monomer mix, dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in Chinese hamster V79 cells in vitro in two independant experiments.

 

The study design was performed as follows:

 

Experiment I: 18 hrs preparation interval, 4 hrs exposure period without and with S9 mix

                      

Experiment II: 18 hrs prep. interval, 18 hrs exposure period without S9 mix and 28 hrs prep. interval, 4 hrs and 28 hr exposure period with S9 mix.

 

In each experimental group, two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

 

The highest applied concentration in Experiment I (3000 µg/ml of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD 473.

 

No clear toxic effects indicated by reduced mitotic indices or cell numbers of below 50% were observed up to the highest required test item concentration. Therefore, concentrasions at the border of test item solubility in culture medium were scored for cytogenetic damage.

 

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiments I and II, in the absence of S9 mix, statistically significant increases (2 % - 2.5 % aberrant cells, exclusive gaps) were observed, but were within our historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and are regarded as being biologically irrelevant.

 

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) 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 chromosomal aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

 

Therefore, the test substance is considered to be non-clastogenic under the experimental conditions reported.

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