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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
11-03-2008 - 12-06-2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 476, GLP. Study according to relevant guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isodecyl methacrylate
- Supplier: Evonik Röhm GmBH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Aug 26, 2008
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml
with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

Experiment II:
without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml
with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: relative non-toxicity towards the cells and solubility properties of the test item
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
concurrent solvent controls (THF)
Positive controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Purity > 98 % ; Supplier :ACROS Organics, Geel, Belgium

Migrated to IUCLID6: without metabolic activation
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Purity 99.5 %; Supplier: Merck, Darmstadt, Germany

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

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 4 hours with and 24 hours without metabolic activation. The experimental parts of the second experiment with
and without metabolic activation were performed in two separate experiments (experiment II and IIA) for technical reasons. The
results are combined and reported as experiment II.



NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation
microscope (Nikon, 40407 Düsseldorf, Germany).
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10exp+6 cells found in the negative and/or solvent controls fall within the laboratory historical control data
range of 2001 – 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies (Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

Evaluation of Results
A test item is regarded as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive
response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test
points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is regarded as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency
at least at one of the concentrations in the experiment.
The test item is regarded as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be
considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low
spontaneous mutation rate in the range normally found ( mutants per 10exp+6 cells) a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
Statistical Analysis
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT
Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the
groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability
value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in forward gene mutations in mammalian cells
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1200 µg/mL with S9 mix; at 1.0 µg/mL and above in Experiment I and 37.5 µg/mL in Experiment II without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: V79
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results and Discussion

Isodecyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

Phase separation of the test item was observed at 300 µg/mL and above in the first experiment with metabolic activation and at 150 µg/mL and above in the second experiment without metabolic activation. In the second experiment with metabolic activation phase separation was noted at 300 µg/mL and above.

Relevant toxic effects indicated by a relative cloning efficiency 1 below 50 % occurred at 1.0 µg/mL and above in the first experiment without metabolic activation and at 1200.0 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 37.5 µg/mL without metabolic activation and at 1200 µg/mL with metabolic activation. The striking difference of toxic concentrations with and without metabolic activation is probably based on protein or lipid binding effects. In the presence of metabolic activation the protein and lipid concentration is higher due to the S9-mix added.This fact is furthermore supported by the considerably less severe cytotoxicity following 24 h treatment without metabolic activation. During long term exposure 10 % FCS have to be added increasing the protein and lipid concentration of the medium.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.

The induction factor reached or exceeded the threshold of three times the corresponding solvent control in experiment I at 37.5 µg/mL in the first culture with metabolic activation and at the same concentration in the first culture in experiment II without metabolic activation. However, both effects were judged as biologically irrelevant fluctuations since no increase was observed at higher concentrations or in the parallel cultures under identical conditions. Furthermore, the effects were not dose-dependent as indicated by the lacking statistical significance.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 106cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 106cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

Conclusion

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

Isodecyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

Phase separation of the test item was observed at 300 µg/mL and above in the first experiment with metabolic activation and at 150 µg/mL and above in the second experiment without metabolic activation. In the second experiment with metabolic activation phase separation was noted at 300 µg/mL and above.

Relevant toxic effects indicated by a relative cloning efficiency 1 below 50 % occurred at 1.0 µg/mL and above in the first experiment without metabolic activation and at 1200.0 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 37.5 µg/mL without metabolic activation and at 1200 µg/mL with metabolic activation. The striking difference of toxic concentrations with and without metabolic activation is probably based on protein or lipid binding effects. In the presence of metabolic activation the protein and lipid concentration is higher due to the S9-mix added.This fact is furthermore supported by the considerably less severe cytotoxicity following 24 h treatment without metabolic activation. During long term exposure 10 % FCS have to be added increasing the protein and lipid concentration of the medium.

 

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.

 

The induction factor reached or exceeded the threshold of three times the corresponding solvent control in experiment I at 37.5 µg/mL in the first culture with metabolic activation and at the same concentration in the first culture in experiment II without metabolic activation. However, both effects were judged as biologically irrelevant fluctuations since no increase was observed at higher concentrations or in the parallel cultures under identical conditions. Furthermore, the effects were not dose-dependent as indicated by the lacking statistical significance.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 106cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 106cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

Conclusion

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.