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EC number: 246-644-8 | CAS number: 25134-21-8
Genetic toxicity: in vitro
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:
- 2012-02-15 to 2012-03-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant study conducted in accordance with internationally recognised test methods
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- 1,2,3,6-tetrahydromethyl-3,6-methanophthalic anhydride
- EC Number:
- 246-644-8
- EC Name:
- 1,2,3,6-tetrahydromethyl-3,6-methanophthalic anhydride
- Cas Number:
- 25134-21-8
- Molecular formula:
- C10H10O3
- IUPAC Name:
- 3a,4,7,7a-tetrahydromethyl-4,7-methano-2-benzofuran-1,3-dione
- Test material form:
- other: liquid
- Details on test material:
- - Name of test material (as cited in study report): METH-E (Methyl-(endo)-5-norbornene-2,3-dicarboxylic anhydride)
- Analytical purity: 99.6 %
- Purity test date: 2011-10-25
- Lot/batch No.: T120211293
- Expiration date of the lot/batch: 1 year from production date
- Stability under test conditions: Stable
- Storage condition of test material: Tightly closed container, protected from humidity in a cool, dry and well ventilated place
Constituent 1
Method
- Target gene:
- no details given
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: provided by ECACC (European Collection of Cells Cultures)
- Properly maintained: yes, -80 +/- 10°C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Experiment A with 3/20 h treatment/sampling time with and without S9 mix: 350, 400, 450, 500, 550 μL/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 100, 150, 200, 250, 300, 350 μL/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 150, 200, 250, 300, 350 μL/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 350, 400, 450, 500, 550, 600 μL/mL test item. - Vehicle / solvent:
- solvent: DMSO
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Ethylmethane sulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5.0 μg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
Experiment A with 3/20 h treatment/sampling time with and without S9 mix: 350, 400, 450, 500, 550 μL/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 100, 150, 200, 250, 300, 350 μL/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 150, 200, 250, 300, 350 μL/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 350, 400, 450, 500, 550, 600 μL/mL test item.
Preparation of cultures
Cell cultures were treated with Colchicine (0.2 μg/mL) two hours prior to harvest. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind. - Evaluation criteria:
- The Chromosome Aberration Assay is considered valid if following criteria are met:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data.
– the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations. - Statistics:
- no details given
Results and discussion
Test results
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no - Remarks on result:
- other: strain/cell type: Chromatid and chromosome
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Mean % cells with structural chromosomal aberrations – Experiment A
Concentration |
S9 mix |
Treatment time |
Harvesting time |
Mean % aberrant cells |
Mean number |
Mean number |
|
(μg/mL) |
|
(hours) |
(hours) |
Incl. gaps |
Incl. gaps |
polyploid cells |
endoreplicated cells |
Solvent control (7 μL DMSO/mL) |
- |
3 |
20 |
4 |
4 |
0.0 |
0.0 |
350 |
- |
3 |
20 |
3 |
3 |
0.0 |
0.0 |
400 |
- |
3 |
20 |
4 |
4 |
0.0 |
0.0 |
450 |
- |
3 |
20 |
5 |
5 |
0.0 |
0.0 |
500 |
- |
3 |
20 |
5 |
5 |
0.0 |
0.0 |
550 |
- |
3 |
20 |
6 |
6 |
0.0 |
0.0 |
Positive control (1.0 μL/mL) |
- |
3 |
20 |
28 ** |
28 ** |
0.0 |
0.0 |
Solvent control (11 μL DMSO/mL) |
+ |
3 |
20 |
4 |
4 |
0.0 |
0.0 |
350 |
+ |
3 |
20 |
5 |
5 |
0.0 |
0.0 |
400 |
+ |
3 |
20 |
4 |
4 |
0.0 |
0.0 |
450 |
+ |
3 |
20 |
5 |
5 |
0.0 |
0.0 |
500 |
+ |
3 |
20 |
5 |
5 |
0.0 |
0.0 |
Positive control (Cyc. 5.0 μg/mL) |
+ |
3 |
20 |
31 ** |
31 ** |
0.0 |
0.0 |
EMS: Ethyl methanesulphonate Cyc. : Cyclophosphamide ** : p <0.1
Mean % cells with structural chromosomal aberrations – Experiment B
Concentration |
S9 mix |
Treatment time |
Harvesting time |
Mean % aberrant cells |
Mean number |
Mean number |
|
(μg/mL) |
|
(hours) |
(hours) |
Incl. gaps |
Excl. gaps |
polyploid cells |
endoreplicated cells |
Solvent control (7 μL DMSO/mL) 4 |
- |
20 |
20 |
5 |
2 |
0.0 |
0.0 |
100 |
- |
20 |
20 |
4 |
1 |
0.0 |
0.0 |
150 |
- |
20 |
20 |
4 |
2 |
0.0 |
0.0 |
200 |
- |
20 |
20 |
6 |
2 |
0.0 |
0.0 |
250 |
- |
20 |
20 |
5 |
2 |
0.0 |
0.0 |
300 |
- |
20 |
20 |
5 |
3 |
0.0 |
0.0 |
Positive control (0.4 μL/mL) |
- |
20 |
20 |
30 ** |
27 ** |
0.0 |
0.0 |
Solvent control (7 μL DMSO/mL) |
- |
20 |
28 |
4 |
2 |
0.0 |
0.0 |
150 |
- |
20 |
28 |
5 |
2 |
0.0 |
0.0 |
200 |
- |
20 |
28 |
5 |
2 |
0.0 |
0.0 |
250 |
- |
20 |
28 |
3 |
1 |
0.0 |
0.0 |
300 |
- |
20 |
28 |
7 |
2 |
0.0 |
0.0 |
Positive control (0.4 μL/mL) |
- |
20 |
28 |
31 ** |
26 ** |
0.0 |
0.0 |
Solvent control (11 μL DMSO/mL) |
+ |
3 |
28 |
4 |
2 |
0.0 |
0.0 |
350 |
+ |
3 |
28 |
3 |
1 |
0.0 |
0.0 |
400 |
+ |
3 |
28 |
5 |
3 |
0.0 |
0.0 |
450 |
+ |
3 |
28 |
5 |
1 |
0.0 |
0.0 |
500 |
+ |
3 |
28 |
5 |
1 |
0.0 |
0.0 |
550 |
+ |
3 |
28 |
4 |
1 |
0.0 |
0.0 |
Positive control (Cyc. 5.0 μg/mL) |
+ |
3 |
28 |
27 ** |
23 ** |
0.0 |
0.0 |
EMS: Ethyl methnesulphonate Cyc. : Cyclophosphamide ** : p <0.1
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The substance, when tested up to cytotoxic concentrations both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. It is therefore considered as not clastogenic in this system. - Executive summary:
The substance has been tested in a Chromosome Aberration Assay in V79 cells using OECD test methods.
There were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases.
The substance is therefore considered as not clastogenic in this test system.
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