Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
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:
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


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.