Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-08-20 to 2014-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
(1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Phenobarbital/β-naphthoflavone induced rat liver)
Test concentrations with justification for top dose:
Pre-experiment: 20.3, 40.6, 162.5, 325.0, 650.0, 1300.0, 2600.0 µg/mL, 5.2 µL/mL (with and without S9 mix)
Experiment I: 0.3, 0.7, 1.6, 4.1, 10.2, 25.6, 64.0, 160.0, 400.0 µg/mL (with and without S9 mix)
Experiment II: 0.1, 0.3, 0.7, 1.6, 4.1, 10.2, 25.6, 64.0, 160.0 µg/mL (18 and 28 hours without S9 mix)
Experiment II: 0.3, 0.7, 1.6, 4.1, 10.2, 25.6, 64.0, 160.0, 320.0 µg/mL (4 hours with S9 mix)
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
EMS
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix at concentrations of 1000.0 (Exp. I), 600.0 (Exp. II, 18 h treatment), 500.0 (Exp. II, 28 h treatment) µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CPA
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix at concentrations of 1.4 (Exp. I) and 2.0 (Exp. II) µg/mL
Details on test system and experimental conditions:
RANGE FINDING EXPERIMENT
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression and/or reduction in cell number in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment.
The pre-test was performed with 9 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 18 hrs after start of the exposure.

MAIN EXPERIMENT
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours ±S9 mix (Exp. I); 4 hours +S9 mix (Exp. II); 18 and 28 hours -S9 mix (Exp. II)
- Expression time (cells in growth medium): 14 and 24 hours, resp.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (Exp. I and II) and 28 h (Exp. II)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
The chromosomal aberration assay will be considered acceptable if it meets the following criteria:
a) The rate of chromosomal aberrations in the solvent controls falls within the historical laboratory control data range.
b) The rate of chromosomal aberrations in the positive controls is statistically significant increased.

A test item can be classified as non-clastogenic if:
− the number of induced structural chromosomal aberrations in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic if:
− the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data and
− either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.
If the above mentioned criteria for the test item are not clearly met, the test item will be classified as equivocal or a confirmatory experiment may be performed. However, results may remain questionable regardless of the number of times the experiment is repeated.

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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH was observed.
- Effects of osmolality: No relevant influence on osmolarity was observed.
- Precipitation: No visible precipitation of the test item in the culture medium was observed in this study. Phase separation was observed at 160.0 μg/mL and above in Experiment I in the absence and presence of S9 mix. In Experiment II phase separation was observed at 25.6 μg/mL and above in the absence of S9 mix after 28 hours continuous treatment and at 64.0 μg/mL and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES:
With regard to the purity (97 %) of the test item, 5.2 μL/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 20.3 μg/mL to 5.2 μL/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures did not fulfill the requirements for cytogenetic evaluation, due to strong test item-induced toxic effects, this preliminary test was repeated with a top dose of 400.0 μg/mL (with and without S9 mix) and designated Experiment I.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results

Experiment

Exposure period [h]

Dose (µg/mL)

S9 mix

Prec.

Aberrant cells in %

Mitotic indices in % of control

1

4

Negative control

-

-

1.0

100.0

 

 

Positive control

-

-

8.5

121.5

 

 

0.7

-

-

1.0

114.6

 

 

1.6

-

-

0.0

92.7

 

 

4.1

-

-

1.5

46.3

 

 

 

 

 

 

 

2

18

Negative control

-

-

1.0

100.0

 

 

Positive control

-

-

19.5

95.6

 

 

0.3

-

-

2.0

125.7

 

 

0.7

-

-

1.5

108.0

 

 

1.6

-

-

0.5

76.5

 

 

 

 

 

 

 

2

28

Negative control

-

-

2.0

100.0

 

 

Positive control

-

-

39.0

32.7

 

 

0.1

-

-

1.0

118.1

 

 

0.3

-

-

1.5

99.4

 

 

0.7

-

-

1.0

56.7

 

 

 

 

 

 

 

1

4

Negative control

+

-

1.5

100.0

 

 

Postive control

+

-

15.5

64.1

 

 

10.2

+

-

2.5

97.7

 

 

25.6

+

-

1.0

117.0

 

 

64

+

-

1.0

61.0

 

 

 

 

 

 

 

2

4

Negative control

+

-

1.0

100.0

 

 

Positive control

+

-

18.5

85.3

 

 

4.1

+

-

3.5

95.8

 

 

10.2

+

-

2.0

85.3

 

 

25.6

+

-

1.0

99.0

Applicant's summary and conclusion