Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following GLP compliant assays have ben performed with the target compound:

OECD TG 471: not mutagenic

OECD TG 473: not clastogenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 13 - Dec 04, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
none
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
His operon (Salmonella), Trp operon (E. coli)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from PB/NF-pretreated rats
Test concentrations with justification for top dose:
1st series: 10, 50, 100, 500, 1000, 5000 µg/plate
2nd series: 313, 625, 1250, 2500, 5000 µg/plate
Vehicle:
DMSO
Purity > 99 %
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: 48-52 hours

NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: other: background clearance

OTHER EXAMINATIONS:
- Determination of polyploidy: na
- Determination of endoreplication: na
- Other:

Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
Statistics:
no
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Additional information on results:
precipitation @50 µg/plate from beginning until end of experiment
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
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:
Aug 30, 2010 - Jan 13, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Minimal Essential Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 after induction using Aroclor 1254
Test concentrations with justification for top dose:
5.00, 8.89, and 15.8 µg / mL
Vehicle:
acetone
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other: griseofulvin
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5, 25, and 31 hours (-S9), 5 hours (+S9)
- Expression time (cells in growth medium): 25 and 31 hours (-S9), 25 hours (+S9)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Solvent control: 4; others: 2

NUMBER OF CELLS EVALUATED: 100 metaphases (structural abberations); 1000 metaphases (polyploidy)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell viability (MTT), cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other:

OTHER:
Evaluation criteria:
The decisive parameter for the evaluation of both, the treated and untreated cultures, is the number of aberrant metaphases (excluding gaps) per 100 cells. A basic prerequisite for the acceptance of a test series is

(a) the correspondence of the actual negative controls with the historic negative controls of the laboratory and
(b) a statistically significant and biologically relevant increase in the number of aberrant metaphases for the respective positive controls in relation to the actual negative controls.

The discussion of biological relevance includes, among other things, considerations concerning the type and time dependent appearance of the observed chromosomal aberrations.

A test material is defined as being negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration. Confirmation of negative results is not considered necessary if these criteria are fulfilled.

A test material is positive or clastogenic in this test system if

• a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
• a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.

There is no requirement for verification of a clear positive response. In both cases, however, the number of aberrant metaphases has to be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
In all other cases, further decisions for testing strategies should be made.
Statistics:
Standard statistical methods have been applied for data processing.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: @15.8 µg/mL
- Other confounding effects: no

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No clear cytotoxic effects (i.e. reduction of mitotic index or cell viability)

see attachment

Conclusions:
The treatment of V79 cell cultures with the test material did not increase the proportion of cells with aberrant chromosomes. The test item was thus not clastogenic in this in vitro test system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, the test item is not considered to be classified for mutagenicity according to Regulation (EC) No 1272/2008.