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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: negative

OECD TG 473: negative

For mutagenicity in mammalian cells (OECD 476) a read across was performed. A detailed justification is provided in chapter 13. Based on the results obtained from the structural analogue substance, the test item is not considered to be mutagenic in mammalian cells.

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
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
Target gene:
His operon (Salmonella), Trp operon (E. coli)
Species / strain / cell type:
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 / solvent:
DMSO
Purity > 99 %
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental 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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
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.
Executive summary:

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537,  and Escherichia coli WP2  uvrA. The plate incorporation test with and without addition of liver S9 mix from NF/PB-pretreated rats was used. Two  independent experimental series were performed.
The procedures used in this study were in accordance with OECD Guideline 471.

The test material was dissolved in acetone and tested at concentrations ranging from 50 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred strting at 50 µg/plate.
9 -aminoacridine, and sodium azide served as strain specific positive control test materials in the absence of S9 mix. 2 -Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In both series performed, there were no treatment related increasing in the mutation frequencies observed with and without metabolic activation.
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
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
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (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 / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other: griseofulvin
Details on test system and experimental 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 / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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.
Executive summary:

The test material was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro with and without S9 mix. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in endoreduplications or polyploidy. The study was performed according to OECD Guideline 473 under GLP conditions.

The positive control materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number of polyploid cells.
The test item precipitated in the culture medium at the highest concentration evaluated, i.e. 15.8 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test), were induced by the test material.
The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (- and + S9 mix) ranged from 0.75 % to 2.50 %. The test item did not show any relevant increase in the number of  aberrant metaphases. Furthermore, no treatment-related increase in  endoreduplications  or polyploid cells was observed. I.e. neither structural nor numerical aberrations were detected.

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:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-03-20 to 2006-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Growth media:
Three media, supplementing RPMI 1640-medium with Glutamax 1 with different serum concentrations were used:

- Exposure medium: RPMI- 5 (RPM 1640 with 5% heat inactivated horse serum) 470 mL RPMI 164025 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

- Culture medium: RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 164050 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin

- Survivor- and selection medium: RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640100 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver homogenate (S9 mix) with standard co-factors
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
see below
Evaluation criteria:
The effects of the test material upon the mutation frequency are defined as

-"No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
-"Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
-All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if:
-the assay is considered valid and -no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
-a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if:
-the assay is considered valid and -a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
-a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
-weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2.81 and 28.1 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
It is concluded that the test item is non-mutagenic and non-clastogenic in this test system under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

The objective of this study was to evaluate the mutagenic activity of the test material by examining its ability to induce TK mutations in L5178Y cells in the absence and presence of a rat liver metabolizing system (S9 mix).

The test material was assayed for its ability to induce mutations at th TK locus (5-trifluorothymidine resistance) in L5178Y mouse lymphoma cells using a fluctuation protocol acording to OECD Guideline 476. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). Acetone was used as the solvent. The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with the test item in the two experimental series in the absence and presence of metabolic activation.

Based on the results it is concluded that the test item is non-mutagenic and non-clastogenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
For this endpoint information from a structural similar compound is available. The study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 476.
See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2.81 and 28.1 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
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.