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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the Bacterial Reverse Mutation Assay according to OECD Guideline 471. Furthermore, the test item was not clasogenic in mammalian cells as examined in the OECD Guideline study 473.

Link to relevant study records

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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 19 - Dec 05, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS Operon (Salmonella typhimurium), 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:
Type and composition of metabolic activation system:
- source of S9 : obtained liver S9 mix obtained from male Wistar rats pre-treated with Aroclor 1254
- method of preparation of S9 mix: intraperitoneal injection of Aroclor 1254; on day 5 to 7 animals were sacrificed, livers were removed and homogenized; homogenate was spun at 9000 x g for 10 minutes; supernatant fluid was decanted and stored at - 196°C
- concentration or volume of S9 mix and S9 in the final culture medium: 10% and 30% in the first and second series, respectively
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic activity
Test concentrations with justification for top dose:
1st series: 0.5, 1.58, 5.00, 15.8, 50.0, 158, and 500 µg/plate
2nd series: 15.8, 28.1, 50.0, 88.9, and 158 µg/plate
Vehicle / solvent:
Acetone- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: standard vehicle with no influence on the number of spontaneous revertants of any strain

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The incubation of plates was performed at (37 +/- 1) °C for 2 to 3 days.

NUMBER OF REPLICATIONS: 3

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; reduction in the number of spontaneous revertants


Evaluation criteria:
Definitions:
The assessment of test material-induced effects is dependent on the number of spontaneous re-vertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies
(Solvent Control) Maximal Mean Number of Colonies over the Actual Solvent Control (Test Material)
≤ 10 ≤ 9 ≥ 30
≤ 30 ≤ 19 ≥ 40
≤ 80 ≤ 29 ≥ 80
≤ 200 ≤ 49 ≥ 120
≤ 500 ≤ 99 ≥ 200
Assessment: "No Increase" "Clear Increase"
All further results, ranging between "no" and "clear", are assessed as "weak increases".
Interpretations:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the test series performed. ("Weak increases" randomly oc-cur 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 rever-tants 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.
In all further cases, a third test series with the bacterial strain in question should be performed and the results of each series be discussed case by case.

Statistics:
not performed
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:
not examined
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:
not examined
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:
not examined
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: at concentration larger or equal to 50 µg/plate

RANGE-FINDING/SCREENING STUDIES: In the first experiment seven concentrations ranging from 0.5 to 500 µg/plate were tested. Due to precipitation and the absence of cytotoxicity the following test material concentrations were used:
0.5, 1.58, 5.0, 15.8, 50.0, 158.0 and 500.0 µg/plate (first experiment)
15.8, 28.1, 50.0, 88.9 and 158 µg/plate (second experiment)

Ames test:
- Signs of toxicity : none
- Mean number of revertant colonies per plate and standard deviation: see attached document 1

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: The positive control should induce a "clear increase" in the number of revertants (see attached document 2)

- Negative (solvent/vehicle) historical control data:
TA 98: 15 - 60
TA 100: 75 - 200
TA 102: 200 - 450
TA 1535: 3 - 37
TA 1537: 4 - 31
WP2 uvrA: 10 -70
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:

This GLP study was performed according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.

The test material was dissolved in acetone and tested at concentrations ranging from 0.50 to 500 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 158 µg/plate. Toxicity to the bacteria was not observed.
Daunomycin, sodium azide, 9 -aminoacridine, 4 -nitroquinolin-N-oxide and cumene hydroperoxide 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.
With and without addition of S9 mix as the external metabolizing system, the test material did not induce any clear or dose dependent increase in the mutation rates under the experimental conditions described.

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:
Nov 10, 2008 - Jan 23, 2009
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)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
COUNCIL REGULATION (EC) No 440/2008 of 30 May 2008
Deviations:
no
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):
CELLS USED
- Type and source of cells: V79 Chinese hamster cells, clone : BAS received from (Hoffmann-La Roche, Pharma, Basel)
- Suitability of cells: V79 cells have been successfully used in mutagenicity testing for many years (Bradley et al. 1981, Chu and Malling 1968, Utesch et al. 1985). This cell line has a high proliferation rate and cloning efficiency.

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes: 22 +/- 1
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Dulbecco's Minimal Essential Medium (DMEM) supplemented with L-glutamine (4 mM), sodium
bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum. All incubations were performed at +37°C in a 4-5 % carbon dioxide atmosphere (100 % humidity).
Cytokinesis block (if used):
colchicine
Metabolic activation:
with and without
Metabolic activation system:
S9 after induction using Aroclor 1254
Test concentrations with justification for top dose:
8.89, 15.8, and 50 µg/mL; the highest concentration was chosen due to the limited solubility of the test item
Vehicle / solvent:
Solvent used: acetone
Justification: Analysis of the historical data of our laboratory and experience of other research groups showed that such amounts of the selected solvents have no influence on the mutation ftequency in this test system.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
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
- Expression time (cells in growth medium): 25 and 31 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

STAIN (for cytogenetic assays): MTT

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; cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
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:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no changes
- Data on osmolality: no changes
- Precipitation: @50 µg/mL
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: Due to the solubility of the test item and the observed precipitation in the culture medium at 50 µg/mL, the following concentrations were used in the main test: 8.89, 15.8 and 50.0 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: done

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No clear cytotoxic effects (i.e. reduction of mitotic index or cell viability)
Conclusions:
The treatment of V79 cell cultures with the test material did not increase the proportion of cells with aberrant chromosomes. Furthermore, the test item did not increase the number of polyploid cells or endoreduplications under the experimental conditions. In conclusion, the test item was not clastogenic in mammalian cells.
Executive summary:

The test material was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. 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 following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254):

No. of slides per concentration:
    Solvent:4
    others: 2
No. of metaphases per slide: 100 (structural abberations)
No. of metaphases per slide: 1000 (polyploidy)
Preparation times:
    -S9 mix: 25, 31 hours
    +S9 mix: 25 hours
Exposure times:
    -S9 mix: 5, 25, 31 hours
    +S9 mix: 5 hours
Solvent: acetone
Concentrations evaluated:
1st series. 8.89, 15.8 and 50 µg/mL
2nd series. 8.89, 15.8 and 50 µg/mL
Positive controls: EMS and Griseofulvin (-S9) and Cyclophosphamide (+S9)

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 473.

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. 50.0 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 1.50 % to 2.75 %. 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

The test item was investigated with regard to its mutagenic potential in a study according to OECD TG 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used.

The test material was dissolved in acetone and tested at concentrations ranging from 0.50 to 500 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 158 µg/plate. Toxicity to the bacteria was not observed.
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.
With and without addition of S9 mix as the external metabolizing system, the test material did not induce any clear or dose dependent increase in the mutation rates under the experimental conditions described.

Chromosome aberration assay

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. This study was performed according to GLP and the methods applied are fully compliant with OECD TG 473.
The test item precipitated in the culture medium at the highest concentration evaluated, i.e. 50.0 µg/mL. No clear cytotoxic effects, i.e. reduction in mitotic index or cell viability (MTT-test), were induced by the test material.
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.



Justification for classification or non-classification

Based on the data provided, which are considered to be suitable and reliable, the test item is not classified for mutagenicity/genotoxicity according to Regulation (EC) No 1272/2008.