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Genetic toxicity in vitro

Description of key information

The mutagenic and clastogenic potential of the test item was investigated in two in vitro gene mutation assays and one chromosome aberration test under GLP and Guideline conform conditions. Based on the results, the test item was neither mutagenic in bacterial cells nor in mammalian cells. Furthermore, the test item did not exhibit a clastogenic potential in mammalian cells.

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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:
Apr 22 - Nov 04, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. E631441
- Expiration date of the lot/batch: 1992-12-20

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stable
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in acetone
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: solution
- Final preparation of a solid: dissolved in acetone
Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Details on mammalian cell type (if applicable):
trp C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The WP2 uvrA-strain not only possesses the tryptophan mutation, but also lacks excision repair (uvrA), so that it is more readily mutated by agents such as UV.
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
his D 3052 mutation
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan: 50-1000 µg/plate have been used in the 1st and 2nd series respectively. Precipitation of the test material accured at concentrations larger or equal to 1000 µg/plate.

Vehicle / solvent:
Acetone
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Positive controls:
yes
Positive control substance:
other: daunomycine
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
The assessment of test material-induced effects is dependent on the number of spontaneous revertants 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 Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".
Interpretations:
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.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation occured at concentrations higher or equal to 1000 µg/plate. Toxicity to bacteria was not observed.
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 471.

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, 2000 - Feb 01, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
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:
mammalian cell line, other: CHL/IU
Details on mammalian cell type (if applicable):
- Type and identity of media: Nine hundreds milliliter of MEM was supplemented with 100 mL of heat-inactivated (56C, 30 min) calf serum (GIBCO BRL, lot no.42100611).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (induction using PB + BNP)
Test concentrations with justification for top dose:
-S9 mix: 920, 1840, 3680 µg / mL
+S9 mix: 920, 1840, 3680 µg / mL
Vehicle / solvent:
Name: Acetone, purity (GC) 99.5 %
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
short term treatment: 6 hours treatment 18 hours recovery
continous treatment: 24 hours

STAIN (for cytogenetic assays): Giemsa

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

NUMBER OF CELLS EVALUATED: 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A cell having at least one structural chromosomal aberration was classified as an aberrant cell. Aberrant cells were counted up excluding cells having only gaps. The test substance was judged to have a potential to induce chromosomal aberration as follows. When both of incidence of structural aberrant cells and that of numerical aberrant cells are less than 5 %, the test substance is negative (-). When either of the incidences is 5 % or more and less than 10 %, the test substance is inconclusive ( ±). When either of the incidences is 10% or more, the test substance is positive ( + ).
Statistics:
Standard statistical methods have been applied for data processing.
Key result
Species / strain:
mammalian cell line, other: CHL/IU
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:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material did not inhibit cell growth more than 50 % in each treatment. In a cytotox screening assay, precipitation was observed at and above 100 µg/mL.

See attachment

Conclusions:
It was concluded that the test item did not have clastogenic potential in this CHL/IU cell line system. The test item was thus not clastogenic in this in vitro test system.

Executive summary:

An in vitro chromosomal aberration study was conducted using the established cell line (CHL/IU) from the lungs of a Chinese hamster to determine the clastogenic potential of the test item. The test performed as short-term test (3 +18 hours) and as continous treatment test for 24 hours. This study was performed according to Japanese GLP and the methods applied are fully compliant with the Japanese Guideline for Mutagenicity Testing.

According to a preliminary test, concentrations selected for the cell growth inhibition test were 10, 20, 30, 100, 200, 500, 1000, 2000, and 3680 µg/mL for the short-term treatment without S9 mix (-S9 mix) and with S9 mix ( +S9 mix). The test substance did not inhibit cell growth more than 50% in any test substance treatment group. Precipitatoin was observed at and above a test material concentration of 100 µg/mL. From these results, concentrations selected for the chromosomal aberration test were 920, 1840, and 3680 µg/mL for the short-term treatment. The incidences of cells with structural and numerical aberrant chromosomes were less than 5% in each test substance treatment group. Therefore, the judgement in the short-term treatment was negative, and the test in the continuous treatment was conducted. According to a preliminary test, concentrations selected for the cell growth inhibition test were 10, 20, 30, 100, 200, 500, 1000, 2000, and 3680 µg/mL for the continuous treatment. The test substance did not inhibit cell growth more than 50 % in any test substance treatment group. Precipitatoin was observed at and above a test material concentration of 100 µg/mL. From this result, concentrations selected for the chromosomal aberration test were 920, 1840, and 3680 µg/mL for the continuous treatment. The incidences of cells with structural and numerical aberrant chromosomes were less than 5% in each test substance treatment group.

It was concluded that the test item did not have clastogenic potential in this CHL/IU cell line system. Thus, the test item was not clastogenic in this in vitro test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 16 - Dec 10, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
The methodology, originally complying with the ICH guidelines, OECD Test Guideline 476 and Annex V, EEC Directive 79-831, was modified in this study in order to screen a large number of test materials. In brief, the main modification is the use of fewer concentrations of the test material, use of only single cultures per concentration and a simplified data reporting. No quality assurance inspection according to GLP guidelines has been performed. Due to these modifications, this report does not fulfill all the requirements for the registration of pharmaceuticals or chemicals.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
fewer concentrations, single cultures, no GLP
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Batch: E98751641
Indication: liquid crystal
Solvent: acetone
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 1640
25 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 1640
50 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 1640
100 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
Test concentrations with justification for top dose:
in a range finding experiment, precipitation of the test material in the cell culture medium was macroscopically visible at the concentrations >/= 281 μg/mL. based on these findings the following
concentrations were selected for the first series in the main study in the presence and absence of S9 mix:
28.1, 88.9 and 281 μg test material/mL medium.
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-nitroquinoline N-oxide
Details on test system and experimental conditions:
see below
Evaluation criteria:
see below
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Conclusions:
It is concluded that the test material is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.
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 as mutagenic according to Regulation (EC) No 1272/2008.