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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

GLP OECD 471: negative

non GLP OECD 476: negative

Read Across to GLP OECD 473: negative

Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 03, 2006 - Jun 22, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
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
- Source of cells:Hoffmann-La Roche, Pharma, Basel, at May 27, 1997
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index: 16 to 17.5 hours
V79 cells have been successfully used in mutagenicity testing for many years. This cell line has a high proliferation rate and cloning efficiency. The cells have a relatively stable karyotype with a modal chromosome number of 22 ± 1, and an aberration rate of about 0-5 % of the metaphases. Since the cell line is not able to metabolize indirect mutagens to reactive forms, the test is performed both in the presence and absence of an external metabolizing system (liver S9 mix of rats pre-treated with Aroclor 1254).



Metabolic activation:
with and without
Metabolic activation system:
S9-mix with S9 from Aroclor 1254 induced rats
Test concentrations with justification for top dose:
In the first experimental series, test material concentrations ranging from 0.50 to 500 µg/mL were tested. The test material precipitated in the culture medium at concentrations ≥158 µg/mL in the absence and presence of S9 mix. No cytotoxic effects were seen. Quality control of the cell preparations did not show a relevant influence of the test material on the structur,e or spreading of the chromosomes in the concentration range investigated. A change in the pH or the osmotic value of the cell culture medium did not occur in the dose range tested.
For these reasons, cultures treated with the following test material concentrations were evaluated in absence and presence of S9 mix for the occurrence of chromosomal aberrations:
1st series: 15.8, 50.0, and 158 µg/mL
2nd series: 50.0, 88.9, 158, 281 and 500 µg/mL
Vehicle / solvent:
Acetone, 0.1%
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:
No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35hours

Exposure times:
- S9 mix: 5, 25 and 35 hours
+ S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated:
Test item first series: 15.8, 50.0 and 158 µg/mL
second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls: - S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL
31.6 and 88.9 µg Griseofulvin (GRIS)/mL

+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL
Rationale for test conditions:
Guideline settings are applied
Evaluation criteria:
Step 1: Evaluation of Slide Quality
Step 2: Evaluation of Chomosomal Aberrations

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 unambiguously 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.
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.
In both cases, however, the number of aberrant metaphases should 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 following the scientific evaluation of all existing data including those of non­ toxicological investigations.
Statistics:
Fisher’s Exact Test
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
Conclusions:
In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.
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 28 - Jul 07, 1997
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRP 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:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G 428, 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):
uvrA pkM101
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors
Test concentrations with justification for top dose:
The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 10 %)
1. Series: 1.58, 5, 15.8, 50, and 158 µg/plate (S9 30 %)
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
cumene hydroperoxide
other: daunomycin
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
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.
Evaluation criteria:
details see results
Statistics:
n.a.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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 gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June - 24 November 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no GLP
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
less concentrations, only single cultures per concentration, and a simplified data reporting
Qualifier:
according to guideline
Guideline:
other: EEC Directive 79-831
Version / remarks:
Annex V, EEC Directive 79-831, Part B, Toxicological Methods of Annex VIII. In vitro Mammalian cell - gene mutation test.
Commission of the European Communities, March 1985:85-7.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995) and ICH Requirements for Registration of Pharmaceuticals for Human Use, Geno¬toxicity: a Standard Battery for Genotoxicity Testing of Pharmaceuticals (Step 4, recommended for adoption 16 July 1997).
Principles of method if other than guideline:
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.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Batch: E97294446

Target gene:
HPRT
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of rats pretreated with Aroclor 1254
Test concentrations with justification for top dose:
In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treat-ment and compared with cell number in the absence of the test material. The test material was weakly cytotoxic (67% cell growth at 28.1 µg/ml in the presence of S9 mix as compared to the actual solvent control) in this range finding test.

Precipitation of the test material in the cell culture medium was macroscopically visible at concentrations >- 28.1 µg/ml.


24 hours treatment without S9 mix: 8.89, 15.8 and 28.1 µg /mL
3 hours treatment with S9 mix: 8.89, 15.8 and 28.1 µg /mL


Vehicle / solvent:
Acetone (0.1% (v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 24 hours without metabolic activation, 3 hours with metabolic activation
- Expression time (cells in growth medium): 48hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine


NUMBER OF REPLICATIONS: 1


NUMBER OF CELLS EVALUATED: >1x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: relative survival

Evaluation criteria:
Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no relevant increase in the mutation frequency (at least a 2-fold) occurs.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear increase in the mutation frequency (at least a 2-fold) occurs dose-dependently (over at least two test material concentrations) or reproducibly at identical concentrations in two independent 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.
Statistics:
No statistics was applied, biological significance was considered as main factor for evaluation.
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: determined at 28.1 µg/mL
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
In a preceding range finding test, cell growth was determined after exposure (3 hours in the presence and 24 hours in the absence of S9 mix) to various test material concentrations. The cell number was determined microscopically 24 hours after start of treatment and compared with cell number in the absence of the test material. The test material was weakly cytotoxic (67% cell growth at 28.1 µg/mL in the presence of S9 mix as compared to the actual solvent control) in this range finding test.

Precipitation of the test material in the cell culture medium was macroscopically visible at concentrations of 28.1 µg/ml.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by an adjusted relative survival below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

Without S9 mix (EZ1144):

Test Material

Conc.
[µg/ml]

Rel.Survival [%]a

Mutation
frequ.
b

Solvent

 

100

253

 

 

 

172

 

 

 

 

Test item

8.89

69

228

 

15.8

82

154

 

28.1PP

77

102

 

 

 

 

NQO

0.10

43

776

 

0.20

56

706

 

 

 

With S9 mix(EZ1144):

Test Material

Conc.
[µg/ml]

Rel.Survival [%] a

Mutation
frequ.
b

Solvent

 

100

188

 

 

 

202

 

 

 

 

Test item

8.89

284

180

 

15.8

230

137

 

28.1PP

189

238

 

 

 

 

BaP

2.00

230

987

 

3.00

218

755

 

a: On day 1 of the experiment                                b: Per 106viable cells

NQO: 4-Nitroquinoline N-oxide;                           BaP: Benzo[a]pyrene

PP: Test material precipitated until end of the exposure time


Conclusions:
In conclusion it can be stated that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.

Executive summary:

OECD 476 screening assay: negative

Endpoint:
in vitro cytogenicity / chromosome aberration 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)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint information from a structural similar compound is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 473. See chapter 13 report for a more detailed justification.
Reason / purpose for cross-reference:
read-across source
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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Based on the available results obtained from Guideline studies, the test item is not considered to be mutagenic in bacteria and mammalian cells. Furthermore, the test item is not considered to be clastogenic in mammalian cells based on the information obtained from a GLP and Guideline conform study with the structural analogue substance.

Justification for classification or non-classification

Based on the data provided, the test item is not classified and labelled as mutagenic according to Regulation (EC) No 1272/2008.