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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint information from two different assays is available. The data were obtained in the following assays:

1) Bacterial in vitro mutagenicity (OECD 471): not mutagenic

2) Chromosomal abberation assay (OECD 473): not clastogenic

3) Mutagenicity in Mammalian Cells for structural analogue (OECD 476): not mutagenic

All these data have been obtained acording to the relevant OECD protocols.

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 08- Dec 08, 1993
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
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:
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):
trp C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
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: 5-250 µg/plate have been used in the 1st and 2nd series respectively. Precipitation of the test material defined the highest test material concentration.

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 102
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 250 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:

Purpose

The purpose of this in vitro reverse gene mutation test is to identify agents that cause mutations in bacteria cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. 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 for all strains. In the series with S9 mix, 10 % or 30 % S9 in the S9 mix were used inn the 1st and 2nd series, respectively.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5.00 to 250 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations larger/equal 250 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitroso-guanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds 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 substances 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.

No increase in revertant colonies was obtained after treatment with the test material up to the highest concentrations investigated, thus showing the absence of mutagenic potential in vitro with and without external metabolizing system.

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:
Jan 16, 2009 - Apr 29, 2009
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)
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):
- 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:
1.58, 5.00, 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
- 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
- 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: @15.8 µg/mL
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

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

Study Design

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. 1.58, 5.00, and 15.8 µg/mL
2nd series. 1.58, 5.00, and 15.8 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.

Results

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 2.0 % to 3.25 %. 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.

Conclusion

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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 16 - Dec 11 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The method applied followed the OECD Guideline for Testing of Chemicals No. 476 (In Vitro Mammalian Cell Gene Mutation Test). No quality assurance inspection according to GLP guidelines has been performed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Growth mediaThree 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/streptomycinCulture 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/streptomycinSurvivor- 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:
2% rat liver homogenate (S9 mix) with standard co-factors
Test concentrations with justification for top dose:
8.89, 28.1 and 88.9 µg/ml
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, acetone
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: without S9
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: with S9
Details on test system and experimental conditions:
see below
Evaluation criteria:
see below
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
@88.9 µg/mL (with S9)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material was weakly toxic to the mouse lymphoma cells in the absence of S9 mix at the concentration of 88.9 µg/ml. No toxicity was seen in the presence of S9 mix. Under the different experimental conditions of this study, the test material precipitated in the cell culture medium at concentrations levels between 28.1 and 158 µg/ml.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negativeIt is concluded that the test material is non-mutagenic in this screening test system under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

Purpose

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).

Study Design

The test material was screened for its ability to induce mutations at the TK locus (5-trifluorothymidine (TFT) resistance) in mouse lymphoma cells using a fluctuation protocol. The study was conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure time was 24 hours in the absence and 3 hours in the presence of S9 mix.

Results

The top doses to determine TFT resistance in this experiment were selected on the basis of the solubility and cytotoxicity characteristics of the test material. The test material was weakly toxic to the mouse lymphoma cells in the absence of S9 mix at the concentration of 88.9 µg/ml. Under the different experimental conditions of this study, the test material precipitated in the cell culture medium at concentration levels between 28.1 and 158 µg/ml. Concentrations ranging from 8.89 to 158 µg/ml were therefore tested.

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 benzo[a]pyrene (with S9 mix). Therefore the study was accepted as valid.

No relevant increases in mutant frequency were observed following treatment with the test material in neither the absence or presence of S9 mix.

Conclusion

It is therefore concluded that the test material is non-mutagenic in this screening 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)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 476. A detailed read across justification is provided in chapter 13 of this dossier.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
For this endpoint a one-to-one read across was performed to a chemical similar compound of the same chemical class with a comparable phys. chem. profile and similar response in biological assays. The relevant study was performed according to GLP and the methods applied are fully compliant with OECD TG 476. A detailed read across justification is provided in chapter 13 of this dossier. The result of the assay was negative with and without metabolic activation.
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 data provided, the test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.