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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471: not mutagenic in bacterial cells

OECD 473: not clastogenic in mammalian cells

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:
Jun 21 - Dec 12, 2000
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: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate (S9 10 %)
2. Series: 5, 15.8, 50, 158 and 500 µg/plate (S9 30 %)
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
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
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
Evaluation criteria:
Please refer to "any other information on materials and methods"
Statistics:
n.a.
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:
not examined
True 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
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
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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.
Executive summary:

Purpose

The purpose of this assay was to provide 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 the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, 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.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred in the concentration range between 158 and 500 µg/plate. Toxicity to the bacteria was not observed.

Each treatment with the test materials used as positive controls (see listed controls above) 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 of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results (see "Details on test system and conditions"), the test material was not mutagenic under the described experimental conditions.

Conclusion

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:
July 16 - November 19, 2002
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
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 Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium including 10% (v/v) FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (induction using Aroclor 1254)
Test concentrations with justification for top dose:
S9 Treatment+Recovery Vehicle Concentration
- 20+0 hours Ethanol 17.75, 20.88, and 24.57 µg / mL
- 44+0 hours Ethanol 15.09 and 17.75 µg / mL
+ 3+17 hours Ethanol 59.09, 65.61, and 81.00 µg / mL
+ 3+41 hours Ethanol 65.61, and 72.90 µg / mL
Vehicle / solvent:
Name: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Remarks:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Continous for 20 and 44 hours
Pulse for 3 + 17 and 3+41 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Controls: 4; others: 2

NUMBER OF CELLS EVALUATED: 100 metaphases (structural abberations)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other:
:
Evaluation criteria:
A test article is considered as positive in this assay if:
1. the proportions of cells with structural aberrations at one or more
concentration exceeds the normal range in both replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural
aberrations (excluding gaps) occurs at these doses.
Increased incidence of cells with gaps or increased proportions of cells with
structural aberrations not exceeding the normal range, or occurring only at very
high or very toxic concentrations are likely to be concluded as "equivocal". Full
assessment of the biological importance of such increases is likely only to be
possible with reference to data from other test systems. Evidence of a dose-related
effect is considered useful but not essential in the evaluation of a positive result.
Cells with exchange aberrations or cells with greater than one structural aberration
occur very infrequently in negative control cultures. Their appearance is therefore
considered to be of particular biological significance.
Statistics:
Standard statistical methods have been applied for data processing.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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: yes (441 µg/mL)
- Cytotoxicity: yes
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Due to the toxicity profiles observed (reductions in cell number), it was not possible to select the same top concentrations for analysis from treatments sampled at 44 hours as those for treatments sampled at 20 hours. The pattern of toxicity for both treatments sampled at 44 hours was such that two concentration choices were possible; one inducing below 50% cytotoxicity and one inducing 60% (or slightly above) cytotoxicity. It was considered prudent to analyse both concentrations from both treatments. Although contrary to the protocol, this was considered to add to the
data set and as such has no adverse effect on the validity of the study.

see attachment

Conclusions:
It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).
Executive summary:

Study Design

The test material was tested in an in vitro cytogenetics assay using duplicate cultures of Chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9). The test article was dissolved in sterile ethanol (ethanol) and the highest dose level used, 400 μg/mL, was in excess of the solubility limit in culture medium. In Experiment 1, treatment in the absence and presence of S-9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of the test material on relative cell number. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 27.49 and 83.89 μg/mL induced approximately 58% and 66% reduction in cell number and 0% and 49% mitotic inhibition (MIH) in the absence and presence of S-9 respectively. In Experiment 2, treatment in the absence of S-9 was continuous for either 20 hours or 44 hours. Treatment in the presence of S-9 was either for 3 hours only followed by a 17-hour recovery period prior to harvest (3+17) or for 3 hours followed by a 41-hour recovery period prior to harvest (3+41). Chromosome aberrations were analysed at two or three dose levels (see overleaf) and the highest concentrations chosen for analysis, 24.57 μg/mL (20+0) or 17.75 μg/mL (44+0) in the absence of S-9 and 81.00 μg/mL (3 +17) or 72.90 μg/mL (3+41) in the presence of S-9, induced approximately 50%, 60%, 54% and 62% reduction in cell number and 81%, 29%, 55% and 7% MIH respectively. Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1 -oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations. Positive controls were included with both treatments in Experiment 1, but only with the 20+0 hour -S-9 and 3+17 +S-9 treatments in Experiment 2.
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 473.

Results

Treatment of cultures with the test material in the absence and the presence of S-9 (both experiments) resulted in frequencies of cells with structural aberrations which were similar to those observed in concurrent vehicle controls for the majority of concentrations analysed. Two exceptions to this were observed: a single culture at the intermediate concentration analysed (67.11 mg/mL) from the 3+17 hour +S-9 treatment in Experiment 1 and a single culture from the lowest concentration analysed (15.09 mg/mL) from the 44+0 hour –S-9 treatment in Experiment 2 both exhibited numbers of aberrant cells that exceeded historical negative control (normal) values. However, in both instances these increases were not observed in the replicate cultures and were not dose-related. Furthermore, the increase observed in the presence of S-9 in Experiment 1 was not observed at similar concentrations analysed in Experiment 2 (performed under identical treatment conditions). The aberrant cell frequency of all other test material treated cultures fell within normal values. It was therefore considered that the increases observed were spurious and of no biological significance. Normal frequencies of cells with numerical aberrations (within historical negative control (normal) ranges) were observed for the large majority of test material treated cultures. The only exception to this was observed in the 3+17 hour –S-9 treatment at the highest concentration tested (27.49 mg/mL) where a single replicate showed an aberrant cell frequency that exceeded the normal range. However, this increase was small and was not observed in the replicate culture. As all other cultures (for all treatments) exhibited normal frequencies of numerical aberrations, this increase was not considered of biological importance.

Conclusion

It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to its limit of cytotoxicity in both the absence and presence of metabolic activation (S-9).

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 provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.