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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the assessment of mutagenicity, in vitro methods have been applied.

The test item was not mutagenic in bacterial cells as determined in a study according toOECD Guideline 471. Additionally, the test item was not clastogenic in mammalian cells in a study according to OECD 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:
Feb 13 - Apr 18, 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)
Version / remarks:
adopted September 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
8 June 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor (pKM101)
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 (pKM101)
Additional strain / cell type characteristics:
other: mutations in the histidine operon
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:
E. coli WP2
Details on mammalian cell type (if applicable):
trp-, uvrA pkM101
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
(his G 428, rfa + R-factor)
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 EEC, OECD and Japanese guidelines for this test system. Solubility experiments using the standard solvents for this assay indicated that the test item showed best solubility performance in Acetone. Based on these data, Acetone was selected as solvent for the current experiment and 281 µg/plate was chosen as the appropriate maximum concentration due to the limited solubility of the test item.
1st series: 0.5, 1.58, 5, 15.8, 50, 158, and 500 μg per plate;
2nd series: 50, 15.8, 50, 158, and 500 μg per plate.
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 cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: -
- Exposure duration/duration of treatment: 2 days


- OTHER:
Rationale for test conditions:
according to Guideline
Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the con-current negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed. However, there is no requirement for verification of a clear positive response according to OECD TG 471.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
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 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:
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
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
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item 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:
August 26, 2005 - March 28, 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)
Deviations:
no
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):
CHO cells, supplied by Dr S Galloway, West Point, PA, USA, are maintained at Covance Laboratories Limited in tissue culture flasks containing McCoy's 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 µg/mL gentamycin. They are subcultured regularly at low density, and before overgrowth occurs, to maintain low aberration frequencies. Stocks of cells preserved in liquid nitrogen are reconstituted for each experiment so as to maintain karyotypic stability. The cells are screened for mycoplasma contamination.
Metabolic activation:
with and without
Metabolic activation system:
S9 after induction using Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1: 12.5, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600 µg / ml medium
Experiment 2: 25, 50, 75, 100, 150, 200, 300, 400, 500, 600 µg / ml medium
Experiment 2 (Trial 2): 50, 75, 100, 150, 175, 200, 250, 275, 300, 325, 350, 400, 500 µg / ml medium
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
-- Treatment
Prior to the start of treatment, the cell sheets from 4 flasks were removed using trypsin/EDTA and a mean cell count obtained. This provided the starting (baseline) count for the calculation of toxicity (expressed as population doublings relative to controls) at the time of cell harvest.
For the cytotoxicity range-finder experiment, S-9 mix or KCl (0.5 mL) was added appropriately as detailed previously. Duplicate cultures (A, B) were treated with the solvent and single cultures treated with the test article at appropriate concentrations (0.1 mL per culture). Positive control treatments were not included.
For the main experiments, S-9 mix or KCl (0.5 mL) was added appropriately as detailed previously. One set of quadruplicate cultures (A, B, C and D) for each of the treatment regimes was then treated with the solvent and one set of duplicate cultures (A, B) with the test article (0.1 mL per culture). Owing to toxicity associated with using acetone for the prolonged 44+0 hour -S-9 treatment, acetone was used as the vehicle at 0.5% final concentration in Experiment 2, Trial 2, and there were 0.05 mL additions for this trial. Additional duplicate cultures were treated with 0.1 mL of the positive control chemicals. All cultures were incubated at 37°C. Treatment media remained on cultures receiving the continuous treatment until sampling, that is, 20 or 44 hours after the beginning of treatment. Cultures received pulse treatments (both in the absence and presence of S-9) for 3 hours only. They were washed twice with sterile saline, and fresh medium containing foetal calf serum and gentamycin added. Cultures were incubated for a further 17 or 41 hours before harvesting.

-- Harvesting
Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase. The monolayers of these cultures were then removed using trypsin/EDTA. An aliquot of cell suspension from all cultures (with the exception of positive control treated cultures) was taken for determination of cell number by using a Coulter Counter.
The remaining suspension from each flask was transferred to a plastic centrifuge tube and the cells pelleted by centrifuging at 200 x 'g' for 5 minutes. The supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37°C for 5 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into an equal volume of fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 200 x 'g', 5 minutes) and resuspension. This procedure was repeated several times (centrifuging at approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean.

-- Preparation of metaphase spreads
Cells were kept in fixative in the refrigerator before slides were prepared but slides were not made on the day of harvest to ensure cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative (if required) so as to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred to clean microscope slides.
After the slides had dried on a warm plate the cells were stained for 5 minutes in 4% (v/v) filtered Giemsa stain in Gurr's pH 6.8 buffer. The slides were rinsed, dried and mounted with coverslips.

--Selection of doses for chromosome analysis
The doses selected for chromosome aberration analysis were selected on the basis of toxicity (expressed as population doublings relative to controls). Toxicity was assessed by two separate measures;

1) Population doublings relative to controls. Population doublings (PD) was calculated for each concentration as follows:
PD = [log (N / Xo)] / log 2, where N = mean final cell count/culture at each concentration Xo = starting (baseline) count

2) Mitotic inhibition relative to controls; that is, percentage of cells in mitosis. Slides from enough dose levels from each treatment group were scored to determine whether chemically induced mitotic inhibition had occurred. This is defined as a clear decrease in mitotic index compared with negative controls, (based on at least 1000 cells counted where possible), preferably dose-related.

Rationale for dose selection
The highest dose for chromosome analysis from cultures sampled at 20 hours should be one at which at least 50% (approximately) reduction in population doublings has occurred or should be the highest dose tested. Analysis of slides from highly cytotoxic concentrations is avoided, if possible. Slides from cultures treated with heavily precipitating doses are checked to confirm that the presence of precipitate does not preclude analysis.

For treatments sampled at 20 hours, slides from the highest selected dose and at least two lower doses, such that a range of cytotoxicity from maximum to little or none is covered, were taken for microscope analysis. For each treatment regime, two solvent control cultures were analysed for chromosome aberrations. Slides from the remaining solvent control cultures were only to be analysed if considered necessary, for example, to help resolve an equivocal result. A single positive control dose level, which gives satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage, is analysed. Untreated controls were only analysed for the continous 44 hour exposure in Experiment 2, Trial 2, where some toxicity appeared to be associated with the use of acetone (although there were no increases in the frequency of chromosome abberations).
Cell count data and the results of dose selection are presented in the results section of this report.

-- Scoring of aberrations
Slides from the selected treatments and from solvent and positive controls were coded using randomly generated letters by a person not connected with the scoring of the slides. The comparison of the cytotoxicity results for the untreated controls and for acetone indicated that acetone was a suitable solvent for the majority of treatments. Since prolonged exposure using acetone as a vehicle appeared to be associated with increased toxicity, cells from untreated control cultures were analysed after the 44 hour treatment in the absence of S-9 (Experiment 2, Trial 2) as a precautinary measure. Labels bearing only the study reference number, experiment number and the code were used to cover treatment details on the slides.
Where possible, one hundred metaphases from each code were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) was noted on a slide, analysis may be terminated. Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately. Classification of structural aberrations was based on the scheme described by ISCN. Under this scheme, a gap is defined as a discontinuity less than the width of the chromatid and no evidence of displacement of the fragment and a deletion is defined as a discontinuity greater than the width of the chromatid and/or evidence of displacement of the fragment. Observations were recorded on raw data sheets with the microscope stage co-ordinates of any aberrant cell.
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories Harrogate (CLEH) standard operating procedures. Although physically located remote from the CLEH facility, all analysts participating in this study were subject to CLEH management and GLP control systems, and all slides and raw data were returned to CLEH for archiving on completion of their analysis.

--Treatment of data
After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorised as follows:
1. cells with structural aberrations including gaps
2. cells with structural aberrations excluding gaps
3. polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures were compared with the current laboratory negative control (normal) ranges to determine whether the assay was acceptable or not. The proportion of cells in category 2 in test article treated cultures were also compared with normal ranges. The statistical significance of any data set was only to be taken into consideration if the frequency of aberrant cells in both replicate cultures at one or more concentration exceeded the normal range. Under this condition, the statistical method used would be Fisher's exact test. Probability values of p < 0.05 were to be accepted as significant. The proportions of cells in categories 1 and 3 were also examined in relation to historical negative control (normal) ranges and statistical analysis by Fisher’s exact test may be used.
The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values of p < 0.05 were to be accepted as significant.
Evaluation criteria:
Acceptance criteria
The CHO assay is considered valid if the following criteria are met:
1. the binomial dispersion test demonstrates acceptable heterogeneity between replicate cultures, and
2. the proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the normal range, and
3. at least 160 cells out of an intended 200 were analysable at each dose level, unless 10 or more cells showing structural aberrations other than gaps only were observed during analysis, and
4. the positive control chemicals induce statistically significant increases in the number of cells with structural aberrations.

Evaluation criteria
A test article iwill be evaluated as to whether:
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.
3. a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
A test article is considered positive in this assay if all of the above criteria are met.
A test article is considered negative in this assay if none of the above criteria are met.

Data that do not fall into either of the above categories are judged on a case by case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance is taken into account, for example consistency of response within and between concentration levels and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.
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
Positive controls validity:
valid

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 acetone. There was a preliminary cytotoxicity range-finding experiment. The highest concentration used in the main experiments, 600.0 µg/mL, was deemed to be the maximum practicable that could be administered to the test system, and was above the level of aqueous solubility.

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 population doubling and mitotic index. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 500.0 µg/mL in the absence and presence of S-9, induced approximately 2% and 69% reduction in population doubling, respectively.

Less than the target 50% cytotoxicity was observed at the highest concentration analysed from the treatment in the absence of S-9 (500.0 µg/mL). this concentration was very close to the maximum that could be tested practicably, and was above the limit of aqueous solubility, with precipitate being evident at harvest. This meant that exposure was longer than the nominal 3 hours. For the above reasons, 500.0 µg/mL was judged a suitable maximum.

In Experiment 2, treatment in the absence of S-9 was continuous for either 20 hours (20+0) or 44 hours (44+0). Treatment in the presence of S-9 was either for 3 hours 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 three or four dose levels (see overleaf). The highest concentrations chosen for analysis were 150.0 µg/mL (20 +0) or 50.0 µg/mL (44 +0) in the absence of S-9 and 300.0 µg/mL (3 +17) or 175.0 µg/mL (3 +41) in the presence of S-9. These concentrations induced approximately 4%, 20%, 50% and 61% reduction in population doubling, respectively.

Less than the target 50% cytotoxicity, as measured by population doubling, was associated with exposure to the highest concentration analysed from the 20 +0 hour -S-9 treatment (150.0 µg/mL). However, this concentration was associated with a 50% reduction in mitotic index, was in excess of the solubility limit in the culture medium, and higher concentrations were not significcantly more toxic. As such, 150.0 g/mL was considered a suitable maximum. Less than the target 50% cytotoxicity, as measured by population doubling, was associated with exposure to the highest concentration analysed from the 44 +0 hour -S-9 treatment (50.0 µg/mL). However, this concentration was associated with a 50% reduction in mitotic index, was in excess of the solubility limit in the culture medium, and higher concentrations were associated with less toxicity. As such, 50.0 g/mL was considered a suitable maximum.

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition and untreated controls were added as acetone is not used a vehicle as frequently as commonly used solvents. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) 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 Experiments 1, but only with the 20 +0 hour -S-9 and 3+17 hour +S-9 treatments in Experiment 2.

Treatment of cultures with the test material in the presence and absence of S-9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to, and not significantly different from, those in concurrent negative controls. The numbers of aberrant cells (excluding gaps) in all test material treated cultures fell within historical vehicle control (normal) ranges.

There were three increases in the frequency of numerical aberrations (polyploidy or endoreduplication) in cultures treated with the test material. These increases were small on two occasions, (one in the absence and the other in the presence of S-9, following a three-hour exposure), and relatively high in one culture, exposed for 44 hours at 25.00 µg/mL in the absence of S-9. These increases occured in single cultures, were scattered throughout the experiments, and were not clearly related to concentration. Therefore, these increases were considered to be sporadic. Moreover, it is noted that relatively few cells were examined for ployploidy and endoreducplication, and this assay is not primarily designed to investigate numerical aberrations. The biological significance of polyploidy is unclear.

It is concluded that the test material did not induce structural chromosome aberrations in cultured Chinese hamster ovary (CHO) cells, either in the absence or presence of metabolic activation (S-9), under the experimental conditions described. Testing was up to the limit of cytotoxicity or the highest practicable concentration, which was above the limit of aqueous solubility. This assay is primarily designed to investigate structural chromosome aberrations, but it is noted that a few instances of increases in numerical aberrations were recorded, which are considered to be sporadic, under the experimental conditions described.

Conclusions:
The test material was not clastogenic in this in vitro test system.
Executive summary:

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

The test material was 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

Justification for classification or non-classification

Based on the provided information the test item is not classified for mutagenicity according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.