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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in the bacterial mutation assay (OECD 471) and not clastogenic in the in vitro chromosome aberration test (OECD 473).

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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:
1999-08-04 to 2000-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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: 0.5, 1.58, 5.00, 15.8, 50, 158 and 500 μg per plate (S9 10 %)
2. Series: 5.00, 15.8, 50.0, 158 and 500 μg per plate (S9 30 %)
Vehicle / solvent:
DMSO
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.
Rationale for test conditions:
according to Guideline
Evaluation criteria:
see Details on test systems and conditions
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 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:
June 14, 2005 - August 30, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests (1995)
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: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17 µg / ml final concentration
Experiment 2: 1, 2, 4, 6, 8, 10, 12, 14, 16, 17 µg / ml final concentration
A top concentration of 17.00 mg/mL (the maximum practicable concentration based on solubility in the primary solvent, DMSO) was chosen as the maximum for the cytotoxicity range-finding experiment. Doses for Experiment 1 were selected based on the results of this cytotoxicity range-finding experiment.
Vehicle / solvent:
DMSO
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 with the test article (0.1 mL per culture for all treatments). 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 then 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. Cell numbers were determined using a Coulter counter (and toxicity was subsequently further assessed by mitotic index).
The 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 cytotoxicity. 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% reduction in population doublings (approximately) 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 were checked to confirm that the presence of precipitate did not preclude analysis.
For treatments sampled at 20 hours, slides from the highest selected dose and two lower doses, such that a range of cytotoxicity from maximum to little or none is covered, were taken for microscope analysis. For treatments sampled at 44 hours, slides from the highest selected dose was taken for microscope analysis. This dose level should be the same as the highest dose taken from the corresponding treatment at the 20-hour sampling time, if possible.
In this study, due to the limited solubility of the test article in the primary solvent (DMSO), the highest dose tested for all 3-hour treatments in the absence and presence of S-9 was the maximum practicable concentration.
For each treatment regime, two solvent control cultures were initially to be 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.
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. 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. 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 and is detailed in Appendix 2. 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 the majority of the analysts participating in this study were physically located remote from the CLEH facility, all analysts 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 are analysable at each dose level, unless 10 or more cells showing structural aberrations other than gaps only have been 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 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
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses
3. there is a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
A test article will be considered positive in this assay if all of these criteria are met.
A test article will be considered negative in this assay if none of these 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. Analysis of additional cells from vehicle / or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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

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 anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest dose level used in the main experiments, 17.00 μg/mL, was the maximum practicable concentration based on solubility in the primary solvent (dimethyl sulphoxide, DMSO) and was determined following a preliminary cytotoxicity range finding experiment.

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. Chromosome aberrations were analysed at three dose levels (see below). The highest concentration chosen for analysis, 17.00 μg/mL, was the maximum practicable concentration and did not induce a reduction in population doubling in the absence and presence of S-9.

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 one or three dose levels (see overleaf). The highest concentration chosen for analysis, 17.00 μg/mL, resulted in the presence of precipitate (for 20+0 and 44+0 –S-9 treatments) or was the maximum practicable concentration (for 3+17 and 3+41 +S-9 treatments). This induced reductions in population doubling of 0% (20+0 -S-9 and 3+17 +S-9 treatments), 1% (3+41 +S-9 treatment) and 7% (44+0 -S-9 treatment).

Appropriate negative (solvent) 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 hour +S-9 treatments in Experiment 2.

Treatment of cultures with the test material in the absence and presence of S-9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to those in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in cells treated with the test item fell within historical negative control (normal) ranges.

No increases in the frequency of cells with numerical aberrations, which exceeded the historical negative control (normal) range, were observed in cultures treated with the test material in the absence and presence of S-9 in both experiments. It is concluded that the test material did not induce structural or numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells in both the absence and presence of metabolic activation (S-9). Cultures were tested up to the maximum practicable concentration (for 3+17 hours in the absence and presence of S-9 and for 3+41 hours in the presence of S-9) and into the precipitating range (for 20+0 hours and 44+0 hours in the absence of S-9).

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 available data, the test item is not classified for mutagenicity according to Regulation (EC) No 1272/2008.