Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

With and without addition of S9-Mix as the external metabolizing system, the test item was not mutagenic in the Bacterial Reverse Mutation Assay (OECD 471).

The test item did not induce structural chromosome aberrations in cultured Chinese Hamster Ovary (CHO) cells in both the absence and presence of metabolic activation (S-9), when tested at concentrations up to its limit of cytotoxicity or solubility. Treatment of cultures with the test item induced numerical aberrations both in the presence and in absence of S-9, however since this assay is not primarily designed to investigate numerical aberrations the biological significance of these observations is not known.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 1997 to 20 March 1997
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:
1983
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
other: Proposal for Replacement of Guidelines 471 and 472: Bacterial Reverse Mutation Test, Revised Draft Document, OECD
Version / remarks:
1995
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix induced by Aroclor 1254
Test concentrations with justification for top dose:
First series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate (maximum recommended concentration according to Guideline)
Second series: 5, 15.8, 50, 158, and 500 µg/plate (highest concentration was choosen due to precipitation at 5000 µg/plate)
Vehicle / solvent:
- Vehicle/solvent used: DMSO
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
benzo(a)pyrene
cumene hydroperoxide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 independent experiments (3 plates per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria



Evaluation criteria:
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 streng precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases should a third test series with the bacterial strain in question be performed. lf 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated 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
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
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations >= 500 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control compounds should induce a "clear increase" in the number of revertants.
- Negative (solvent/vehicle) historical control data: TA98: 15- 60, TA 1535: 3- 37, TA 100: 75 - 200, TA 1537: 4 - 31, TA 102: 200 - 450, WP2: 50 - 200

Conclusions:
With and without addition of S9-Mix as the external metabolizing system, the test item was not mutagenic in the Bacterial Reverse Mutation Assay.
Executive summary:

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. The test item was dissolved in dimethyl sulfoxide and tested at concentrations ranging between 5 and 5000 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 500 μg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 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.

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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2005 - 21 November 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)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Tripartite Hannonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test article stock solutions were prepared by dissolving the test item in DMSO, with the aid of sonication, warming at 37°C and vortex mixing (where required), to give the top concentrations as specified. The stock solutions were membrane filter-sterilised (Pall Acrodisc CR, pore size, 0.2 μm) and subsequent dilutions made using sterile DMSO. The test article solutions were protected from light and used within 3.5 hours.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr S Galloway, West Point, PA, USA
- Suitability of cells: The cells have a low number of chromosomes which simplifies chromosome analysis.
- Methods for maintenance in cell culture if applicable: They are subcultured regularly at low density, and before overgrowth occurs, to maintain low aberration frequencies.
- Modal number of chromosomes: 21

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's SA medium including 10"/o (v/v) foetal calf senun (FCS), and 100 μg/mL gentamycin (5% CO2)
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from male Sprague Dawley rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 (- S9); 3 + 17 hours: 25, 50, 100 µg/mL
Experiment 1 (+ S9); 3 + 17 hours: 300, 400, 500 µg/mL

The highest concentrations chosen for analysis, 100.0 μg/mL in the absence of S-9 and 500.0 μg/mL in the presence of S-9, induced approximately 56% and 64% reduction in population doubling respectively.

Experiment 2 (- S9); 20 + 0 hours: 16.38, 20.48, 25.60 µg/mL
Experiment 2 (- S9); 44 + 0 hours: 8.389, 10.49, 13.11 µg/mL
Experiment 2 (+ S9); 3 + 17 hours: 125, 150, 250 µg/mL
Experiment 2 (+ S9); 3 + 41 hours: 180.4, 225.5, 281.9, 2100 µg/mL

The highest concentrations chosen for analysis, 25.60 μg/mL (20+0) or 13.11 μg/mL (44+0) in the absence of S-9 and 250.0 μg/mL (3+17) or 2100 μg/mL (3+41) in the presence of S-9, induced approximately 65%, 57%, 59% and 11% reduction in population doubling,
respectively. Although less than the target 50% cytotoxicity observed at the highest concentration analysed from the 3+41 hour +S-9 treatment (2100 μg/mL), this concentration was observed to be in excess of the solubility limit in the culture medium (at the end ofthe treatment period) and was the maximum practicable concentration achievable in this test system.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 6E+5 cells per flask (20 hour treatments) or 3E+5 cells per flask (44 hour treatments) corresponding to 9.4 mL cell suspension per 75 cm2 flask

DURATION
- Exposure duration: Continuous treatment: 20 or 44 hours (- S9 mix only). Pulse treatment: 3 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The pelleted cells were resuspended in 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 to fresh, ice-cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation and resuspension. The procedure was repeated several times until the cell pellets were clean. For 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. After fixation the slides dried on a warm plate and 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.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 per dose (100 per slide)

DETERMINATION OF CYTOTOXICITY
- Method: population doubling, cell counts, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes


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.
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. Analysis of additional cells from vehicle / or treated cultures or further experimental work may be deemed necessary to aid evaluation of the data.
Statistics:
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 aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. Probability values ofp <= 0.05 were to be accepted as significant.
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: Yes

RANGE-FINDING/SCREENING STUDIES: Based on the results of the range-finding study the following doses were selected for analysis:
Experiment 1 (- S9); 3 + 17 hours: 25, 50, 100 µg/mL
Experiment 1 (+ S9); 3 + 17 hours: 300, 400, 500 µg/mL

Experiment 2 (- S9); 20 + 0 hours: 16.38, 20.48, 25.60 µg/mL
Experiment 2 (- S9); 44 + 0 hours: 8.389, 10.49, 13.11 µg/mL
Experiment 2 (+ S9); 3 + 17 hours: 125, 150, 250 µg/mL
Experiment 2 (+ S9); 3 + 41 hours: 180.4, 225.5, 281.9, 2100 µg/mL
Although less than the target 50% cytotoxicity observed at the highest concentration analysed from the 3+41 hour +S-9 treatment (2100 μg/mL), this concentration was observed to be in excess of the solubility limit in the culture medium (at the end ofthe treatment period) and was the maximum practicable concentration achievable in this test system.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
A) -S9-mix: mean of structural aberrations excluding gaps = 1.01 (SD: 1.37, 95% reference range: 0-4); mean of endoreduplicated cells = 0.03 (SD: 0.17, 95% reference range: 0-1); mean of polyploid cells = 2.94 (SD: 1.66, 95% reference range: 0-6); mean of numerical aberrations = 3.02 (SD: 1.68, 95% reference range: 0-6)

B) +S9-mix: mean of structural aberrations excluding gaps = 1.25 (SD: 1.23, 95% reference range: 0-4); mean of endoreduplicated cells = 0.47 (SD: 0.85, 95% reference range: 0-3); mean of polyploid cells = 2.86 (SD: 1.84, 95% reference range: 0-7); mean of numerical aberrations = 3.39 (SD: 2.03, 95% reference range: 0-8)

Less than 50% cytotoxicity was observed at the highest concentration analysed from 3 +41 hour +S-9 treatment (2100 µg/mL). However, this concentration was observed to be in excess of the solubility limit in the culture medium (at the end of the treatment period) and was the maximum practicable concentration achievable in this test system.

Conclusions:
lt is concluded that the test item did not induce structural chromosome aberrations in cultured Chinese hamster ovary (CHQ) cells in both the absence and presence of metabolic activation (S-9), when tested at concentrations up to its limit of cytotoxicity or solubility. Treatment of cultures with the test item induced numerical aberrations both in the presence and in absence of S-9, however since this assay is not primarily designed to investigate numerical aberrations the biological significance of these observations is not known.
Executive summary:

The test item was tested in an in vitro cytogenetics assay according to OECD Guideline 473 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) and the highest dose level used in the main experiments, 2100 μg/mL, 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 test article dose levels for chromosome analysis were selected by evaluating the effect of the test item on population doubling. Chromosome aberrations were analysed at three dose levels. The highest concentrations chosen for analysis, 100.0 μg/mL in the absence of S-9 and 500.0 μg/mL in the presence of S-9, induced approximately 56% and 64% reduction in population doubling respectively.

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 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 up to four dose levels and the highest concentrations chosen for analysis, 25.60 μg/mL (20+0) or 13.11 μg/mL (44+0) in the absence of S-9 and 250.0 μg/mL (3+17) or2100 μg/mL (3+41) in the presence of S-9, induced approximately 65%, 57%, 59% and 11 % reduction in population doubling, respectively. Although less than the target 50% cytotoxicity observed at the highest concentration analysed from the 3+41 hour +S-9 treatment (2100 μg/mL), this concentration was observed to be in excess of the solubility limit in the culture medium (at the end of the treatment period). As such, this was considered a suitable maximum.

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 (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 Experiment 1, but only with the 20+0 hour -S-9 and 3+17 +S-9 treatments in Experiment 2.

Treatment of cultures with the test item in the absence and presence of S-9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to, and not significantly (p ≤ 0.05) different from those seen in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all test item treated cultures fell within historical vehicle control (normal) ranges.

Treatment of cultures with the test item induced numerical aberrations both in the presence and in absence of S-9. Pulse treatments in the absence and presence of S-9 in Experiment 1 resulted in an increase in the frequencies of cells with endoreduplication at all concentrations analysed. No such increases in endoreduplicated cells were observed following treatments in Experiment 2 (20+0 -S-9, 3+ 17 +S-9, 44+0 -S-9 and 3+41 +S-9). However, there were some instances (single cultures at 225.5 and 281.9 μg/mL and both cultures at 2100 μg/mL) of increases in frequencies of polyploid cells following 3+41 hour +S-9 treatments in Experiment 2. However, since this assay is not primarily designed to investigate numerical aberrations the biological significance of these oberservations is not known.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed. The number of normochromatic cells with micronuclei was not increased.

In conclusion, the test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 2006 - 04 April 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
8 June 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests
Version / remarks:
1995
Qualifier:
according to
Guideline:
other: CH Requirements for Registration of Pharmaceuticals for Human Use, Genotoxicity: a Standard Battery for Genotoxicity Testing of Pharmaceuticals (Step 4, recommended for adoption 16 July 1997).
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recommended species according to Guideline.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 223 - 239 g
- Assigned to test groups randomly: yes, under following basis: according to a random !ist which had been provided by the Rando 96 program developed and used at Merck KGaA, Darmstadt
- Housing: individually in Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm) on softwood chippings
- Diet: ad libitum (Standard diet from Provimi Kliba SA)
- Water: ad libitum (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 45 - 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Miglyol 812 neutral oil
- Concentration of test material in vehicle: 20 mL/kg bw
Details on exposure:
To achieve maximal sensitivity of the test system, the test material was administered once intraperitoneally in this investigation. This procedure is in compliant with the OECD Guideline (dated on 1997) and EU Method B.12 (dated 2000).
Duration of treatment / exposure:
24 and 48 hours after administration of the test material preparation of bone marrow smears took place for the high dose group. Bone marrow smears were prepared 24 hours after administration for the low and mid dose group.
Frequency of treatment:
once
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
633 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): With this dose a statistically significant increase in the number of polychromatic erythrocytes with micronuclei, as compared to the negative control, was to be expected.
- Route of administration: oral
- Doses / concentrations: 16.5 mg/kg bw
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose (2000 mg/kg bw) was selected to produce signs of toxicity but no mortality. The mid (633 mg/kg bw) and low dose (200 mg/kg bw) is obtained by dilution in half-log ranges, i.e. 3.16.

TREATMENT AND SAMPLING TIMES: The test material was administered once. Preparation of bone marrow smears took place 24 and 48 hours after administration.

DETAILS OF SLIDE PREPARATION: The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum with the aid of a syringe, and suspended in the serum. This suspension was filtered through cellulose according to Romagna (1988) and centrifuged for 5 in at 150 x g. The sediment was then resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension. After 3 hours of drying, the slides were stained according to a modified Giemsa- staining method described by Gollapudi and Kamra (1979).

METHOD OF ANALYSIS: A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics. Round particles with about 1120 - 115 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic. For determination of the quotient of nonnochromatic to polychromatic erythrocytes, both erythrocyte stages were screened for micronuclei and counted separately up to a total of 1000 erythrocytes per animal. Then counting was limited to polychromatic erythrocytes. Nonnochromatic erythrocytes with micronuclei, however, observed during scoring were registered also. Thus, based on this value and on the quotient, the number of micronucleated nonnochromatic erythrocytes per 1000 could be extrapolated.
Evaluation criteria:
The primary parameter for evaluation of the results of this test system is the number of micronucleated, polychromatic erythrocytes (MN-PCE). This number is increased by treatment of cells with mutagenic test materials as compared to the respective negative controls.

- A positive effect in this test system is defined by the occurrence of mean MNPCE values of a treatment group which are statistically significantly higher than those of the actual negative control. A prerequisite for this is, however, that these values are above those predetermined as historical negative controls of our laboratory (cf. Historical data).
An indispensable prerequisite for evaluating the results of such investigations is the occurrence of significant positive effects in the actual positive control group.

- A test material showing no positive effect in the main study is defined as a non-mutagen in this test system. In this case the study is terminated.

- If a positive effect in a single test group occurs (i.e. dose-independently), a repeat experiment has to be considered. In case that no positive effects occur in that experiment the test material is defined as a non-mutagen. The single positive effect of the first experiment is interpreted as a randomly occurring event of no biological significance.

- A test material is defined as mutagenic in this system if dose-related and/or single, reproducible (in independent experiments) positive effects occur. Establishment of dose-dependent effects of the test material is preferable. For this reason, if a positive effect occurs in a study in which a single, limit dose of 2000 mg/kg bw has been applicated, 3 different test material doses have to be administered in the supplementary experiment. The criteria mentioned above for a negative or positive test result apply for this experimental design likewise.



Statistics:
Descriptive statistics
For all groups, mean values were calculated ofthe following parameters:
NCE/PCE - number ofnonnochromatic erythrocytes (NCE)/ number of polychromatic erythrocytes (PCE)/ animal
MN-NCE - number of micronuclei-containing cells/ 1000 NCE/ animal
MN-PCE number ofmicronuclei-containing cells / 1000 PCE/ animal
For the parameter body weight the mean values and the relative body weight gains to the preceding mean values were calculated.

Statistical tests
For further statistical analysis, the numbers of micronuclei-containing polychromatic erythrocytes (MN-PCE), nonnochromatic erythrocytes (MNNCE) and the quotient of NCE/PCE per animal were used.

Pairwise comparison
Bach treatment group was compared to the negative control.

For comparisons of micronuclei-containing polychromatic and normochromatic erythrocytes, the exact Mann-Whitney-test was used against one-sided alternatives. The p-values ( exact significance one-sided) of these comparisons are presented.

For comparisons of the quotient of NCE/PCE, the Dunnett's t-test was used against two-sided alternatives. The p-values (significance) of these comparisons are presented for those dose groups that showed a higher mean value than the negative control.

Software
The numerical calculation was performed using the computer-aided micronucleus test pro gram (Version 2.3c) and for the exact Mann Whitney test and Dunnett's t-test the SPSS-System (Version 9.0), running under Windows NT was used.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
in the highest dose group
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: The test material was diluted in Miglyol 812 neutral oil.
- Clinical signs of toxicity in test animals: Clear toxic effects (ptosis, abdominal position and a loss in body weight) but no mortality were observed in the high dose group (2000 mg/kg bw).
- Rationale for exposure: Dose was selected to produce signs of toxicity but no mortality.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): No treatment-related variation was observed.
- Appropriateness of dose levels and route: The route chosen is in compliant with the OECD Guideline (dated on 1997) and EU Method B.12 (dated 2000). Appropriate dose levels were selected as the highest dose group induced some clinical signs but no mortality. No clinical signs were observed in the mid and low dose group, however, a decrease in body weight was observed.
Conclusions:
The test material did not cause a relevant increase in the number of polychromatic erythrocytes with micronuclei and did not increase the number of normochromatic cells with micronuclei. Therefore, the test item is not considered to be mutagenic in the micronucleus test in rats under conditions wher the positive control exerted potent mutagenic effects.
Executive summary:

The micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No. 474 under GLP conditions

In the current experiment the test material was given once intraperitoneally to male rats. Rats of the negative control group received the solvent for the test material alone, i.e. an intraperitoneal dose of 20 mL/kg body weight Miglyol 812 neutral oil. The animals of the positive control group were treated with an oral dose of 16.5 mg /kg body weight cyclophosphamide.

Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the high dose group (2000 mg/kg bw), preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the low and mid dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. A total of 30 animals (5 male rats per dose group) were used.

For microscopic investigation one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined.

The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micronucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.

 The highest test material dose induced some clinical signs of toxicity and, in addition, a weak decrease in body weight. No relevant treatment-related variation was observed for the quotient of normochromatic : polychromatic erythrocytes.

The mean numbers of polychromatic erythrocytes with micronuclei for the negative control (solvent) were all in or very close to the expected range predetermined by historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.

No biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed.

The number of normochromatic cells with micronuclei was not increased.

In conclusion, the test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

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