Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, gene mutation in bacteria, OECD 471, GLP, with and without S9: negative with and without S9

Key, chromosomal aberration, OECD 473, GLP, with and without S9: negative without S9, positive with S9

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 AUG 2005 - 14 JUL 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted according to Good Laboratory Practice (GLP) and followed the OECD Guideline for the testing of Chemicals: Genetic Toxicology: 473 In vitro mammalian chromosome aberration test.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
21 Jul 1997
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.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 after induction using Aroclor 1254
Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rats induced with Aroclor 1254
- method of preparation of S9 mix : The batches of MolTox S-9 were stored frozen in aliquots at -80°C and thawed just prior to use. Glucose-6-phosphate (180 mg/mL), NADP (25 mg/mL), 150 mM KCl and rat liver S-9 were mixed in the ratio 1:1:1:2. An aliquot of the resulting S-9 mix was added to each cell culture designated for treatment in the presence of S-9 to achieve the required final concentration in a total of 10 mL. The final concentration of liver homogenate in the test system was 2%. Cultures treated in the absence of S-9 received an equal volume of 150 mM KCl.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-Odealkylase activities).
Test concentrations with justification for top dose:
Top concentration in experiment was determined based on range-finding test

concentrations used for cell analysis:
Experiment I:
200.0, 300.0, 350.0 µg/mL (-S9)
200.0, 250.0 , 300.0 µg/mL (+S9)
The highest concentrations chosen for analysis (3 treatment + 17 recovery hours), 350.0 µg/mL in the absence of S-9 and 300.0 µg/mL in the presence of S-9, induced approximately 50% and 42% reduction in population doubling, respectively.

Experiment II
5, 100, 1500, 3.355 µg/mL (-S9)
50, 250, 300 µg/mL (+S9)
The highest concentrations chosen for analysis (treatment + recovery hours), 1500 µg/mL (20+0) and 3.355 µg/mL (44+0) in the absence of S-9 and 300.0 µg/mL (3+17 and 3+41) in the presence of S-9, induced approximately 39%, 47%, 59% and 25% reduction in population doubling, respectively.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile DMSO was added to cultures designated as negative controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.929 x 10E6 (20+0, -S9, 3+17, +S9), 1.097 x 10E6 (44+0, -S9, 3+41, +S9)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
2 continuous treatments (treatment+recovery time +/-S9: 20+0 -S9; 44+0 -S9): Treatment media remained on cultures receiving the continuous treatment until sampling, that is, 20 or 44 hours after the beginning of treatment.
3 pulse treatments (treatment+recovery time +/-S9: 3+17 +S9; 3+17 - S9; 3+41 +S9): 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.
- Harvest time after the end of treatment (sampling/recovery times):
2 continuous treatments (treatment+recovery time +/-S9: 20+0 -S9; 44+0 -S9): 0 h
3 pulse treatments (treatment+recovery time +/-S9: 3+17 +S9; 3+17 - S9; 3+41 +S9): 17 h , 41 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Colchicine (1 µg/mL) for 2 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cells were resuspended in 4mL 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 for 5 minutes) and resuspension. This procedure was repeated twice (centrifuging at approximately 1250 x 'g', 2-3 minutes) until the cell pellets were clean.
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.
- Number of cells spread analysed per concentration (number of replicate cultures and total number of cells scored): Where possible, one hundred metaphases from each treatment/control were analysed for chromosome aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only cells with 19-23 chromosomes were considered acceptable for analysis of structural aberrations. Classification of structural aberrations was based on the scheme described by ISCN (ISCN (1995) An International System for Human Cytogenetic Nomenclature. Editor Felix Mitelman; S Karger, Switzerland.). 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
- Determination of polyploidy, endoreplication: Any cell with more than 23 chromosomes, that is polyploid, endoreduplicated and hyperdiploid cells, observed during this search was noted and recorded separately.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative population doubling (RPD), mitotic index (MI)
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 concentrations 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.
Statistics:
Descriptive statistics using Fisher's exact test

Richardson C, Williams D A, Allen J A, Amphlett G, Chanter D 4 and Phillips B (1989) Analysis of data from in vitro cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Sub-committee Report, Part III), Ed D J Kirkland, Cambridge University Press, pp 141-154.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH, osmolality: Measurements on post-treatment media from the range-finder in the absence and presence of S-9 indicated that the test article had no marked effect on osmolality (greater than a shift of 50 mOsm/kg) or pH (shift of greater than 1 pH unit) as compared to concurrent vehicle controls
- Precipitation and time of the determination: It was noted that precipitate was visible at the time of harvest at a concentration of 350.0 pg/mL. As such, the test article was present during the recovery stage of treatment.
- Definition of acceptable cells for analysis: 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.

RANGE-FINDING/SCREENING STUDIES (if applicable): It should be noted that two separate range-finder treatments were performed due to limited culture availability. In the first range-finder, cultures were treated at 3+17 hours, - and +S-9 and 20+0 hours -S-9. In the second, cultures wisre treated at 44+0 hours -S-9 and 3+41 hours +S-9.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range. The positive control chemicals NQO and CPA induced statistically significant increases in the number of cells with structural aberrations.

For all test methods and criteria for data analysis and interpretation:
please refer to result tables attched in 'Attached background material'

Chromosome aberration test (CA) in mammalian cells:
- please refer to result tables attched in 'Attached background material'
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : Classification of structural aberrations was based on the scheme described by ISCN (ISCN (1995) An International System for Human Cytogenetic Nomenclature. Editor Felix Mitelman; S Karger, Switzerland.). 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.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : At least 160 cells out of an intended 200 were analysable at each dose level, unless 10 or more cells showed structural aberrations other than gaps, observed during analysis.
please refer to result tables attched in 'Attached background material'
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : A single culture at the highest concentration analysed (3.355 pg/mL) following 44+0 hour -S-9 treatment exhibited an increase in numbers of polyploid cells, but this was not reproduced between replicate cultures.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Negative (solvent/vehicle) historical control data:
-S9: 25 studies, 113 cultures
range 0-8, mean 1.5, SD 1.74 (structural aberrations including gaps); range 0-7, mean 1.01, SD 1.37 (structural aberrations excluding gaps)
+S9: 17 studies, 64 cultures
range 0-6, mean 1.66, SD 1.45 (structural aberrations including gaps); range 0-5, mean 1.25, SD 1.23 (structural aberrations excluding gaps)
Conclusions:
It is concluded that under the experimental conditions employed, the test material induced both structural and numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells following pulse 3+17 hour treatment in the presence of metabolic activation (S9). No such increases in structural or numerical aberrations were observed following 3+41 hour +S9 treatment at an equivalent concentration, nor following treatments in the absence of S9 (3+17, 20+0 and 44+0 hour treatments) at concentrations either up to the limit of cytotoxicity, or, limited by solubility, in the test system.
Executive summary:

The registered substance was tested for cytogenicity according to OECD Guideline 473 following GLP.

Purpose
The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells thus providing information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in human.

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 (S9). The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level used in the main experiments, 1500 µg/mL, was determined following a preliminary cytotoxicity range-finding experiment.

Results
Cytotoxicity based on PD
In Experiment 1, treatment in the absence and presence of S9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S9 mix used was prepared from a rat liver post-mitochondrial fraction from Aroclor 1254 induced animals. 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, 350.0 µg/mL in the absence of S9 and 300.0 µg/mL in the presence of S9, induced approximately 50% and 42% reduction in population doubling, respectively.

In Experiment 2, treatment in the absence of S9 was continuous for either 20 hours (20+0) or 44 hours (44+0). Treatment in the presence of S9 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 three dose levels and the highest concentrations chosen for analysis, 1500 µg/mL (20+0) and 3.355 µg/mL (44+0) in the absence of S9 and 300.0 µg/mL (3+17 and 3+41) in the presence of S9, induced approximately 39%, 47%, 59% and 25% reduction in population doubling, respectively.

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 S9, 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 -S9 and 3+17 +S9 treatments in Experiment 2.

Structural Chromosome aberration analysis
Treatment of cultures with test substance in the absence of S9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls for the majority of concentrations analysed. Numbers of aberrant cells (excluding gaps) for the majority of  treated cultures fell within historical negative control (normal) ranges.

Pulse 3+17 hour treatment of cultures with the test substance in the presence of S9 resulted in frequencies of cells with structural aberrations that were significantly higher (p≤0.05) than those observed in concurrent vehicle controls for the highest two concentrations analysed (250 and 300 µg/mL) in both treatments. These increases were observed to be dose-related. Large increases in cells with structural aberrations, exceeding historical vehicle control values were observed in both replicate cultures at the concentration of 300 µg/mL in both treatments. Single cultures at 200 and also at 250 µg/mL in Experiment I also exhibited aberrant cell frequencies exceeding historical vehicle control values, although such increases were not observed in the replicate cultures at either concentration. All other treated cultures exhibited numbers of aberrant cells that fell within historical vehicle control (normal) values. The high concentration analysed (300 µg/mL) induced 42% and 59% cytotoxicity (based on population doublings [PD]) in Experiments 1 and 2, respectively.

Pulse 3+41 hour treatment of cultures with the test item in the presence of S9 (Experiment 2) resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent vehicle controls at a concentration of 300 µg/mL (a concentration that induced 25% cytotoxicity based on PD). The aberrant cell frequency of both replicate cultures at this concentration fell within normal values.

Numerical Chromosome aberration analysis
Frequencies of cells with numerical aberrations fell within the historical negative control (normal) range for the majority of cultures treated in the absence of S9 (both experiments).

Pulse 3+17 hour treatments in the presence of S9 resulted in large increases in numerical aberrations for the majority of concentrations analysed (both experiments). These increases were attributable to endoreduplicated cells and clearly exceeded historical vehicle control ranges for the majority of treated cultures. Dose related increased toxicity was observed in both treatments over the concentration range analysed (200-300 µg/mL and 50-300 µg/mL, Experiments 1 and 2 respectively).

No such increases in numerical aberrations were observed following 3+41 hour +S9 treatment at a concentration of 300 µg/mL.

Conclusion
It is concluded that under the experimental conditions employed, the test material induced both structural and numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells following pulse 3+17 hour treatment in the presence of metabolic activation (S9). No such increases in structural or numerical aberrations were observed following 3+41 hour +S9 treatment at an equivalent concentration, nor following treatments in the absence of S9 (3+17, 20+0 and 44+0 hour treatments) at concentrations either up to the limit of cytotoxicity, or, limited by solubility, in the test system.

It should be noted that this particular assay is primarily designed to detect structural rather than numerical chromosome aberrations and the biological relevance of endoreduplication in vitro is questionable.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 SEP 1996 - 10 DEC 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) standards. The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
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
Remarks:
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
Remarks:
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Remarks:
mutations on the trpE locus , uvrB
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
- source of S9 : Young male CD rats, ca 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KCl, then homogenised with more of the same medium (approximately 3 ml/g wet liver) in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9000 x g for 10 minutes and supenatants collected and stored at -80°C until required for preparation of the S9 mix. Supernatant is used within 6 months of preparation.
- method of preparation of S9 mix :
0.1 M NADP, sodium salt, in aqueous solution: 0.4 ml
0.1 M glucose-6-phosphate, sodium salt, in aqueous solution: 0.5 ml
0.4 M MgCl2.6H2O/1.65 M KCl aqueous solution: 0.2 ml
Supernatant from liver homogenate: 1.0 ml
0.2 M Sodium phosphate buffer (pH 7.4): 5.0 ml
Purified water to a final volume of 10.0 ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 mix sterility check plates were used to assure that the S9 mix was free of microbial contamination. Appropriate positive control chemicals were used to confirm the enzymatic activity of the S9 mix.
Test concentrations with justification for top dose:
The test was performed in two series with: 5, 15, 50, 150, 500, 1500, and 5000 µg/plate. In the first test no visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test item at concentrations from 5 to 5000 µg/plate. A top exposure concentration of 5 mg/plate was therefore selected for use in the second test.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: 2-aminoanthracene (+S9; TA1535 (2 µg/plate), WP2uvrA (10 µg/plate)), danthron (+S9; TA102 (30 µg/plate))
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (experiment 1); pre-incubation (experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none (experiment 1), 20 min (experiment 2)
- Exposure duration/duration of treatment: 2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After incubation, numbers of revertant colonies were counted with an automated colony counter. Total colonies on nutrient plates were counted in the same way.
Evaluation criteria:
A test material is considered to have mutagenic potential if it induces a dose-related increase in revertants over the concurrent solvent or vehicle controls, and if this increase reaches at least a doubling of the control values. If an increase of 2-fold or greater is recorded only at the highest tested dose (or the highest not showing clear toxicity) and this increase is reproducible, this too would be considered a positive result. Such a positive result may be observed under one or more sets of test conditions (i.e. in one or more tester strains, with or without S9 mix).
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Particles of precipitated test material were observed on plates containing concentrations of 1500 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES (also referred to experiment 1):
Sterility checks, spontaneous reversion rate and viability checks: The absence of colonies on test item and S9 mix sterility check plates indicates that these preparations were free of microbial contamination. The total colony counts confirmed the viability and high cell density of the cultures. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects of DMSO inclusion on these rates.
Mutagenic activity of positive control chemicals: Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.
Effect of the test item: No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at concentrations from 5 to 5000 µg/plate. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test item. A top exposure concentration of 5 mg/plate was therefore selected for use in the second test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
Sterility checks, spontaneous reversion rate and viability checks
The absence of colonies on test item and S9 mix sterility check plates indicates that these preparations were free of microbial contamination.
The total colony counts confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects of DMSO inclusion on these rates.

Mutagenic activity of positive control chemicals
Appropriate positive control chemicals (with S9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

Ames test:
- Signs of toxicity : No signs of toxicity were observed (no thinning of the background lawn)
- Individual plate counts : please refer to attached pdf
- Mean number of revertant colonies per plate and standard deviation : please refer to attached pdf
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.
Executive summary:

The registered substance was tested for gene mutation according to OECD Guideline 471 following GLP.

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 human.

Study Design

The test material was examined for mutagenic activity in five histidine dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA102, TA1535 and TA1537, and one tryptophan dependent auxotroph of Escherichia coli, strain WP2uvrA, using pour-plate tests.

The tests, which were conducted in the presence and absence of an activating system derived from rat liver (S9 mix), employed a range of concentrations up to 5000 µg/plate. All tests included solvent (dimethyl sulphoxide) controls with and without S9 mix.

Results

No increases in reversion to prototrophy were obtained with any of the six bacterial strains at concentrations tested, either in the presence or absence of S9 mix.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine, ENNG, danthron and cumene hydroperoxide when examined under similar conditions.

Conclusion

It was concluded that the test material did not exhibit any mutagenic activity under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Key, micronucleus test, rat, i.p., OECD 474, GLP: negative

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:
16 MAR 2006 - 17 MAY 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in compliance with the OECD Principles of Good Laboratory Practice and was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No.474 and the OECD Principles of Good Laboratory Practice.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 218 - 235 g
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to the cages according to a random list which had been provided by a computer program developed in house. The rats were identified by metal earmarks.
- Housing: The animals were kept individually in Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm) on softwood chippings
- Diet (e.g. ad libitum): ad libitum (Standard diet from Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum (tap water from Makrolon drinking bottles)
- Acclimation period: The animals were received from the breeding center and were adapted to the laboratory conditions for a period of at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 45 - 55
- Photoperiod (hrs dark / hrs light): 12/12
- Atmospheric pressure: 751 - 759 mm Hg
Route of administration:
intraperitoneal
Vehicle:
Miglyol 812 neutral oil (20 mL/kg body weight) Batch: ZDP K34243307
Details on exposure:
As proposed in the EEC Directive and in the OECD Guideline, the animals were treated with the test compounds once. To achieve maximal sensitivity of the test system, the test material was administered intraperitoneally in this investigation. Dosing of the animals of different groups was staggered over one to three day intervals to allow for sufficient sample preparation time.
Duration of treatment / exposure:
single administration
Frequency of treatment:
1
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
316 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5 per dose and 5 per control
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
Cyclophosphamide (CPA) = Endoxan® (Baxter Oncology, Frankfurt, Germany) provided as ampouls containing:
Cyclophosphamide x H2O 106.9 mg
Cyclophosphamide anhydrous form 100.0 mg
NaCl 45.0 mg
Art. No.: E 432-1
Batch: 4GH156D
Stable until: August, 2007

25 mg Endoxan® (= 16.5 mg CPA) were dissolved in 10 mL Aqua pro injectione directly before use.
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments, 5 male rats treated intraperitoneally with 1000 mg/kg body weight of the test item (in 20 mL/kg body weight of Miglyol 812 neutral oil) showed clear toxic effects (abdominal position, ptosis and body weight loss) but no mortality.
For these reasons, the dose of 1000 mg/kg body weight was selected as the high dose for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of √10 = 3.16.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were treated with the test compounds once. To achieve maximal sensitivity of the test system, the test material was administered intraperitoneally in this investigation. Dosing of the animals of different groups was staggered over one to three day intervals to allow for sufficient sample preparation time.

DETAILS OF SLIDE PREPARATION: Immediately after the rats had been killed by CO2, one femur of each animal was dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum (Biochrom, Berlin, Germany) with the aid of a syringe, and suspended in the serum. This suspension was filtered through cellulose and centrifuged for 5 min 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) using Giemsa's solution with Weise buffer solution and mounted in Entellan.

METHOD OF ANALYSIS: A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250 x).
Evaluation criteria:
A positive effect in this test system is defined by the occurrence of mean MN-PCE values of a treatment group which are statistically significantly higher than those of the actual negative control.

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 has been applicated, 3 different test material doses have to be administered in the supplementary experiment. The above mentioned criteria for a negative or positive test result apply for this experimental design likewise.
Statistics:
Descriptive statistics
For all groups, mean values were calculated of the following parameters:
NCE/PCE - number of normochromatic erythrocytes (NCE) /
number of polychromatic erythrocytes (PCE) / animal
MN-NCE - number of micronuclei-containing cells / 1000 NCE /
animal
MN-PCE - number of micronuclei-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), normochromatic erythrocytes (MN-NCE) and the quotient of NCE/PCE per animal were used.

Pairwise comparison
Each 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 program (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.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
some clinical signs at highest dose
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: The high dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments, 5 male rats treated intraperitoneally with 1000 mg/kg body weight of the test item (in 20 mL/kg body weight of Miglyol 812 neutral oil) showed clear toxic effects (abdominal position, ptosis and body weight loss) but no mortality.
For these reasons, the dose of 1000 mg/kg body weight was selected as the high dose for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of √10 = 3.16.
- Clinical signs of toxicity in test animals: at 1000 mg/kg bw clinical symptoms were bevelling (4/10 animals), ptosis (1/10 animals), abdominal position (6/10 animals)
- Rationale for exposure: As proposed in the OECD Guideline, the animals were treated with the test compounds once.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 2000 polychromatic erythrocytes per animal, using 5 males per group, were evaluated. The negative control (solvent) values were all in the expected range predetermined as historical controls of the laboratory. For the positive control, a statistically significant increase in polychromatic erythrocytes with micronuclei was established (p < 0.01). No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed for the test item. For the number of normochromatic cells with micronuclei, calculated per 1000 normochromatic erythrocytes by means of the above mentioned quotient, no increase was observed.
- Ratio of PCE/NCE (for Micronucleus assay): No relevant treatment-related variation was observed.
- Appropriateness of dose levels and route: The highest dose revealed toxicity but no mortality.
- Statistical evaluation: No statistically significant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item treated groups (p> 0.05). For statistical methods, please refer to methods section.

Table 1: Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE in different dose groups


Test material Dose Preparation MN-PCEa
[mg/kg] time [h] males
Solvent - 24
1.60
test material 100 24 1.90ns
test material 316 24 1.10ns
test material 1000 24 2.10ns
test material 1000 48 1.00ns
Cyclophosphamide 16.5 24 21.2**


a:     Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
Statistical significance:         ns: not significant (p > 0.05)    *:  0.01  <  p  ≤  0.05    **:  p  ≤  0.01

Conclusions:
The test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.
Executive summary:

The registered substance was tested for cytogenicity according to OECD Guideline 474 following GLP.

Purpose
The purpose of the in vivo micronucleus test is to identify agents that cause chromosomal damage or damage to the mitotic apparatus thus providing information on possible mutagenic effects of  the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in human.

Study Design
The micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No.474 and the OECD Principles of Good Laboratory Practice.
In the current experiment, the test chemical was given once intraperitoneally to male rats at doses of 100, 316 or 1000 mg/kg body weight. Rats of the negative control group received the solvent 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 groups (1000 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.

Results
Under the conditions of the present study the following results were obtained:

Test material Dose Preparation MN-PCEa
[mg/kg] time [h] males
Solvent - 24
1.60
test material 100 24 1.90ns
test material 316 24 1.10ns
test material 1000 24 2.10ns
test material 1000 48 1.00ns
Cyclophosphamide 16.5 24 21.2**


a:     Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
Statistical significance:         ns: not significant (p > 0.05)    *:  0.01  <  p  ≤  0.05    **:  p  ≤  0.01

The highest test material dose induced some clinical signs of toxicity. A 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 statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

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

in vitro

Gene mutation in bacteria, OECD 471

The registered substance was tested for gene mutation according to OECD Guideline 471 following GLP.

Study Design

The test material was examined for mutagenic activity in five histidine dependent auxotrophs of Salmonella typhimurium, strains TA98, TA100, TA102, TA1535 and TA1537, and one tryptophan dependent auxotroph of Escherichia coli, strain WP2uvrA, using pour-plate tests.

The tests, which were conducted in the presence and absence of an activating system derived from rat liver (S9 mix), employed a range of concentrations up to 5000 µg/plate. All tests included solvent (dimethyl sulphoxide) controls with and without S9 mix.

Results

No increases in reversion to prototrophy were obtained with any of the six bacterial strains at concentrations tested, either in the presence or absence of S9 mix.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine, ENNG, danthron and cumene hydroperoxide when examined under similar conditions.

Conclusion

It was concluded that the test material did not exhibit any mutagenic activity under the conditions of this test.

Chromosomal aberration, OECD 473

The registered substance was tested for in vitro cytogenicity according to OECD Guideline 473 following GLP.


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 (S9). The test article was dissolved in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest dose level used in the main experiments, 1500 µg/mL, was determined following a preliminary cytotoxicity range-finding experiment.

Results
Cytotoxicity based on PD
In Experiment 1, treatment in the absence and presence of S9 was for 3 hours followed by a 17-hour recovery period prior to harvest (3+17). The S9 mix used was prepared from a rat liver post-mitochondrial fraction from Aroclor 1254 induced animals. 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, 350.0 µg/mL in the absence of S9 and 300.0 µg/mL in the presence of S9, induced approximately 50% and 42% reduction in population doubling respectively.

In Experiment 2, treatment in the absence of S9 was continuous for either 20 hours (20+0) or 44 hours (44+0). Treatment in the presence of S9 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 three dose levels and the highest concentrations chosen for analysis, 1500 µg/mL (20+0) and 3.355 µg/mL (44+0) in the absence of S9 and 300.0 pg/mL (3+17 and 3+41) in the presence of S9, induced approximately 39%, 47%, 59% and 25% reduction in population doubling, respectively.

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 S9 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 -S9 and 3+17 +S9 treatments in Experiment 2.

Structural Chromosome aberration analysis
Treatment of cultures with test substance in the absence of S9 in both experiments resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls for the majority of concentrations analysed. Numbers of aberrant cells (excluding gaps) for the majority of  treated cultures fell within historical negative control (normal) ranges.

Pulse 3+17 hour treatment of cultures with the test substance in the presence of S9 resulted in frequencies of cells with structural aberrations that were significantly higher (p≤0.05) than those observed in concurrent vehicle controls for the highest two concentrations analysed (250 and 300 µg/mL) in both treatments. These increases were observed to be dose-related. Large increases in cells with structural aberrations, exceeding historical vehicle control values were observed in both replicate cultures at the high concentration of 300 µg/mL in both treatments. Single cultures at 200 and also at 250 µg/mL in Experiment I also exhibited aberrant cell frequencies exceeding historical vehicle control values, although such increases were not observed in the replicate cultures at either concentration. All other treated cultures exhibited numbers of aberrant cells that fell within historical vehicle control (normal) values. The high concentration analysed (300 µg/mL) induced 42% and 59% cytotoxicity (based on population doublings [PD]) in Experiments 1 and 2, respectively.

Pulse 3+41 hour treatment of cultures with the test item in the presence of S9 (Experiment 2) resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent vehicle controls at a concentration of 300 µg/mL (a concentration that induced 25% cytotoxicity based on PD). The aberrant cell frequency of both replicate cultures at this concentration fell within normal values.

Numerical Chromosome aberration analysis
Frequencies of cells with numerical aberrations fell within the historical negative control (normal) range for the majority of cultures treated in the absence of S9 (both experiments).

Pulse 3+17 hour treatments in the presence of S9 resulted in large increases in numerical aberrations for the majority of concentrations analysed (both experiments). These increases were attributable to endoreduplicated cells and clearly exceeded historical vehicle control ranges for the majority of treated cultures. Dose related increased toxicity was observed in both treatments over the concentration range analysed (200-300 µg/mL and 50-300 µg/mL, Experiments 1 and 2 respectively).

No such increases in numerical aberrations were observed following 3+41 hour +S9 treatment at a concentration of 300 µg/mL.

Conclusion
It is concluded that under the experimental conditions employed, the test material induced both structural and numerical chromosome aberrations in cultured Chinese hamster ovary (CHO) cells following pulse 3+17 hour treatment in the presence of metabolic activation (S9). No such increases in structural or numerical aberrations were observed following 3+41 hour +S9 treatment at an equivalent concentration, nor following treatments in the absence of S9 (3+17, 20+0 and 44+0 hour treatments) at concentrations either up to the limit of cytotoxicity, or, limited by solubility, in the test system.

It should be noted that this particular assay is primarily designed to detect structural rather than numerical chromosome aberrations and the biological relevance of endoreduplication in vitro is questionable.

in vivo

Mammalian Erythrocyte Micronucleus Test, OECD 474

The registered substance was tested for in vivo cytogenicity according to OECD Guideline 474 following GLP.

Purpose
The purpose of the in vivo micronucleus test is to identify agents that cause chromosomal damage or damage to the mitotic apparatus thus providing information on possible mutagenic effects of  the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in human.

Study Design
The micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No.474 and the OECD Principles of Good Laboratory Practice.
In the current experiment, the test chemical was given once intraperitoneally to male rats at doses of 100, 316 or 1000 mg/kg body weight. Rats of the negative control group received the solvent 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 groups (1000 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.

Results
Under the conditions of the present study the following results were obtained:

Test material Dose Preparation MN-PCEa
[mg/kg] time [h] males
Solvent - 24
1.60
test material 100 24 1.90ns
test material 316 24 1.10ns
test material 1000 24 2.10ns
test material 1000 48 1.00ns
Cyclophosphamide 16.5 24 21.2**


a:     Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
Statistical significance:         ns: not significant (p > 0.05)    *:  0.01  <  p  ≤  0.05    **:  p  ≤  0.01

The highest test material dose induced some clinical signs of toxicity. A 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 statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

Conclusion
The test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.

Overall conclusion

The test material was not mutagenic in assays in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and in E. coli wp2 in the presence or absence of exogenous metabolic activation system.

The test maetrial exposed Chinese hamster ovary cells show significant reproducible increase in chromosomal aberrations at cytotoxic concentrations in the presence but not the absence of S9 activation.

These in vitro findings have triggered further in vivo investigations, showing that the test material was not mutagenic in the micronucleus test in rats after intraperitoneal administration under conditions where the positive control exerted potent mutagenic effect.

 

Justification for classification or non-classification

The key studies indicate no genotoxic potential present in vivo, thus no classification for mutagenicity is triggered in accordance with Regulation (EC) No 1272/2008.