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Genetic toxicity in vitro

Description of key information

key; M-554147-01-1; Ames (OECD 471); GLP; S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 102; 33 – 5000 µg/plate; negative

key; M-758173-01-1; MNT in vitro (OECD 487); GLP; Human Lymphocytes; 500 – 2000 µg/mL (± S9 for 3+21 h) negative; 200 – 1300 µg/mL (- S9 for 24+24 h) positive

key; M-027630-01-1; HGPRT in vitro (no OECD, but similar to 476); GLP; Chinese Hamster Ovary cells; 40 – 1222 µg/mL, negative

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:
19 Jan - 05 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 2020
Deviations:
yes
Remarks:
solubility and stability of test substance in vehicle not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received triple treatments of 80 mg/kg bw phenobarbital/ß-naphthoflavone orally on consecutive days.

The protein concentration of the S9 preparation was 30.8 mg/mL in the pre-experiment/experiment I and 35.0 mg/mL in experiment II.

100 mL cofactor solution are composed as follows: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM sodium phosphate buffer (pH 7.4). S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix.

Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Experiment I (pre-experiment): 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate (with and without metabolic activation)

Since no toxic effects were observed in experiment I, 5000 µg/plate was chosen as maximal concentration.

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle/solvent used: Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

- Justification for percentage of solvent in the final culture medium: not reported
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine 4-NOPD: TA 1537 50 µg/plate and TA 98 10 µg/plate in DMSO; -S9 2-aminoanthracene 2-AA: TA 1535, TA 1537, TA 98, TA 100 2.5 µg/plate and TA 102 10 µg/plate in DMSO; + S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for pre-incubation test (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 h
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and clearing or diminution of the background lawn
Rationale for test conditions:
according to OECD guideline
Evaluation criteria:
Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
A dose dependent increase is considered biologically relevant if the threshold is exceeded at
more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Acceptability:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the
following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the
historical data
- the positive control substances should produce an increase above the threshold of twice
(strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony
count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels
showing no signs of toxic effects, evident as a reduction in the number of revertants below
the indication factor of 0.5.
Statistics:
Mean values and standard deviation were calculated. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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: No precipitation of the test item occurred up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The preexperiment is reported as experiment I.

STUDY RESULTS
No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed in both experiments neither with nor without S9 mix. No substantial increase in revertant colony numbers of any of the five strains investigated was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. For results of both experiment I and experiment II, please refer to the attached background material under "Overall remarks, attachments"

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent, untreated and positive controls were within the range of the laboratory's historical control data. For details, please refer to the attached background material under "Overall remarks, attachments".
Conclusions:
The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 Jun - 01 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 2016
Deviations:
yes
Remarks:
percentage of solvent in the final culture medium not provided, the used concentration of the positive control (VIN) differs from the concentration used for historical positive control data
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and source of cells: Human blood was collected from two healthy, non-smoking male donors.
- Whether whole blood or separated lymphocytes were used: Whole blood was used.
- Whether blood from different donors were pooled or not: Cells from the different donors were pooled.
- Mitogen used for lymphocytes: 2 % Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media: HEPES-buffered RPMI medium containing 10 % (v/v) heat inactivated foetal calf serum and 0.52 % penicillin / streptomycin
- CO2 concentration, humidity level, temperature: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats (175-199 g) which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254. The S9 fraction was obtained commercially.

The protein concentration of the S9 preparation was 4 mg/mL.
The S9 fraction was tested for its ability to activate benzo(a)pyrene (BP) and 2-aminoanthracene (2-AA) by the provider.

1 mL S9 supplemented cofactor solution was composed as follows: 100 µmol sodium phosphate buffer (pH 7.4), 8 µmol MgCI2, 33 µmol KCI, 5 µmol Glucose-6-phosphate (disodium salt), 4 µmol NADP (disodium salt) and 100 µL S9 fraction (final concentration 10 %).
Test concentrations with justification for top dose:
A maximum concentration of 2000 µg/mL was selected for the cytotoxicity range-finder experiment in order that treatments were performed up to the maximum recommended concentration according to current regulatory test guidelines.

Range-Finding study:
3+21 h, +/- S9, 7.256 - 2000 µg/mL
24+24 h, - S9, 7.256 - 2000 µg/mL

Experiment 1:
3+21 h, - S9, 62.5, 125.0, 250.0, 500.0*, 1000* and 2000* µg/mL
24+24 h, - S9, 200.0*, 400.0, 800.0*, 1000, 1200, 1300*, 1400, 1500, 1600, 1800, 1900 and 2000 µg/mL

Experiment 2:
3+21 h, + S9, 62.5, 125.0, 250.0, 500.0*, 1000* and 2000* µg/mL

* = Concentrations selected for analysis.
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 h with and without S9 mix and additionally 24 h without S9 mix.
- Harvest time: 21-24 h after the end of treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B (Cyto-B) was added to a final concentration of 6 µg/mL to the culture medium 20-22 h prior to harvest.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
At the defined sampling time, cultures were centrifuged, the supernatant removed and the cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 37±1°C for 4 minutes. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation and resuspension, which was repeated as necessary. For slide preparation, cells were again centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. Slides were air-dried and stored. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2000 per concentration
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Replication index (RI)
Rationale for test conditions:
The tests were performed according to current regulatory test guidelines.
Evaluation criteria:
The test article was considered to induce clastogenic and/or aneugenic events by the following observations:
1. statistically significant increase in the frequency of binucleate cells with micronuclei (MNBN cells) at one or more concentrations
2. incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates
3. concentration-related increase in the proportion of MNBN cells (positive trend test)

The test article was considered:
- positive if all of the above criteria were met
- negative if none of the above criteria were met
Results which only partially satisfied the above criteria were dealt with on a case-bycase basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account.

Acceptance criteria:
The assay was considered valid if the following criteria were met:
1. binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures
2. frequency of MNBN cells in vehicle controls fell within the 95th percentile of the current observed historical vehicle control (normal) ranges
3. positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei that clearly exceeded the normal range
4. minimum of 50 % of cells had gone through at least one cell in vehicle control cultures at the time of harvest
5. maximum concentration analysed under each treatment condition met the criteria specified
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable
heterogeneity between replicates by means of a binomial dispersion test. The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test. A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
exposure conditions 24 + 24 h
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 800 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
exposure conditions 3 + 21 h
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
exposure conditions 3 + 21 h
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2000 µg/mL
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 and osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested (2000 µg/mL) as compared to the concurrent vehicle controls.
- Precipitation and time of the determination: The solubility limit of the test item in culture medium was in the range of 1150 to 2301 µg/mL, as indicated by precipitation at the higher concentration which persisted following 24 hours of incubation at approximately 37°C.

RANGE-FINDING/SCREENING STUDIES:
The results of the cytotoxicity range-finder experiment were used to select suitable concentrations for the main micronucleus experiment.

CYTOTOXICITY:
Cytotoxicity was determined based on the relative RI. After exposure for 3 + 21 h, the test item caused a reduction in RI of more than 20 % at 2000 µg/mL both in the absence and presence of a metabolic activation system. After 24 + 24 h exposure, the test item led to a reduced RI of more than 20 % at a concentration of 800 µg/mL and above in the absence of metabolic activation.

STUDY RESULTS
Treatment of cells with the test item for 3 + 21 h in the absence and presence of S9 resulted in frequencies of MNBN cells which were similar to and not significantly higher (at the p≤0.05 level) than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all cultures treated with the test item fell within the normal ranges and there was no statistically significant linear trend.
These data indicate that the test item did not induce micronuclei after 3 + 24 h exposure, neither in the presence nor absence of metabolic activation.

Following 24 + 24 h treatment with the test item in the absence of S9, frequencies of MNBN cells were significantly higher (p≤0.001) than those observed in concurrent vehicle controls at the highest two concentrations analysed (800 and 1300 µg/mL, giving 30 % and 50 % reduction in RI, respectively). The MNBN cell frequency of treated cultures at these two concentrations exceeded the normal range and a statistically significant linear trend (p≤0.001) was observed.
These data are therefore indicative of a positive response.
For details on the results, please refer to attached background material under "Overall remarks, attachments".

- Concurrent vehicle negative and positive control data:
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition, which did not lead to statistically significant increase of micronuclei. All positive control compounds (CPA, MMC for 24 h and VIN for 48 h) induced statistically significant increases in the proportion of cells with micronuclei.
The study is therefore accepted as valid.

HISTORICAL CONTROL DATA
The proportion of micronucleated binucleate (MNBN) cells in the vehicle control cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. The number of MNBN cells in the positive controls MMC and VIN fell within the historical control data. For the positive control CPA, only historical data for a concentration of 3 µg/mL were currently available. However, during the test, 7 µg/mL CPA were selected for analysis. Therefore, the number of MNBN cells were above the historical positive control data for 3 µg/mL CPA. For details on the historical control data, please refer to attached background material under "Overall remarks, attachments".
Conclusions:
The study was performed according to OECD guideline 487 and compliant with GLP. The test item was found not to induce micronuclei in cultured human peripheral blood lymphocytes following 3 + 21 h treatment neither in the presence nor absence of a metabolic activation system (S9), when tested up to the maximum recommended concentration of 2000 µg/mL.
In contrast, the test item induced micronuclei in the same test system following 24 + 24 h treatment in the absence of a metabolic activation system (S9). The concentrations of the test item inducing micronuclei where accompanied by reduction of replication index.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Apr - 04 Jul 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
adopted 2016
Deviations:
yes
Remarks:
test conduct based on the method described by Hsie et al. (Mutation Res. 86: 193-214, 1981), O'Neill et al. (Mutation Res. 45: 91-101, 1977a) with deviations as compared to the OECD TG; for details refer to "Principles of method if other than guideline"
Principles of method if other than guideline:
replicate culture not pooled for the data analysis, the highest concentration caused less than 10 % of relative survival, less than 2 million cells were cultured during the expression period and plated for mutant selection, expression period was only 6 days, justification for choice of solvent not provided
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-Kl-BH subclone, Oak Ridge National Laboratory, Tennessee, USA

For cell lines:
- Absence of Mycoplasma contamination: yes
- Cell cycle length, doubling time or proliferation index: 12-14 h
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
Medium: hypoxanthine-free Ham's F12 medium or complete F12 nutrient mixture supplemented with L-glutamine (1 mM), penicillin (50 units/mL), streptomycin (50 µg/mL) and heat-inactivated fetal calf serum (final concentration: 10 %)
Culture conditions: 5 % CO2 at 37 °C in a humidified atmosphere
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254. The S9 fraction was obtained commercially.

The protein concentration of the S9 preparation was 41.3 mg/mL.

The cofactor solution was composed as follows: 8 mM MgCI2, 33 mM KCI, 5 mM glucose-6-phosphate, 1 mM NADP in sodium phosphate buffer (pH 7.4). S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of 40 % in the S9 mix.
Test concentrations with justification for top dose:
Concentrations for mutagenicity study were chosen based on a cytotoxicity test.

Cytotoxicity test:
Without S9 mix: 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100, 200, 400 µg/mL
With S9 mix: 6.25, 12.5, 25.0, 50.0, 100, 150, 200, 400, 800 µg/mL

Main test:
Experiment 1
Without S9 mix: 40, 50, 60, 70, 80, 90 µg/mL

Experiment 2
Without S9 mix: 60, 70, 80, 90, 100, 125 µg/mL
With S9 mix: 102, 204, 407, 815, 1222 µg/mL

Experiment 3
Without S9 mix: 60, 70, 80, 90, 100, 125 µg/mL
With S9 mix: 100, 199, 399, 798, 1196 µg/mL
Vehicle / solvent:
Dimethyl sulfoxide (DMSO), 1 % in the final culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylbenzanthracene (DMBA) +S9 mix, 20 µg/mL
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4x10^6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment (sampling/recovery times): approximately 13 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 6 days
- Fixation time (start of exposure up to fixation or harvest of cells): approximately 13 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 6-TG, final concentration 10 µg/mL, 7 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment, relative population growth and cloning efficiency (CE)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Mutation frequency = (number of mutation colonies/number of cells seeded corrected by absolute CE)
Rationale for test conditions:
Test was performed similar to OECD guideline.
Evaluation criteria:
An assay is considered
- positive if a dose-dependent and reproducible increase in mutant frequency is observed. It is desirable to obtain this dose-relation for at least 3 doses. The mutagenic response should be at least twice that of the negative controls. If a reproducible increase greater than two times the minimum criterion is observed for a single dose near the highest testable concentration, the test article is also considered mutagenic.
- suspicious if there is no dose-relation but if one or more doses induce a mutant frequency which is considered significant and/or is at least twice that of the negative control
- negative if none of the doses tested (for a range of applied concentrations which extends to toxicity causing about 30 % survival or less) induces a reproducible mutant frequency which is considered significant

Acceptance criteria
An assay normally is considered acceptable for evaluation of the results only if the following criteria are satisfied:
- activation and non-activation assays were repeated independently in a second assay
- average cloning efficiency of the negative controls should be at least 50 %
- background mutant frequency (average of the negative controls) should not exceed 25x10 cells. Assays with higher spontaneous mutant frequencies, however, are not necessarily invalid if all other criteria are fulfilled.
- experimental mutant frequency is considered acceptable only if the absolute cloning efficiency is 10 % or greater
- mutant frequencies for at least five treated cultures are normally determined in each assay
- mutant frequencies are normally derived from sets of 8-10 dishes for each dose level. To allow for contamination losses, an acceptable mutant frequency can be calculated from a minimum of 5 dishes.
- positive control must induce a mutant frequency of at least three times that of the negative control
Statistics:
The POISSON heterogeneity test is used to determine whether or not there are statistically significant increases in mutant frequency.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A clear solution was obtained up to a concentration of 200 mg/mL.

CYTOTOXICITY:
Cytotoxicity test: With and without metabolic activation conditions, the test item induced concentration-related cytotoxicity as seen by decreases in relative population growth and cloning efficiency.
Main tests: In the absence of S9 dose-related decreases were observed in relative survival to treatment. In the presence of S9 dose-related decreases in both relative survival to treatment and relative population growth were noted.

STUDY RESULTS (MAIN TESTS)
Experiment 1:
In this trial the spontaneous mutant frequency of the vehicle controls was significantly elevated over the range of variation for vehicle controls among different trials. Therefore, the results of this trial were not used for evaluation of the genotoxicity of the test item.

Experiment 2 and 3:
Without metabolic activation, 80 µg/mL test item induced a statistically significant increase in mutant frequency over the concurrent vehicle controls. This was only observed in experiment 3 and in one culture, which was neither confirmed by the duplicate treatment or the other trial nor was this increase dose-related.
Therefore, the test article was evaluated as non-mutagenic in the absence of metabolic activation.

With metabolic activation 2 cultures in experiment 2 showed a statistically significant increase in mutant frequency over the concurrent vehicle controls at test article concentrations of 407 µg/mL and 815 µg/mL. However, this increase was not dose-related and it was not confirmed by the duplicate treatment or in the second trial. Moreover, it is in a range which is typical of vehicle control variation and the statistsical significance seemed to be the result of the extremely low spontaneous mutant frequency of the vehicle controls. Therefore, the test material was evaluated as non-mutagenic in the presence of metabolic activation in this test system.
For details on the results, please refer to attached background material under "Overall remarks, attachments".

- Concurrent vehicle negative and positive control data:
Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition, which did not lead to statistically significant increase of mutant frequencies. The positive controls EMS and DMBA revealed a clear mutagenic effect in all experiments.

HISTORICAL CONTROL DATA
- In experiment 2 and 3, the vehicle and positive control mutant frequencies were all in the normal range of the historical control data.
Conclusions:
The study was performed according to OECD guideline 476 and compliant with GLP. The test item is considered to be non-mutagenic in the HPRT test using Chinese hamster Ovary cells with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

key; M-758872-01-1; MNT in vivo (OECD 474); GLP; Rat peripheral blood; 50 – 200 mg/kg b., negative

key; M-27591-01-1; MNT in vivo (OECD 474); GLP; Mouse bone marrow; 80 mg/kg bw, negative

key; M-026551-01-1; Germ-cell cytogenetic in vivo (OECD 483); GLP; Mouse spermatogonia; 80 mg/kg bw, negative

key; M-025903-01-1; CA in vivo (no OECD, but similar to 475); GLP; Chinese hamster bone marrow; 2000 mg/kg bw, negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Nov 2018 - 15 Jan 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Kent, England
- Age at study initiation: approximately 5 to 6 weeks old
- Weight at study initiation: 128-174 g (males), 116-150 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were housed in groups of up to 3, by sex, in solid floor cages with appropriate bedding.
- Diet: pelleted rodent diet (Teklad 2014C Rodent Maintenance Diet, Envigo RMS (UK) Limited) ad libitum
- Water: mains tap water (in bottles) ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): room was air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Nov 2018 To: 11 Jan 2019
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: methylcellulose with 0.1 % v/v Tween 80
- Justification for choice of solvent/vehicle: Based on a formulation trail, where the suitability of the vehicle was confirmed.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
The test item was formulated on the day of use as a suspension in the vehicle methylcellulose (medium viscosity) supplemented with 0.1 % v/v Tween 80. Separate formulations were prepared for each dose level, with the weighed quantity of test item being suspended in the appropriate quantity of vehicle.
Duration of treatment / exposure:
24 h
Frequency of treatment:
Rats were dosed twice, approximately 24 hours apart.
Post exposure period:
Animals were observed periodically for 48 hours after the last dosing.
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
No. of animals per sex per dose:
Range-finder: 3/sex/dose
Main test: 6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
6 Samples from an existing positive control bank of blood obtained from rats dosed with CPA were considered.
- Doses / concentrations: 15 mg/kg bw
Tissues and cell types examined:
Blood
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding study was carried out in order to select the highest dose of the test item that did not produce mortality or severe signs of clinical toxicity up to the maximum tolerated dose level (MTD). Groups of 3 male or 3 female rats were dosed with the test substance in a range of 200-500 mg/kg bw/day. The results of the range-finder study revealed no gender-related difference in toxicity and therefore, the main study was conducted with male rats only.

TREATMENT AND SAMPLING TIMES:
Groups of 6 male rats were dosed twice, approximately 24 h apart, with the vehicle alone (negative control) or the test item (50, 100 or 200 mg/kg bw/day). At the end of the observation period (approximately 48 h after the second test item administration), animals had a terminal blood sample taken for micronucleus scoring. At least 0.2 mL whole blood was taken from each main study animal into tubes containing K2EDTA. Samples were gently flicked to mix the blood and anticoagulant and then mixed, by inverting several times, before being placed on a roller and then held on water ice until storedat between 2 °C and 8 °C prior to fixing.

DETAILS OF SLIDE PREPARATION:
Blood samples were diluted, fixed, stored, labelled and analysed according to the biomarker method BMK034FC. In brief, blood samples were fixed in two separate methanol aliquots and stored at ≤ -70 ˚C for at least 3 days. One set of samples was then washed out of fixative and analysed. The remaining set of samples was transferred to long term storage solution for continued storage at ≤ -70 ˚C. The six samples from the positive control bank were washed out of fixative and analysed according to the biomarker method BMK034FC.

METHOD OF ANALYSIS:
The analysis of micronuclei (MN) was performed by the biomarker method BMK034FC. It is a flow cytometric method designed by Litron Laboratories to detect micronuclei based on expression of CD71 (identifies reticulocytes, RET), CD61 (identifies platelets) and propidium iodide (PI, DNA stain). The machine (FACSVerse flow cytometer) was calibrated for the detection of MN at the beginning of each run using the kit supplied Malaria Biostandard. A minimum of 4000 and a maximum of approximately 20000 RET were scored for the presence of MN for each animal.
Evaluation criteria:
For the test to have been considered positive if the following criteria needed to be met:
1. A statistically significant increase in the frequency of MN-RET occurred at 1 or more
dose levels.
2. The incidence and distribution of MN-RET in individual animals at the dose level(s)
showing statistical significance exceeded the laboratory’s historical negative Control
data (e.g. exceeded control limits).
3. A dose-related increase in the frequency of MN-RET (where more than 2 dose levels
are analysed) was observed.
Results which only partially satisfy the above criteria were dealt with on a case-by-case basis.
Evidence of a dose-related effect was considered useful but not essential in the evaluation of a
positive result. Biological relevance was also taken into account.

For a test to be considered negative if the following criteria needed to be met:
1. None of the dose levels exhibited a statistically significant increase in the frequency
of MN RET, compared with the concurrent negative Control.
2. There was no dose-related increase when evaluated by an appropriate trend test.
3. All results were within Sequani’s historical negative Control data (e.g. within Control
limits).
4. Bone marrow exposure to the test item has occurred.
Statistics:
For the % RET data, the treated group(s) and the positive control were compared with the negative control using one-tailed exact Wilcoxon Rank Sum tests. A Jonckheere trend test was conducted for dose-response relationship investigation.

Micronucleated reticulocytes (MN-RET) data was analysed using a Poisson model with a log link function using GENMOD procedure in SAS. The effect of the test item was evaluated using two statistical approaches:
1. A linear trend test, built as a linear combination of the logs of the proportions of micronuclei, for dose-response relationship investigation.
2. Pairwise comparisons to the negative control, using the one-sided Likelihood Ratio test, to detect the doses significantly different from the control.

All the tests were one-sided, a decreasing trend being not of interest. So, in case of decrease and calculated p value < 0.5, the final p value was estimated as 1-[p value].
Overdispersion of the data was estimated by the deviance of the model divided by the associated degrees of freedom. Where overdispersion was detected (dispersion parameter >1), a correction was applied on the model.
The positive control group was not included in the model with the treated groups, but was calculated separately in a model that contained only the two control groups.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at and above 100 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 200-500 mg/kg bw/day
- Clinical signs of toxicity in test animals: Minor body weight loss was observed in all animals. Clinical signs were observed at 320 and 500 mg/kg bw/day. For details, please refer to the attached background material 1. There were no clinical signs observed in males or females following administration at 200 mg/kg bw/day. Based on the results, the MTD was considered to be 200 mg/kg bw/day in males and females.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: clinical signs were observed at 100 and 200 mg/kg bw/day. For details, please refer to the attached background material 2.
- Induction of micronuclei: no statistically significant increases in MN-RET frequency was noticed at any dose level (please also refer to the attached background material 3).
- % RET: statistically significant decrease in the % RET at 100 and 200 mg/kg bw/day, indicating toxicity to the bone marrow and proving exposure of the bone marrow to the test substance.
- Positive control: the samples from the positive control bank had statistically significant increases in the number of MN-RET compared with the concurrent control group which demonstrated that the test system was capable of detecting a known clastogen. There was a statistically significant decrease in the % RET in the positive control group, indicating toxicity to the bone marrow.
- Historical control data: the negative and positive control are within the historical controls from 2015 - 2018 (refer to the attached background material 4).
Conclusions:
The study was performed according to OECD guideline 474 and compliant with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the MTD of 200 mg/kg bw/day in male rats.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Sep - 30 Nov 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 2016
Deviations:
yes
Remarks:
1000 instead of 4000 immature erythrocytes scored, only one dose tested, relative humidity and age of mice slightly different to the guideline, historical control data lacking standard deviations and 95 % control distribution limit
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: approximately 8-12 weeks
- Weight at study initiation: 28-41 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Mice were kept in groups of a maximum of three to five mice, split by sex and test group, in Makrolon cages type I and type II respectively, using a bedding of soft wood granules type S 8/15 (Fa. Ssniff, Spezialdiäten GmbH, Soest, Germany).
- Diet: fixed-formula food (Altromin 1324 Standard Diet; Altromin GmbH, Lage, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 35-43
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: 0.5 % Cremophor emulsion
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
The test substance was suspended in the vehicle, treated for ten minutes with supersonics, and stirred with a magnetic mixer until administration. For negative control, the vehicle was likewise stirred with a magnetic mixer until administration.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24, 48 and 72 h after administration of test substance
24 h after administration of positive/negative controls
Dose / conc.:
80 mg/kg bw/day
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 20 mg/kg bw in deionized water
Tissues and cell types examined:
Tissue: bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose selection was based on a pilot study, in which groups of five animals, including both males
and females, were orally administered 50 mg/kg bw, 100 mg/kg bw (two groups), and 150 mg/kg bw of the test item respectively.

TREATMENT AND SAMPLING TIMES:
The test substance was administered once at a dose of 80 mg/kg bw 24, 48 and 72 h after the administration, the animals were sacrificed by decapitation, and the femoral marrow was prepared. Sacrifice of negative and positive controls was performed, only after 24 h.

DETAILS OF SLIDE PREPARATION:
Schmid's method was used to produce the smears. In brief, the femur was separated and the proximal end was opened. Fetal calf serum was pushed into the open end of the marrow cavity. The contents were then flushed several times, and the bone marrow passed into the serum as a fine suspension. This was repeated from the other end of the femur. The tube containing the serum and bone marrow was centrifuged (1000 rpm, 5 min) and the supernatant was removed except for a small resediue. The sediment was mixed until the suspension was homogenous. One drop of the suspension was placed on a slide, which were dried overnight.
The smears were stained automatically with an Ames Hema-Tek Slide Stainer. Slides were then destained with methanol and rised with deionized water and dried. For covering, the slides were transferred to xylene for 10 min. Subsequently, slides were covered.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. The micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. The ratio of polychromatic to normochromatic erythrocytes was established. Therefore, the number of normochromatic erythrocytes per 1000 polychromatic was noted. In addition, the number of normochromatic erythrocytes showing micronuclei was also established.
Evaluation criteria:
Acceptance critria
An assay was considered acceptable if the figures of the negative and positive control were within the expected range in accordance with the laboratory's experience.

Assessment criteria
A test was considered positive if there was a relevant and significant increase in the numbers of polychromatic erythrocytes with micronuclei at any of the intervals in comparison to the negative control.

A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time point.

In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of historical negative controls provided the increase was not significant.
A test was also considered equivocal if there was a significant increase in that rate which was in the range of negative controls according to the laboratory’s experience. In either case, a second test will performed at the most sensitive interval.
Statistics:
The test group with the highest mean, if this superceded the negative control mean, and the positive control were checked by Wilcoxon's non-parametric rank sum test in respect to the count of polychromatic erythrocytes with micronuclei and the rate of normochromatic erythrocytes.
The rate of normochromatic erythrocytes with micronuclei is examined if the micronucleus rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean is compared with the negative control using the one-sided chi2 -test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reversible symptoms occurred, including apathy, reduced motility and difficulty in breathing
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 50-150 mg/kg bw
- Clinical signs of toxicity in test animals: clinical signs of toxicity were observed in all dose groups. These symptoms were apathy, reduced motility, shivering and difficulty in breathing. Two of ten mice died in the 100 mg/kg bw group and five out of five mice died in the 150 mg/kg bw group. Based on these results, 80 mg/kg bw was chosen for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: the treated animals showed compound-related symptoms for up to six hours, which consisted of apathy, reduced motility and difficulty in breathing. Thereafter, their external appearance and physical activity remained unaffected. Their eating behavior was normal. There were no substance-induced mortalities.
- Induction of micronuclei: the ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment. There were no biologically important or statistically significant
variations in regard to the incidence of micronucleated polychromatic erythrocytes between the negative control and the treated groups. For details, please refer to the attached background material.
- Positive control: The effect of the positive control cyclophosphamide consisted of a clear increase in polychromatic erythrocytes with micronuclei.
- Historical control data: historical data for the positive and negative controls were provided without information on the range or standard deviations. However, the negative controls revealed similar results when compared to the historical control data, whereas the positive control led to partially less polychromatic erythrocytes with micronuclei compared to the historical control. Please also refer to the attached background material.
Conclusions:
The present study was conducted according to the OECD TG 474 under GLP conditions. Based on the in vivo micronucleus test, there were no indications of a clastogenic effect after single oral treatment of male and female mice by gavage with the test substance at a dose level of 80 mg/kg bw.
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Sep 1989 - 27 Feb 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
Version / remarks:
adopted 2016
Deviations:
yes
Remarks:
relative humidity slightly different from guideline, only one dose investigated, only 100 metaphases scored for each animal, no historical control data provided, evaluation criteria different from guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
Version / remarks:
adopted 1986
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian spermatogonial chromosomal aberration test
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: Approximately 5 hours before treatment
- Housing: Individually in Makrolon Type I, with wire mesh top including granulated soft wood bedding
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, Germany)
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Ropdorf, Germany) ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Apr 1989 To: 27 Feb 1990
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: 0.5 % Cremophor
- Justification for choice of solvent/vehicle: Vehicle was chosen due to its relative nontoxicity for the animals.
- Concentration of test material in vehicle: not specified
- Amount of vehicle: 10 mg/kg bw
Details on exposure:
The test item was applied, suspended in the vehicle.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
Single oral treatment
Post exposure period:
6, 24, 48 h
Dose / conc.:
80 mg/kg bw/day
No. of animals per sex per dose:
6 males were treated, but only 5 males were evaluated
Control animals:
yes, concurrent vehicle
Positive control(s):
ADRIBLASTIN - Doxorubicin-sulfat hydrochloride
- Route of administration: intraperitoneally, single
- Doses / concentrations: 10 mL/kg bw
Tissues and cell types examined:
Spermatogonia
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose selection was based on a preliminary study which was performed with the same strain and under identical conditions as the main study. In this preliminary study the toxicity of the test item in male and female mice was investigated by oral treatment of up to 150 mg/kg bw. In fact, 3 tests were done. In the first one, two animals per dose level received a single oral dose of 60, 80 or 100 mg/kg bw. In the second test, two animals per dose level received a single oral dose of 125 or 150 mg/kg bw of the test item. In the third test, 10 animals per dose level received a single oral dose of 100 mg/kg bw. The maximum tolerated dose for the main study was set, based on the results from these tests.

DETAILS OF SLIDE PREPARATION:
About 4 hours prior to sacrifice, animals were injected intraperitoneally with the spindle inhibitor colcemid (4.0 mg/kg bw) to arrest cells in metaphase.The animals were sacrificed by cervical dislocation. Both testes were collected, the tunicas were removed, the tubules were separated mechanically in physiological saline and dissociated enzymatically with collagenase (0.1 % w/v) resulting in a single cell suspension. After 30 minutes incubation at 37° C the enzyme solution was removed by a brief centrifugation and the cells were resuspended in hypotonic trisodium citrat solution (1 % w/v, prewarmed to 37 °C). The cell suspension was then incubated for 20 min. at 37 °C. The cells were sedimented by a brief centrifugation, the hypotonic supernatant was discarded and the cell pellet was fixed with 3:1 absolute methanol:glacial acetic acid fixative for 30 min. Then the cell pellet was gently resuspended. Prior to preparing the slides the fixative was changed and enough fixative was added to get a relatively thin cell suspension. The fixative-cell suspension was spread by flame-drying and stained with Giemsa solution. Cover slips were mounted. At least one slide was prepared from each animal.

METHOD OF ANALYSIS:
At least 100 well spread metaphases per animal were scored for cytogenetic damage on coded slides. Only metaphases with the characteristic chromosome number of 40 were included in the analysis. Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Five animals per test group were evaluated as described. The remaining animal of each test group was evaluated in case animals died within the test group.

OTHER:
To describe a cytotoxic effect the mitotic index (% cells in mitosis; 500 cells scored) was determined.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically or biologically significant increase in the number of structural chromosomal aberrations.

A test article producing no increase in the number of structural chromosomal aberrations is considered as non-mutagenic in this system.

Both biological and statistical significance should be considered together.
Statistics:
Statistical significance was evaluated by means of the nonparametric Mann-Whitney test.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduction of spontaneous activity, eyelid closure, apathy and convulsion as reported from the preliminary study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY STUDY:
The preliminary study comprised 3 tests, with the compound being applied via the oral route at doses up to 150 mg/kg bw.
Clinical signs of toxicity were noticed at all dose levels up to and including 150 mg/kg bw; these almost consisted of reduced spontaneous activity, abdominal position, eyelid closure and apathy.
With respect to mortality, none of the animals of the first test died; in the second test, one of the two animals treated with 125 mg/kg bw died whereas the two animals treated with 150 mg/kg bw survived. In the third test two of the 10 animals treated with 100 mg/kg bw of the test item died.
Based on these findings, the maximum tolerated dose level was set at 80 mg/kg bw with respect to the main study.

RESULTS OF DEFINITIVE STUDY
The chromosome aberration frequency was not significantly increased as compared to the negative control.
- Positive control: The positive control (Adriblastin 10 mg/kg bw) showed a distinct increase of induced aberration frequency.
- Historical control data: Historical control data were not provided.
For details on the results, please refer to the attached background material.
Conclusions:
The present study was conducted according to the OECD TG 483 under GLP conditions. In the in vivo mouse germ-cell cytogenetic assay the test article did not induce chromosome aberrations in spermatogonia of the mouse under the experimental conditions reported.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Apr - 23 Aug 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosomal Aberration Test)
Version / remarks:
adopted 2016
Deviations:
yes
Remarks:
scientific justification for using species other than rats and mice not provided, no historical control data provided, only one dose tested, only 100 metaphases analysed, mitotic index not evaluated
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
hamster, Chinese
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: conventionally homebred
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 26-35 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Animals were individually kept in Makrolon type I cages, using bedding of soft wood granules type S 8/15 (Fa. Ssniff, Spezialdiaten GmbH, Soest, Germany)
- Diet: fixed-formula feed (Ssniff H Standard Diet), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24
- Humidity (%): 35-49
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: 0.5 % aqueous Cremophor
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
The test item was suspended in 0.5 % aqueous Cremophor emulsion using a Dismembrator, and then stirred with a magnetic mixer until administration. The negative control received 0.5 % agueous Cremophor emulsion by the same method.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
6, 24, 48 h after administration of test substance
24 h after administration of positive/negative controls
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 30 mg/kg bw in deionized water
Tissues and cell types examined:
Tissue: bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose selection was based on a preliminary study, in which groups of 4 animals, including both males and females, were orally treated with 80, 160, 320, 640, 1000, 2000, 2500, 5000 and 10000 mg/kg bw of the test item, respectively.

TREATMENT AND SAMPLING TIMES:
Five male and five female Chinese hamsters were orally treated with 2000 mg/kg bw of the test substance once. Animals were sacrificed 6, 24 and 48 h after administration.

DETAILS OF SLIDE PREPARATION:
Two hours before the sacrifice, an intraperitoneal injection of colcemid was given at a dose of 3.3 mg/kg bw, to arrest the mitoses in the metaphase stage. The microscopic slide preparation was carried out according to Boiler and Schmid (1970). In brief, the animals were sacrificed individually shortly before they were prepared. The femur was then dissected, separated and freed from adhering tissue. The marrow channel was then opened. About 2 mL of warm (30°C) physiological NaCl solution was pulled into a syringe with a needle. The needle was inserted into the marrow channel at one open end, and marrow rinsed into a tube. This was repeated.
The tubes were then centrifuged (1500 rpm, 5 min) and the supernatant was discarded. The pellet was repeatedly mixed with 2 mL of hypertonic medium and the tubes were incubated at 30 °C for 20 min. After centrifugation (1500 rpm, 5 min), the supernatant was discarded and the pellet mixed. 2 mL fixing agent was added and incubated for 30 minutes which was repeated twice. About 5-7 drops of the suspension was transferred to a slide and dried. Slides were dripped in methanol for 3 min and stained with 5 % Giemsa stain for 20-25 min. Slides were rinsed with water and dewatered with acetone, transferred to xylene for 30 min and subsequently covered after drying.

METHOD OF ANALYSIS:
Light microscope at approximately 1000x magnification was used for the evaluation. Approximately 100 metaphases of each experimental animal were examined for structural changes in the chromosomes including gaps, breaks, fragments, deletions, exchanges and multiple aberrations. In addition metaphases showing chromosome disintegration as an indication of a cytotoxic effect were also recorded.
Evaluation criteria:
Acceptance criteria
An assay was acceptable if there was a biologically relevant increase in chromosome aberrations induced by the positive controls and if the negative control rate was in the expected range according to the laboratory's experience.

Assessment criteria
A test was considered positive if there was a relevant and statistically significant increase in the aberration rate.

A test was considered negative if there was no such increase at any time interval.

A test was considered equivocal if there was an increase which was statistically significant but not considered relevant, or if an increase occurred, which was considered relevant, but which was not statistically significant.
Statistics:
The one-sided corrected chi2 test was used for the statistical evaluation. A difference was considered as significant if the probability of error was below 5 %. The test was only performed if this rate was higher in the treated group compared to the negative control.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
four of 34 treated animals died, all other animals showed no symptoms
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose: 80-10000 mg/kg bw
- Clinical signs of toxicity in test animals:
5000 and 10000 mg/kg bw: all animals died
2500 mg/kg bw: two out of four animals died
at and above 640 mg/kg bw: apathy, reduced motility, reduced reflexes, digging and grooming movements, bloody nose, palmospasm and shivering.
Based on these results, the dose chosen for the main test was 2000 mg/kg bw.

RESULTS OF THE MAIN STUDY
- Clinical signs: the treated animals showed no symptoms. Their external appearance and physical activity remained unaffected. Their eating behavior was normal. Four of 34 treated animals died during the conduct of the test due to acute toxicity of 2000 mg/kg bw.
- Types of structural aberrations for significant dose levels: Metaphases with aberrations including gaps, metaphases with aberrations exluding gaps and metaphases with exchanges were compared. There was no significant deviation from the negative control for all time points investigated. Please also refer to the attached background material.
- Positive control: The positive control induced a clear increase in chromosome aberrations and therefore a clear clastogenic effect, which documented the system's sensitivity.
- Historical control data: Historical negative or positive control data were not provided.
Conclusions:
The present study was conducted according to the OECD TG 475 under GLP conditions. In the cytogenetic study of bone marrow in Chinese hamster the test substance did not show clastogenic effects at the aute oral dose of 2000 mg/kg bw.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

To characterize the mutagenic/genotoxic potential of the present test substance, several in vitro and in vivo studies have been conducted, almost according to currently acknowledged test guidelines. 

 

In-vitro testing

Bacterial mutagenicity

With respect to bacterial mutagenicity, the key study refers to an Ames test conducted according to OECD Guideline 471 and in compliance with GLP with a standard battery of tester strains including 5 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537, TA 102; M-554147-01-1). The test substance was completely soluble in Dimethyl sulfoxide (DMSO) and no precipitation was observed at any concentration tested. In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I (plate incorporation test). Since no toxic effects were observed in experiment I, six concentrations (33, 100, 333, 1000, 2500 and 5000 µg/plate) were tested in experiment II (pre-incubation test). Both experiments were performed with and without metabolic activation system (S9 mix). The test substance did not induce relevant increases in revertant colony numbers for any tester strain neither in the absence nor presence of metabolic activation. Further, no cytotoxicity was observed. Untreated, solvent and positive controls were considered as valid as the mean numbers of revertant colonies of the solvent controls fell within the range of historical data, and positive control treatments increased the number of revertants significantly. Under the conditions of the assay, the test item was not mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.

 

A further Ames test (M-028670-01-1) was conducted according to OECD Guideline 471 and in compliance with GLP with a standard battery of tester strains including 4 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537) and the E.coli strain (WP2 uvrA). Five concentrations of the test substance (312.5, 625, 1250, 2500 and 500 µg/plate) were tested in the presence and absence of a metabolic activation system (S9 mix). The test substance did not induce relevant increases in revertant colony numbers for any tester strain neither in the absence nor presence of metabolic activation. No cytotoxicity was observed up to the highest concentration tested. The controls were considered valid, as a marked increase in the revertant colonies was observed for the positive controls whereas the spontaneous revertant colonies of the solvent control was within the variance of the laboratories background data. However, no details on the historical control data for vehicle and positive control were provided. Under the conditions of the assay, the test item was not mutagenic in S. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in E. coli WP2 uvrA with and without metabolic activation.

 

Further bacterial mutagenicity data are available, with results consistent with those reported above; these will only be shortly mentioned here, for purpose of data completeness.

In three further GLP-compliant Ames tests, up to 5000 µg/plate of the test substance yielded negative results in the four S. typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 both with and without metabolic activation (M-029085-01-1, M-027611-01-1 and M-025825-01-1). In a Rec- Assay with spores using B. subtilis strains H17 (Rec+) and M45 (Rec-), the test item showed no property of DNA-damaging effects up to 5000 µg/disk neither in the presence nor absence of a metabolic activation (M-028627-01-1).

 

In conclusion, the test substance is considered as non-mutagenic in bacteria.

 

With respect to mammalian cells, several in vitro studies are available with the in vitro MNT and the in vitro mammalian cell hprt gene mutation studies being selected as key studies.

 

Cytogenetic assay in mammalian cells

With respect to the first key study, the test substance was assessed for its potential to induce micronuclei in human lymphocyte in vitro (clastogenic or aneugenic activity) by a GLP-compliant study according to OECD Guideline 487 (M-758173-01-1). Two independent experiments were carried out: During the first experiment, cells were treated with 0 – 2000 µg/mL of the test substance for 3 + 21 h in the presence and absence of metabolic activation (S9 mix). In the second experiment, the cells were treated with the test item in the same concentration range for 24 + 24 h in the absence of a metabolic activation system. 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range. The positive control substances, mitomycin C, cyclophosphamide, and vinblastine led to the expected increase in the number of cells containing micronuclei. The solubility limit of the test substance in culture medium was in the range of 1150 to 2301 µg/mL, indicated by precipitation ≥ 1300 µg/mL. The test substance caused a reduced proliferation index, observed at ≥ 800 µg/mL (24 + 24 h exposure without S9) and ≥ 2000 µg/mL (3 h + 21 h exposure with and without S9). The test substance did not induced micronuclei in human peripheral blood lymphocytes following 3 + 21 h treatment neither in the presence nor in the absence of metabolic activation. Further treatment with the test item for 24 + 24 h, led to significantly higher frequencies of micronucleated binucleate cells at 800 and 1300 µg/mL (giving 30% and 50% reduction in RI, respectively) in the absence of metabolic activation when compared to the vehicle control. Therefore, the test item is considered to induce micronuclei in cultured human peripheral blood lymphocytes following 24+24 h treatment in the absence of a metabolic activation system (S9).

 

Further, a GLP-compliant chromosome aberration study considered for support is available, which was performed to determine clastogenic properties of the test substance (M-028377-02-1). The study conducted was similar to OECD Guideline 473. Human lymphocytes obtained from 2 donors were exposed to the test substance in the presence and absence of metabolic activation in two independent experiments. Thereby, cells were treated with up to 5200 µg/mL for 2.5 h with and without metabolic activation. The mitotic indices in the treated cultures fell from 500 µg/mL onwards in the absence of metabolic activation and from 1300 µg/mL onwards in the presence of metabolic activation. In experiment 1, there was a statistically significant increase in the number of cells with structural chromosomal aberrations at 500 and 5000 µg/mL in the absence of S9 mix. In the presence of the metabolic activation, no structural chromosomal aberration was noted in the first experiment. In the second experiment, a different batch of the test item with a higher purity was used. In this experiment, there was a statistically significant increase in the number of structurally aberrant cells at 1300, 2600 and 5200 µg/mL in the absence of metabolic activation. In the presence of metabolic activation, a significant structural chromosomal aberration was only noted at 1300 µg/mL. The positive controls were considered valid, as mitomycin C and cyclophosphamide had a clear clastogenic effect. In total, the test item showed a clastogenic effect without S9 mix at cytotoxic concentrations of 500 - 5200 µg/mL. With S9 mix a weak clastogenic effect could not be ruled out.

 

In a GLP-conform study, the test substance was investigated in regards to clastrogenic activity by measuring sister chromatid exchange (SCE) in Chinese hamster ovary cells (M-026488-01-1). Therefore, the cells were treated with up to 5000 µg/mL of the test substance for 24 h in the absence and for 2 h in the presence of metabolic activation. Cytotoxicity was observed at 500.0 µg/mL through 5.0 mg/mL by reduction in monolayer confluence and by a decrease in mitotic cells starting at 166.7 µg/mL without metabolic activation and at 3000 µg/mL with metabolic activation. A statistically significant increase in SCE was observed at a dose level of 250.0 µg/mL in the absence of metabolic activation system, which increased with rising concentrations of the test item. At lower concentrations, the SCE frequencies were elevated but not statistically significant as compared to the solvent controls. Statistically significant increase of SCE frequencies was also noted at the two highest dose levels of 2.0 mg/mL and 3.0 mg/mL with a dose related increase in the presence of metabolic activation. The positive controls mitomycin C and cyclophosphamide resulted in a clear and statistically significant rise in SCE frequencies and was considered valid. Under the condition of the test, the test item is considered positive for SCE induction both in the presence and absence of metabolic activation.

 

A further SCE study is available, which only is shortly mentioned here, for purpose of data completness. In this GLP-compliant sister chromatid exchange (SCE) assay with Chinese hamster ovary cells, 200 µg/mL of the test item induced a significant, but marginal increase in SCE in the absence of metabolic activation (M-025499-01-1). All other tested concentrations (ranging from 25-400 µg/mL without S9 and 157-1250 µg/mL with S9), did not increase SCE in the cells. The test item is therefore considered not to induce clastogenic effects in Chinese hamster ovary cells under the conditions of the assay.

 

In an unscheduled DNA Synthesis (UDS) Assay, primary rat hepatocytes were exposed to the test substance in a concentrations range of 5.00 µg/mL to 750 µg/mL (M-026493-01-1). The test item did not induce significant increases in UDS. Therefore, the test substance is considered to be non-mutagenic in the rat primary hepatocyte UDS. 

Thus, with respect to cytogenicity, the findings from different studies appear inconsistent in their entirety. 

 

A yeast study is also available, which only is mentioned here for purpose of data completeness. In this study, Saccharomyces cerevisiae strain D7 was investigated for induction of mitotic recombination at concentrations of up to 10000 µg/mL (M-027595-01-1). No evidence for induction of mitotic recombination by the test item was found neither in the absence nor presence of metabolic activation.

 

Thus, with respects to mammalian cells, a cytogenic potential of the test item cannot definitively ruled out under in vitro testing conditions.

 

Gene mutations in mammalian cells

The second in vitro key study with respect to mammalian cells refers to gene mutation. Within this study, the potential of the test substance to induce genotoxicity in mammalian cells was determined in a test performed equivalent to OECD Guideline 476 (M-027630-01-1). Chinese hamster ovary cells were treated with 60-125 µg/mL in the absence and with 100-1222 µg/mL in the presence of metabolic activation for 5 hours. The test substance induced dose-dependent cytotoxicity both in the presence and in the absence of S9. In the absence of metabolic activation dose-related decreases were observed in relative survival to treatment at and above 80 µg/mL. In the presence of metabolic activation, the test substance induced a decrease in relative survival at and above 100 µg/mL and relative population growth. In the first experiment, the spontaneous mutant frequency of the vehicle controls was significantly elevated over the range of variation for vehicle controls among different trials. Therefore, the results of this trial were not used for evaluation of the genotoxicity of the test item. In experiments 2 and 3, statistically significantly increased mutant frequencies were observed, which, however, did not fulfil the evaluation criteria for a positive finding: Without metabolic activation, 80 µg/mL of the test item induced a statistically significant increase in mutant frequency over the concurrent vehicle controls. This was only observed in experiment 3 and in one culture, which was neither confirmed by the duplicate treatment or the other trial nor was this increase dose-related. With metabolic activation, two cultures in experiment 2 showed a statistically significant increase in mutant frequency over the concurrent vehicle controls at test article concentrations of 407 µg/mL and 815 µg/mL. However, this increase was not dose-related and it was not confirmed by the duplicate treatment or in the second trial. Moreover, it is in a range which is typical of vehicle control variation and the statistical significance seemed to be the result of the extremely low spontaneous mutant frequency of the vehicle controls. All other investigated concentrations of the test item did not cause a significant increase in mutant frequencies. Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition, which did not lead to statistically significant increase of mutant frequencies. The positive controls ethylmethanesulphonate and dimethylbenzanthracene revealed a clear mutagenic effect in all experiments. The vehicle and positive control mutant frequencies were all in the normal range of the historical control data in the experiment 2 and 3. In total, the test item is considered to be non-mutagenic in the HPRT test using Chinese hamster ovary cells with and without metabolic activation.

In conclusion, the test substance is considered as non-mutagenic in the mammalian gene mutation tests in vitro.

 

Conclusion on genetic toxicity in vitro:

All in vitro tests for gene mutation effects afforded negative results both in bacteria and in mammalian cells. Controversial results were obtained by in vitro tests investigating possible clastogenic effects of the test substance. Positive findings were observed in human lymphocytes (micronucleus and chromosome aberration assay) and in Chinese hamster ovary cells (sister chromatid exchange assay), which were predominantly found at cytotoxic concentrations. However, based on the available data, clastogenic properties of the test substances in mammalian cells cannot be ruled out. Accordingly, further in vivo studies have to be regarded.

 

In-vivo testing:

Regarding the genetic toxicity in vivo, several cytogenetic assays in rat, mouse and hamster are available. Of these studies, a MNT with rat, a MNT with mouse, both conducted according to OECD Guideline 474, as well as a chromosome aberration assay with Chinese hamster conducted according to OECD Guideline 475 and a mouse spermatogonial chromosome aberration test according to OECD Guideline 483, fulfilled the criteria of a key study. 

 

In the first MNT key study, the test substance was assessed for its potential to induce micronuclei in peripheral blood of rats by a GLP-compliant test according to OECD Guideline 474 (M-758872-01-1). Male rats were orally treated with vehicle alone (negative controls) or 50, 100 or 200 mg/kg bw/day of the test substance on 2 successive days, approximately 24 hours apart. Blood samples were taken from all animals approximately 48 hours after the final dose administration. Samples from an existing positive control bank of blood obtained from rats dosed with cyclophosphamide revealed statistically significant increases in the number of micronucleated reticulocytes (MN-RET) compared with the concurrent negative control group. Both the vehicle and positive controls gave frequencies of micronucleated cells within the historical control data range. In addition, there were no statistically significant increases in MN-RET frequency in male rats given any dose level of the test substance compared with the negative control group. At 100 and 200 mg/kg bw clinical signs and a statistically significant decrease of reticulocytes were observed. The latter indicating toxicity to the bone marrow and demonstrating exposure of the bone marrow to the test substance. In total, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the maximum tolerated dose level of 200 mg/kg bw/day in male rats. The test substance is considered to be neither clastogenic nor aneugenic in the rat bone marrow micronucleus assay.

 

In the second MNT key study, the test substance was assessed in male and female mice for a possible clastogenic effect on the chromosomes of bone marrow erythroblasts by a GLP-compliant test according to OECD Guideline 474 (M-027591-01-1). Mice received a single oral administration of 80 mg/kg bw test substance. 24, 48 and 72 hours after the treatment, the femoral marrow were prepared. Animals, which received either the vehicle control or the positive control, were sacrificed after 24 hours. After single treatment with the test substance, the treated animals showed compound-related symptoms for up to 6 hours. Thereafter, animals remained unaffected. The positive control was considered valid, as it had a clear clastogenic effect evident by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The test substance did not led to a relevant increase of the number of micronucleated normochromatic erythrocytes in any of the groups. Based on the in vivo micronucleus test, there were no indications of a clastogenic effect after single oral treatment of the mouse with the test substance.

 

In the third key study, the test substance was also assessed for clastogenic effects in the bone marrow of Chinese hamster following a single oral treatment with 2000 mg/kg bw (M-025903-01-1). The study was performed according to GLP and equivalent to OECD Guideline 475. The specimens of bone marrow were prepared 6, 24 and 48 hours after substance administration whereas the negative and positive control specimens were prepared after 24 hours. Although the treated animals showed no clinical symptoms, four out of 34 animals died during the conduct of the test due to acute toxicity. The test substance did not induce an increase in structural aberrations compared to the negative control in any of the groups. The results for the positive control indicated a clear clastogenic effect and documented the system's sensitivity. However, historical negative or positive control data were not provided. In total, the test substance did not show clastogenic effects at the acute oral dose of 2000 mg/kg bw in the cytogenetic study of bone marrow in Chinese hamster.

 

The following, non-key study, is only mentiones for purpose of data completeness. Negative results were also obtained in a GLP-compliant sister chromatid exchange (SCE) assay in bone marrow of Chinese hamster following single oral doses of 500-2000 mg/kg bw (M-028379-01-1). During the study, the treated animals showed no symptoms of toxicity at any dose level. The test substance caused a slight but significant reduction of the mitotic index at 1000 and 2000 mg/kg bw, but no statistically significant increase of SCE frequencies was observed. The positive control was considered valid, as a clear increase in SCE frequencies was noted. In summary, there was no indication of a DNA-modifying effect of the test substance after single oral doses of up to and including 2000 mg/kg bw in the sister-chromatid exchange test on the Chinese hamster.

 

In the fourth key study, negative results were also obtained in a GLP-compliant mouse germ-cell cytogenetic assay (OECD 483, M-026551-01-1). For investigating the potential of the test substance to induce chromosome aberration in spermatogonial mitoses, mice received a single oral dose of 80 mg/kg bw of test substance. The animals were sacrificed after 6, 24 and 48 h following treatment, and both testes were collected for examination. The test article did not enhance the chromosome aberration frequency significantly as compared to the negative control. The appropriate reference mutagen (positive control) showed a distinct increase of induced aberration frequency. Historical negative and positive control data were not provided. In total, the test item did induce chromosome aberrations in spermatogonia in the in vivo mouse germ-cell cytogenetic assay under the experimental conditions.

 

Conclusion on genetic toxicity in vivo:

The positive findings regarding clastogenic effects in vitro could not be confirmed by in vivo testing. The fact that all in vivo experiments yielded negative results justifies the conclusion that the test article has no genotoxic or mutagenic potential.

 

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to CLP Regulation (EC) No. 1272/2008, and are therefore conclusive and do not warrant classification.