Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

See "additional information"

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:
From 2003-09-11 to 2003-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dated May 19, 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch No.: 00410155
Aggregate State at Room Temperature: solid
Colour: brown powder
Purity: 96 %
Stability in Solvent: Not indicated by the sponsor
Storage: Room temperature
Expiration Date: 2004-05-05
Target gene:
The histidine dependant strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide (DMSO)
Justification for choice of solvent: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, at 10 μg/plate with TA1535 and TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation, at 10 μg/plate with TA 98, at 50 μg/plate with TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, at 4µL/plate with WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation, at 2.5 μg/plate with TA 1535, TA 1537, TA 98 and TA 100, at 10 μg/plate with WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay

Experimental performance:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL: Overlay agar

In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

DURATION:
- Pre-incubation period: 60 minutes (experiment II)
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicates.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
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 WP2 uvrA) 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.
Statistics:
No statistical evaluation of the data was required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I since relevant toxic effects were only observed at high concentrations and 5000 μg/plate were chosen as maximum concentration of the
main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
- Negative (solvent/vehicle) historical control data: In experiment I with metabolic activation, the data in the negative control of strain TA 98 and in the solvent control of strain TA 100 were slightly above the laboratory historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strong toxic effects reduced the background growth in the second experiment at 2500 and 5000 μg/plate in strain TA 98 without metabolic activation and in strain TA 100 with and without metabolic activation. Relevant toxic effects, evident as a reduction in the number of revertants below 0.5 times the corresponding control, occurred at the concentrations presented in the section "any additional information including tables".
Remarks on result:
other: all strains/cell types tested

Strong toxic effects occurred at the following concentrations (μg/plate):

Strain

Experiment 1

Experiment 2

 

Without S9 mix

With S9 mix

Without S9 mix

With S9 mix

TA1535

2500-5000

2500-5000

2500-5000

1000-5000

TA1537

2500-5000

2500-5000

2500-5000

5000

TA98

2500-5000

5000

2500-5000

/

TA100

2500-5000

2500-5000

2500-5000

2500-5000

WP2uvrA

2500-5000

1000-5000

1000-5000

1000-5000

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Guideline adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch No.: 00410155
Aggregate State at Room Temperature: Solid
Colour: Brown
Molecular Weight: 147.18
Purity: 96 %
Stability in Solvent: Not indicated by the Sponsor
Storage: At room temperature
Expiration Date: May 05, 2004
Species / strain / cell type:
other: cell line V79
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphthoflavone induced rat liver S9 was used as the metabolic activation system
Test concentrations with justification for top dose:
Range-finder test: 0, 11.7, 23.4, 46.9, 93.8, 187.5, 375.0, 750.0 and 1500.0 µg/mL

Experiment I
4h exposure - 18h preparation time, without S9 mix: 0,12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL (50.0, 100.0 and 200.0 µg/mL were selected for metaphase analysis)
4h exposure - 18h preparation time, with S9 mix: 0,12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL ((2.5, 25.0 and 50.0 µg/mL were selected for metaphase analysis)

Experiment II
18h exposure - 18h preparation time, without S9 mix: 0, 1.6, 3.1, 6.3, 12.5, 25.0, and 50.0 µg/mL (12.5, 25.0 and 50.0 µg/mL were selected for metaphase analysis)
28h exposure - 28h preparation time, without S9 mix: 0, 6.3, 12.5, 25.0, and 50.0 µg/mL (25.0 and 50.0 µg/mL were selected for metaphase analysis)
4h exposure - 28h preparation time, with S9 mix: 0,12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/mL (12.5, 25.0 and 50.0 µg/mL were selected for metaphase analysis)
Vehicle / solvent:
Vehicle chosen: dimùehtylsulfoxide (DMSO). The final concentration of DMSO in the culture medium was 0.5 % (v/v).
Justification for choice of vehicle:The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
200 µg/mL (1.6 mM) without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
0.7 - 1.0 µg/mL (2.5 - 3.5 µM) with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

EXPERIMENTAL PERFORMANCE:
Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-freemedium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL medium were used. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.

All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

DURATION
- Exposure duration:
Without S9 mix: Experiment I: 4 hours, Experiment II: 18 and 28 hours (continuous exposure)
With S9 mix: Experiment I and II: 4 hours
- Expression time (cells in growth medium):
Without S9 mix: Experiment I: 11.5 hours, Experiment II: none (continuous exposure)
With S9 mix: Experiment II: 11.5 hours and 21.5 hours for the 18 and 28 hour preparation intertvals, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours in Experiment I and 18 hours and 28 hours in Experiment II

SPINDLE INHIBITOR (cytogenetic assays): 15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 μg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts respectively). Per experiment two slides per group were prepared. After preparation the cells were stained.
STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except in experiment II with metabolic activation at the 28 hrs preparation interval with 50 µg/mL, where 200 metaphase plates were scored. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and/or reduced cell number

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
- Determination of endoreplication: investigated
- Other: Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates.
Evaluation criteria:
A test item is classified as non-clastogenic if:
− the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
− no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
− the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
− either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if:
− the number of induced numerical aberrations is not in the range of the laboratory historical control data (0.0 - 8.5 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see details below
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 412 mOsm, pH 7.3 versus 402 mOsm and pH 7.2 at 1500 µg/mL).
- Precipitation:
In the pre-test, precipitation of the test item in culture medium was observed after treatment with 750 μg/mL and above in the absence and the presence of S9 mix.
In the cytogenetic experiments, precipitation of the test item in culture medium was observed in both experiments in the presence of S9 mix with 100 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 11.7 and 1500 µg/mL were applied. Clear toxic effects were observed after treatment with 375 µg/mL and above in the absence and the presence of S9 mix. In addition, 24 hrs continuous treatment with 46.9 µg/mL and above in the absence of S9 mix induced strong toxic effects.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments, EMS (200 μg/mL) and CPA (0.7 and 1.0 μg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects indicated by reduced cell numbers and/or mitotic indices of below 50 % of control were observed in both experiments in the absence and the presence of S9 mix. In detail reduced cell numbers were observed in experiment II in the absence of S9 mix after 28 hrs continuous treatment with 50 µg/mL (30 % of control) and in experiment II in the presence of S9 mix after 4 hrs treatment with 100 µg/mL (33 % of control). In addition, in the absence of S9 mix the mitotic indices were reduced in experiment I after 18 hrs continuous treatment with 50 µg/mL (49 % of control) and in experiment II after 28 hrs continuous treatment with 50 µg/mL (44 % of control).
In experiment I the concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Remarks on result:
other: with S9 at the 28 hour preparation time

In experiment I in the absence and the presence of S9 mix and in experiment II in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

In experiment II, in the presence of S9 mix at preparation interval 28 hrs a biologically relevant dose-related increase in the number of cells carrying structural chromosome aberrations (0.0 %, 1.8 %, and 8.0 %) was observed in the concentration range evaluated (25 to 100 μg/mL). In addition, the aberration rates (1.8 % and 8.0 %) were significantly increased after treatment with 50 and 100 μg/mL as compared to the corresponding solvent control (0.0 %). However, only the value after treatment with 100 μg/mL clearly exceeded our historical control data range (0.0 - 4.0 % aberrant cells). Also, the number of cells carrying exchanges after treatment with 100 μg/mL was distinctly increased (4.0 %) as compared to the solvent control (0.0 %). Therefore, the dose-dependency and the statistical significance have to be regarded as being biologically relevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.7 - 4.9 %) as compared to the rates of the solvent controls (1.9 - 3.2 %).

Conclusions:
Under the experimental conditions reported, the test item T002251 induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) at prolonged 28 hours preparation interval at a cytotoxic and precipitating concentration only. The biological significance of the increase in this study under only one exposure condition (4 h, +S9, 28 h preparation period) is suspect due to confounding factors at the highest concentration level evaluated (100 mg/mL). At this level, mitotic index and cell count were 75% and 33%, respectively. Mitotic index is an indirect measurement of cytotoxicity, and known to be less sensitive monitor of cellular health. Therefore, following the Weight-of-Evidence (WOE) approach the relevance of chromosomal aberrations induced under high cytotoxic (>50%) and precipitating conditions is questionable. Furthermore, the response at the mid dose (50 mg/mL) is within historical range, suggesting that clastogenicity is only seen under extreme culture conditions. Additional WOE factors include lack of corroborative clastogenic response in the 4 hour, +S9, 18 hour preparation interval, and absence of in vivo clastogenic potential in a mouse micronucleus assay.

Genetic toxicity in vivo

Description of key information

See "additional information"

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:
2004-01-06 to 2004-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C (dated 2000-05-19)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-Harmonised Tripartite Guideline S2A and S2B (CPMP/ICH/141/95; CPMP/ICH/174/95)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
Batch No.: 00410155
Aggregate State at Room Temperature: Solid
Colour: Brown
Purity: 96%
Stability in Solvent: Not indicated by the Sponsor
Storage: At room temperature
Expiration Date: May 05, 2004
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 5-7 weeks (males); 7-9 weeks (females)
- Weight at study initiation: males mean value 29.7g (SD +/- 2.2g); females mean value 25.0g (SD +/- 1.7g)
- Assigned to test groups randomly: yes, and identified by cage number
- Fasting period before study: the animals were starved over night but received water ad libitum
- Housing: single, Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 25 - 72%
- Photoperiod (hrs dark / hrs light): 12h light (artificial) / 12h dark
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals
- Concentration of test material in vehicle: adequate spaced dose levels
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 20 ml/kg body weight orally.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was formulated in PEG 400.
Duration of treatment / exposure:
24h and 48h (only highest dose level)
Frequency of treatment:
The animals received the test item, the vehicle or the positive control substance once.
Post exposure period:
Examination of animals of all dose groups for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Negative control
24h sampling time
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Test group
24h sampling time
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Test group
24h sampling time
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Test group
24h and 48h sampling time
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Positive control
24h sampling time
No. of animals per sex per dose:
Six males and six females were assigned to eaeh test group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: single oral administration
- Doses / concentrations: 40 mg/kg b.w. (volume administered 10 ml/kg b.w.)
- Other: dissolved in deionised water; solution prepared on day of administration
Tissues and cell types examined:
bone marrow tissue - at least 2000 polychromatic erythrocytes were analysed per animal for micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24h after treatment. For the highest dose level an additional sample was taken at 48h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved over night but received water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24h after administration of the test item.
Sampling of the bone marrow was done 24h and 48h after treatment, respectively.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed with CO2 and cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resispended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide ws made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males / 5 females) per test group were evaluated as described. The remaining 6th animal of each group and sex is evaluated in case an animal of that group dies.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 males and 1 female died after treatment with 600 mg/kg b.w. in main experiment (no deaths at 600 mg/kg b.w. in pre-experiment)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 600, 750 and 1000 mg/kg b.w.
- Solubility: soluble
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, adbominal position, eyelid closure, ruffled fur, apathy, death
- Evidence of cytotoxicity in tissue analyzed: To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sampie and reported as the number of PCEs per 2000 erythrocytes.
- Harvest times: 24h and 48h (for highest dose level)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with T002251 were below or near to the value of the vehicle control group.
The following percentages of PCEs with micronuclei were calculated:
a) vehicle (24h): 0.055%
b) test item 150 mg/kg b.w. (24h): 0.075%
c) test item 300 mg/kg b.w. (24h): 0.070%
d) test item 600 mg/kg b.w. (24h): 0.060%
e) positive control 40 mg/kg b.w. (24h): 2.185%
f) test item 600 mg/kg b.w. (48h): 0.030%
- Ratio of PCE/NCE (for Micronucleus assay):
a) vehicle (24h): 1103/2000
b) test item 150 mg/kg b.w. (24h): 1042/2000
c) test item 300 mg/kg b.w. (24h): 1082/2000
d) test item 600 mg/kg b.w. (24h): 1088/2000
e) positive control 40 mg/kg b.w. (24h): 1123/2000
f) test item 600 mg/kg b.w. (48h): 863/2000
- Appropriateness of dose levels and route: determined via pre-experiment
- Statistical evaluation: no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level
Conclusions:
Interpretation of results: negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Additional information

T002251, a starting material, is without alerts in DEREK. Initial classification = non genotoxic.

Additionally, an Ames test, a CAT test and an in vivo MNT have been performed.

 

IN VITRO BACTERIAL REVERSE MUTATION ASSAY, AMES TEST (GLP)

T002251 formulated in DMSO, was tested in a bacterial reverse mutation assays (Ames test) with 4 histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and with Escherichia coli WP2uvrA. Concentrations up to 5000 µg/plate were tested in 2 independent assays, in the absence or presence of S9-metabolic activation (Aroclor-induced rat liver S9-mix). Strong toxic effects reduced the background growth at 2500 and 5000 µg/plate in strains TA98 and TA100 as evidenced by a decrease in the number of revertants observed. T002251 did not induce a dose-related, more than 2- or 3-fold increase in the number of revertant colonies in any of the tester strains in the absence or presence of S9-metabolic activation. The vehicle control and appropriate positive control reference articles confirmed the adequacy of the test system. It was concluded that T002251 was not mutagenic under the conditions of this assay.

IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION ASSAY (GLP)

T002251 formulated in DMSO, was tested in an in vitro mammalian chromosome aberration test in Chinese hamster V79 cells. Following a dose range finding test, T002251 was tested, in the absence or presence of S9-metabolic activation (Aroclor-induced rat liver S9-mix) up to 400 µg/mL following 4, 18 or 28 hours treatment; precipitation occurred at 100 µg/mL and above. The chromosomes were prepared 18 or 28 hours after the start of treatment. The tests were performed to the limit of toxicity. In the presence of S9-mix at a prolonged (28 hours) preparation interval, a statistically significant response in aberrant cells was seen at the highest concentration level evaluated, where precipitation and substantial cytotoxicity was observed (33% cell number). The vehicle control and appropriate positive control reference article confirmed the adequacy of the test system. It was concluded that T002251 induced structural chromosome aberrations in presence of S9 mix at a prolonged (28 h) preparation interval at a cytotoxic and precipitating concentration under the conditions of this assa

IN VIVO MAMMALIAN MICRONUCLEUS TEST (GLP)

To evaluate the possibility of clastogenic potential in vivo, a mouse micronucleus assay was performed. T002251 formulated in PEG400, was administered to mice in an in vivo micronucleus test. Single oral (gavage) doses of 0 (vehicle), 150, 300 and 600 mg/kg T002251 and 40 mg/kg cyclophosphamide (positive reference article) were administered to NMRI mice (6 males and 6 females at each time point for each dose) and bone marrow was sampled 24 hours or 48 hours (600 mg/kg only) after dosing. Although the high dose was considered suitable on the basis of a pilot study, 3 animals (2 male and 1 female) died 6 hours after dosing at 600 mg/kg, indicating that this dose level was at or beyond the maximum tolerated dose level for this assay. No increase in the frequency of micronucleated cells was observed in the polychromatic erythrocytes of the bone marrow of animals tested with T002251. The mean number of polychromatic erythrocytes was decreased at the 48 hours preparation after treatment with the test item, a cytotoxic effect of the compound to the bone marrow, and target tissue exposure. The vehicle control and the positive reference articles confirmed the adequacy of the test system. It was concluded that T002251 was not an in vivo clastogen under the conditions of this assay.

ASSESSMENT OF T002251 GENOTOXICITY

There is no evidence for mutagenic potential of T002251 in bacteria. The biological significance of the increase in chromosome aberrations observed in Chinese hamster V79 cells under only one exposure condition (4 h, +S9, 28 h preparation period) is suspect due to confounding factors at the highest concentration level evaluated (100 mg/mL). At this level, mitotic index and cell count were 75% and 33%, respectively. Mitotic index is an indirect measurement of cytotoxicity, and known to be less sensitive monitor of cellular health. Therefore, following the Weight-of-Evidence (WOE) approach the relevance of chromosomal aberrations induced under high cytotoxic (>50%) and precipitating conditions is questionable. Furthermore, the response at the mid dose (50 mg/mL) is within historical range, suggesting that clastogenicity is only seen under extreme culture conditions. Additional WOE factors include lack of corroborative clastogenic response in the 4 hour, +S9, 18 hour preparation interval, and absence of in vivo clastogenic potential in a mouse micronucleus assay.

T002251 is considered non-genotoxic intermediate.

Justification for classification or non-classification

Based on the weight of evidence described above and the criteria of the CLP Regulation (EC) 1272/2008, T002251 should not be classified for mutagenicity.