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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Test

The test substance is not mutagenic in the bacterial reverse mutation test, either in the absence or presence of a metabolic activation system.

Chromosome Abberation Test

The test substance did not induce either structural or numerical chromosome aberrations in the absence or presence of a metabolic activation system.

HPRT Test

Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

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 16 March 2015 to 26 March 2015
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch no. COD-001545
Description: Solid, yellowish
Purity: 97.3%
Stability of test compound: stable until 31 Jan 2016
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix induced with phenobarbital/ β-naphthoflavone
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2600 and 5200 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, DMSO was used as the vehicle. DMSO had been demonstrated to be suitable in bacterial reverse mutation tests and is a substance for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
Without S9 mix: : TA100, TA 1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix: WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix: TA 1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD)
Remarks:
Without S9 mix: TA 98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2-AA)
Remarks:
With S9 mix: All strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation method

DURATION:
- pre-incubation period: 20 min
- Expression time: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

Further information:
- method of cells evaluation
The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System


Evaluation criteria:
Reproducibility of results was confirmed by two independent experiments (Experiment I and II).
The results are judged positive without statistical analysis if the criteria listed below are all met based on the mean number of colonies on each plate.
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
On the other hand, the results are judged negative if the mean number of revertant colonies for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
No statistical tests were performed.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the doses of 2600 and 5200 μg/plate in all assays
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the doses of 2600 and 5200 μg/plate in all assays
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the doses of 2600 and 5200 μg/plate in all assays
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the doses of 2600 and 5200 μg/plate in all assays
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the doses of 2600 and 5200 μg/plate in all assays
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at the doses of 2600 and 5200 μg/plate in the test substance group with and without metabolic activation in both assays.

RANGE-FINDING/SCREENING STUDIES: Not conducted prior to test

HISTORICAL CONTROL DATA
- Positive historical control data: Valid, within the range
- Negative (solvent/vehicle) historical control data: Valid, within the range

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 February 2015 to 20 May 2015
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro mammalian gene mutation test
Specific details on test material used for the study:
Batch no.: 080722
Description: Solid, yellowish
Stability of test compound: The test substance was stable over the study period under the storage conditions.
The homogeneity of the test substance was ensured by mixing prior to preparation of test substance formulations.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length: 12 - 14 hours
- Methods for maintenance in cell culture: Stocks of the CHO cell line (1-mL portions) are maintained at negative 196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant.

MEDIA USED
- Type and identity of media: Culture medium: Ham's F12 medium
Metabolic activation:
with and without
Metabolic activation system:
S9 mix- induced with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity assay: 3500 μg/mL (top concentration)

Mutation assay:
Experiment 1
With (2.3 μg/mL)/ Without S9 mix 4-hour exposure
4.7 μg/mL, 9.4 μg/mL, 18.8 μg/mL, 37.5 μg/mL, 75.0 μg/mL, 150.0 μg/mL, 300.0 μg/mL

Experiment 2
With/Without S9 mix 4-hour exposure
3.9 μg/mL, 7.8 μg/mL, 15.6 μg/mL, 31.3 μg/mL, 31.3 μg/mL, 125.0 μg/mL, 250.0 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle. DMSO has been demonstrated to be suitable in the test and is a vehicle for which historical control data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours treatment
- Expression time (cells in growth medium): entire expression period of 7 – 9 days
- Selection time: 6 - 7 days
- Fixation time: 5 - 8 days

SELECTION AGENT: 10 mL selection medium ("TG" medium)

SPINDLE INHIBITOR: Methanol

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells per dose group

DETERMINATION OF CYTOTOXICITY
- Method: Coning efficiency survival and viability


Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose- response relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance were considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: In this study, in the absence and in the presence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 150.0 μg/mL and above in the 1st Experiment and at 125.0 μg/mL and above in the 2nd Experiment.

RANGE-FINDING/SCREENING STUDIES:
After 4 hours treatment in the absence of S9 mix, cytotoxicity was observed as indicated by reduced relative cloning efficiency of about or below 20% relative survival at 1750.0 μg/mL and above. In contrast, in the presence of S9 mix and after 24 hours treatment in the absence of S9 mix, clearly reduced relative cloning efficiency was observed after treatment with 109.4 μg/mL and above.

HISTORICAL CONTROL DATA
- Positive historical control data: Valid
- Negative (vehicle) historical control data: Valid

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 16, 2008 to February 6, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF
Version / remarks:
No 12 Nousan No 8147
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch no. 080722
Description: Pale yellow green powder
Species / strain / cell type:
mammalian cell line, other: Chinese hamster Lung (CHL) cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: CHL cells established from the lung of Chinese hamster

MEDIA USED
- Type and identity of media: MEM (Gibco BRL), supplemented with 10% inactivated newborn calf serum, 100 IU/ml Penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes, Stock confirmed to the Mycoplasma-free
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary toxicity assay: 19.5 to 5000 μg/mL
Assay: 1st experiment (6 hr exposure): concentrations of 78.1, 156, 313 or 625 μg/mL without and with metabolic activation
2nd experiment (24 hr exposure): 30.9, 46.3, 69.4, 104 or 156 μg/mL without metabolic activation
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, and has been demonstrated to be suitable in the in vitro cytogenetic test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 6 hours (short term treatment, absence and presence of a metabolic activation system) or 24 and 48 hours (continuous treatment, absence of a metabolic activation system)

SPINDLE INHIBITOR: 0.2 μg/mL colcemid was added 2 hours before cell harvest

STAIN: 2% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared by dropping the harvest cultures on clean glass slides. The slides were stained with 2% Giemsa.

NUMBER OF CELLS EVALUATED: 100 cells were examined per replicate culture (200 per dose)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
Slides were coded prior to analysis. 100 cells were examined per replicate culture (200 per dose) and were scored for structural aberrations and for numerical aberrations (polyploidy).

DETERMINATION OF CYTOTOXICITY
- Method: Previously performed HPRT study

OTHER EXAMINATIONS:
- Determination of polyploidy: Only polyploid cells having 3 or more copies of haploid number of chromosomes were scored as a numerical chromosome aberration cell. Since the CHL cells have 25 chromosomes in the modal number, the cell with 37 or more chromosomes was recorded as a polyploid cell.
- Determination of endoreplication: Endoreduplication was classified as polyploidy.
Evaluation criteria:
The test material was considered positive if reproducible and significant Increases in the frequencies of structurally (excluding gaps) or numerically aberrant metaphases were observed with a dose-related response.
Statistics:
The number of structurally aberrant metaphases and the number of polyploid metaphases at each concentration were statistically compared with those of corresponding solvent controls using a chi-square test.
Key result
Species / strain:
mammalian cell line, other: Chinese hamster Lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
continuous treatment: 104 and 156 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at 313 and 625 μg/mL

RANGE-FINDING/SCREENING STUDIES:
Nine doses of the test material ranging from 19.5 to 5000 μg/mL in the absence and presence of S9-mix were evaluated in the preliminary growth inhibition test. Precipitation was observed at 313 μg/mL and above both in the absence and presence of a metabolic activation system, Over 50 % cell growth inhibition was not observed in the short-term test but was observed in the continuous test at the concentrations of 156 μg/mL and above.

HISTORICAL CONTROL DATA
- Positive historical control data: Valid
- Negative (vehicle) historical control data: Valid

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

Genetic toxicity in vivo

Description of key information

Micronucleus Assay

It was concluded that the test item did not induce micronuclei in the bone marrow cells of NMRI mice.

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:
From 10 October 2014 to 14 November 2014
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:
2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Micronucleus assay, bone marrow cells
Specific details on test material used for the study:
Lot/Batch #: COD-002002
Description: Yellowish solid
Purity: 95.7%
Stability of test compound: Stability in solvent confirmed
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: About 6 - 10 weeks
- Weight at study initiation: About 38.5 g
- Assigned to test groups randomly: Yes
- Housing: The mice were singly housed in Makrolon Type II/III with wire mesh tops and granulated soft wood bedding.
- Diet: Pelleted standard diet (certified), ad libitum
- Water: Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45% - 65%
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle (on at 6 am)

IN-LIFE DATES: From 5-Oct-2014 to 14-Nov-2014
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Aqueous 0.5% methyl cellulose
- Justification for choice of vehicle: 0.5 % methyl cellulose was selected in order to compare the results with previous toxicity data; it is also a common vehicle in toxicity studies.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ultraturrax treatment was used to formulate the test item. A correction factor of 1.04 was applied to the formulations.

Dose volume applied: 10 mL/kg bodyweight

Preliminary range finding test:
Mice (2 per/sex/dose group) were orally treated once at 1000, 1500 and 2000 mg/kg bw; the animals were observed for clinical signs of toxicity at intervals of approx. 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration

Duration of treatment / exposure:
24 hours
48 hours (additional control and high dose groups)
Frequency of treatment:
once
Post exposure period:
24 and 48 hours
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7
Control animals:
yes
Positive control(s):
Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg bodyweight
Tissues and cell types examined:
Polychromatic erythrocytes (PCEs)
Normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the Preliminary Range Finding Test.

TREATMENT AND SAMPLING TIMES
Sampling of the bone marrow cells from all test substance groups, negative and positive control groups were conducted 24 hours after administration. Additional negative control and high dose animals were allocated to a 48 hours sampling time point.
To confirm exposure, additional satellite animals were allocated to the study and blood samples (0.5 mL) were taken from 3 males/per time point as follows: 1 and 4 hours for the high dose group and 1 hour for the negative control group.

DETAILS OF SLIDE PREPARATION
The cells prepared earlier were re-suspended in a small residual volume of serum and a small drop of the cell suspension was smeared onto a glass slide. At least one slide was prepared from each animal. The smear slides were thoroughly air-dried and then stained with May-Grünwald/Giemsa; coverslips were mounted with EUKITT. Slides were coded for identification.

METHOD OF ANALYSIS
Slides were examined for each animal under NIKON microspores with 100x oil immersion objectives. Per animal 4000 polychromatic erythrocytes (PCE) were analyzed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed as the ratio of polychromatic to total erythrocytes.
Evaluation criteria:
Definitions of micronuclei stained with Giemsa were as follows: Micronuclei exist in an erythrocyte; the diameter of micronuclei is half or less than mature erythrocytes; color of the micronuclei is same as the nuclei of adjacent leukocytes. Criteria for distinguishing polychromatic erythrocytes from normochromatic ones in smears stained with Giemsa were taken as follows. Polychromatic erythrocytes are blue-violet in color and normochromatic erythrocytes are grayish pale red in color. Therefore erythrocytes that were apparently red in color in a field of the view were classified as normochromatic erythrocytes and all the other erythrocytes were scored as polychromatic erythrocytes.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test, except for the 48 h high dose group for which significance was evaluated by means of the ANOVA.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: There was a single mortality at 1500 and 2000 mg/kg bw, but no mortality at 1000 mg/kg bw. Clinical signs included dyspnea, hunched posture, abdominal posture, tumbling, reduced activity and ruffled fur; there was no notable difference in the response between males and females. The presence of clinical signs confirms the systemic availability of the test material.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Males given 1000 mg/kg bw had reduced activity for one hour after treatment and ruffled fur for up to 48 hours after treatment. There were no clinical signs of toxicity at 500 or 250 mg/kg bw.
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated polychromatic erythrocytes at any dose level. In the positive control group dosed with cyclophosphamide, the frequency of micronucleated polychromatic erythrocytes showed a highly significant increase.
- Ratio of PCE/NCE: 54- 57; The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow. There was a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes in the positive control group.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic Toxicity in vitro

 

Key study: Bacterial reverse mutation assay 2015/1099097

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone) using the standard plate and the pre-incubation methods. The test substance was dissolved in dimethyl sulfoxide. The test substance was tested in both experiments at concentrations of 33, 100, 333, 1000, 2600 and 5200 μg/plate.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The positive controls induced the appropriate responses in the corresponding strains. The test substance is not mutagenic in the bacterial strains tested, either in the absence or presence of a metabolic activation system.

 

Supporting studies: Bacterial reverse mutation assay

All studies are GLP and guideline compliant.

In the supporting studies the test substance was also determined to be not mutagenic in the bacterial strains tested, either in the absence or presence of a metabolic activation system.

 

 

Key study: Chromosome aberration test 2009/8000063

The test substance was tested in the in vitro cytogenetic test using cultured Chinese hamster lung (CHL) cells. The test was performed in two independent experiments without and with a metabolic activation system (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The test substance was dissolved in dimethyl sulfoxide. In the short-term treatment, the test substance was treated at a concentration of 78.1, 156, 313 or 625 μg/ml for 6 hours with or without metabolic activation. Eighteen hours after the termination of the treatment, chromosome preparations were made. In the continuous treatment, the test substance was treated without metabolic activation at a concentration of 30.9, 46.3, 69.4, 104 or 156 μg/ml for 24 hours; severe toxicity at the two highest doses prevented analysis. As a result of the metaphase analysis in the cytogenetics tests both with the short-term (with and without metabolic activation) and the continuous treatment (without metabolic activation), there were no significant increases in the frequencies of the metaphases with structural chromosome aberrations and polyploid metaphases at any concentrations of the test substance when compared with concurrent solvent control.

Positive control chemicals, mitomycin C and benzo(a)pyrene, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. The test substance did not induce either structural or numerical chromosome aberrations in the absence or presence of a metabolic activation system.

 

 

Key study: HPRT 2015/111062

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested.

1st Experiment

without S9 mix : 0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0; 300.0 μg/mL

with S9 mix : 0; 2.3; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0; 300.0 μg/mL

2nd Experiment

without S9 mix : 0; 3.9; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0 μg/mL

with S9 mix : 0; 3.9; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0 μg/mL

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In both experiments in the absence and in the presence of metabolic activation no cytotoxicity was observed up to the highest tested concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Genetic Toxicity in vivo

Key study: Micronucleus Assay 2014/1313074

The bone marrow micronucleus test was performed with the test substance in NMRI male mice. Three dose levels of 250, 500 and 1000 mg/kg bw were orally administered once only. Bone marrow smears were obtained from all groups at 24 hours and from the controls and 1000 mg/kg group after 48 hours. Animals given 1000 mg/kg showed signs of reduced activity and ruffled fur.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test substance. In the positive control group dosed with cyclophosphamide, the frequency of micronucleated polychromatic erythrocytes showed a highly significant increase.

It was concluded that the test substance did not induce micronuclei in the bone marrow cells of NMRI mice. 

 

Supporting studies: Micronucleus Assay

All studies are GLP and guideline compliant.

In the supporting studies the test substance was also determined to be not mutagenic in the micronucleus assay.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No mutagenic effects were observed in the in vitro bacterial reverse mutation test, chromosome aberration test, HPRT test and in the in vivo micronucleus assay. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.