Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro gene mutation study in mammalian cells according to OECD guideline 476, in a bacterial reverse mutation assay according to OECD guideline 471 and in an in vitro mammalian chromosome aberration test according to OECD guideline 473, the test item had no mutagenic or clastogenic potential (references 7.6.1-1, 7.6.1-2 and 7.6.1-3).

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:
May 07, 2003 - October 22, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Communities L136, 8. June 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
HIS operon (S. thyphimurium)
TRP operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
uvrA pkM101
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:

- Source of S9: Liver S9 from Aroclor 1254-pretreated rats

- Method of preparation of S9 mix: Male Wistar, HSdCpb:Wu rats aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of 0.15 M KCI per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about + 4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C

- Volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 10% (1st test series) or 30% (2nd test series S9) in 3.11 - 3.1316 mL final culture medium

- Quality controls of S9: Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch
Test concentrations with justification for top dose:
The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 2.81, 8.89, 28.1, 88.9, 281, 889 and 2810 µg/plate (with and without S9).
2nd series: 8.89, 28.1, 88.9, 281 and 889 μg/plate (with and without S9).
In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.
Vehicle / solvent:
Tetrahydrofuran (test item), H2O (daunomycin), DMSO (N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-aminoanthracene, benzo[a]pyrene), ethanol (9-amonoacridine)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
For TA 100 (5 µg/plate), TA 1535 (10 µg/plate) and E.coli WP2 uvrA (5 µg/plate), without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
For TA 102, (10 µg/plate), with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA 98 (2 µg/plate), 100 (2 µg/plate), 1535 (2 µg/plate), 1537 (5 µg/plate) and E.coli WP2 uvrA (10 µg/plate), with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
For TA 102, without S9, 200 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For TA 1537, without S9, 50 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin
Remarks:
For TA 98, without S9, 4 µg/plate
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:
- Plate incorporation method

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Scoring of revertant colonies using an Artek MiniCount colony counter or manually
Evaluation criteria:
See "Any other information on materials and methods incl. tables".
Statistics:
n.a.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 281 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation: See "Any other information on materials and methods incl. tables".

Ames test:
- Signs of toxicity: No signs of toxicity were observed.
- Individual plate counts: See "attached background material".
- Mean number of revertant colonies per plate and standard deviation: See "attached background material"

HISTORICAL CONTROL DATA
See "Any other information on materials and methods incl. tables".
Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

The purpose of this assay according to OECD guideline 471 was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in tetrahydrofuran and tested at concentrations ranging from 2.81 to 2810 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations of >= 281 μg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2017 until 22 February 2018
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
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes have been used successfully for a long time in in vitro experiments. It has been shown that they are an extremely sensitive indicator of in vitro induced chromosome structural changes. These changes in chromosome structure offer readily scored morphological evidence of damage to the genetic material. A proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture; they are easy to culture and thus provide a ready source of dividing cells for the scoring of chromosome aberrations. Furthermore human lymphocytes have a low spontaneous chromosome aberration frequency.

For lymphocytes:
- Sex, age and number of blood donors: 1 male, 20 years old (experiment I) and 1 male, 28 years old (experiment II)
- Whole blood used: Yes
- Blood from different donors pooled: No
- Mitogen used for lymphocytes: PHA (3 µg/mL)

MEDIA USED
- Type and composition of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL)
- Culture conditions: At 37 °C with 5.5% CO2 in humidified air
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Experiment I (with and without metabolic activation): 4.8, 8.4, 14.6, 25.6, 44.8, 78.4, 137, 240, 420, 1260 µg/mL

Experiment II (without metabolic activation): 1.3, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0, 200 µg/mL

With regard to the solubility properties of the test item, 1260 μg/mL were applied as top concentration for treatment of the cultures in experiment I. Considering the precipitation data of Experiment I, 200 μg/mL were chosen as top treatment concentration for Experiment II.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9, 825 µg/mL (experiment I), 550 µg/mL (experiment II)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9, 10 µg/mL
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (experiment I, with and without S9 mix) and 22 hours (experiment II)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: 0.2 µg/mL colcemid for 3 hours
- Methods of slide preparation and staining technique used including the stain used: Cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, and, after drying, covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.
- Number of cells analysed per concentration: 1000 cells (cytotoxicity) and 150 well-spread metaphases (clastogenicity)
- Criteria for scoring micronucleated cells: Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis.
- Criteria for scoring chromosome aberrations: Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations.
- Determination of polyploidy: If multiple copies of the haploid chromosome number (other than diploid) are scored then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is scored as endoreduplicated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (MI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Microscopic examination using 100 x oil immersion objectives.
Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. 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. 150 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
The statistical significance is confirmed by the Fisher’s exact test (modified) (p < 0.05). The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance was considered together.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.6
- Data on osmolality: 390 mOsm (solvent control) and 420 mOsm (maximum concentration of the test item)
- Precipitation and time of the determination: Precipitation of the test item was observed at the end of treatment at 14.6 μg/mL and above in the absence of S9 mix and at 25.6 μg/mL and above in the presence of S9 mix.
- Definition of acceptable cells for analysis: See "Evaluation criteria"

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Using reduced mitotic indices as an indicator for toxicity, no cytotoxic effects were observed in experiment I after 4 hours treatment in the absence and presence of S9 mix. Considering the precipitation data of Experiment I, 200 μg/mL were chosen as top treatment concentration for experiment II.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation: See "Any other information on material and methods incl. tables"

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
- Genotoxicity results: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"
Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is condsidered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item suspended in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:


 



































 



Without S9 mix



With S9 mix



 



Exp. I



Exp. II



Exp. I



Exposure period



4 hrs



22 hrs



4 hrs



Recovery



18 hrs



-



18 hrs



Preparation interval



22 hrs



22 hrs



22 hrs



In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (1260 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance OECD Guideline 473. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In the presence of S9 mix, however, two statistically significant increases in chromosomal aberrations (1.7 and 2.3 % aberrant cells, excluding gaps) were observed after treatment with 14.6 and 25.6 µg/mL. Dose dependency, tested by a trend test, was statistically significant. Since the values are clearly within the range of the laboratory control data (0.4 – 3.2 % aberrant cells, excluding gaps), these findings can be regarded as biologically irrelevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-26 until 2017-11-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016-07-29
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008-05-30
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cells, supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: High proliferation rate, good cloning efficiency of untreated cells (as a rule more than 50%), stable karyotype with a modal chromosome number of 22
- Normal cell cycle time: 12 - 16 h in stock culture

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3x10^6 cells were seeded into each flask with 15 mL of MEM containing Hank's salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% CO2 in humidified air.
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes


MEDIA USED
- Type and composition of media: Minimal essential medium (MEM) containing Hank's salts, neomycin (5 u.g/mL), 10% FBS, and amphotericin B (1%). During treatment no FBS was added to the medium. For the selection of mutant cells, the complete medium was supplemented with 11 µg/mL 6-thioguanine.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: Phenobarbital-/beta-naphtoflavone- induced rat liver S9
- Method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation.
- Concentration of S9 in the final culture medium: A final protein concentration of 0.75 mg/mL was used in the cultures.
- Quality controls of S9: . 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:
Pre-experiment: Concentrations between 9.8 µg/mL and 1260 µg/mL

Main experiment: 2.5; 5.0; 10.0; 20.0; 40.0; 80.0; 160(P) µg/mL with and without metabolic activation
The highest concentration was limited by precipitation.

(P) = Precipitation visible at the end of treatment

The highest concentration was chosen based on the precipitation determined in the range-finding pre-experiment in accordance to the OECD guideline 476.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties of the test substance in DMSO and aqueous media.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
150 and 300 µg/mL, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
1.1 and 2.3 µg/mL, with metabolic activation
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: 0.7 to 1.2x10^7

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 hours
- Exposure duration:
Pre-experiment: 4 hours with and without metabolic activation
Main experiment: 4 hours with and without metabolic activation

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 days
- Fixation time: 6-8 days (for colonies used to determine the cloning efficiency I+II)
- Selective agent: 11 µg/mL 6-thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4-5x10^5 (mutagenicity) and 500 cells (cloning efficiency)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant colonies/ 10^6 cells
Evaluation criteria:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concen¬trations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
Statistical Analysis:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.
A t-Test was not performed since the mutant frequency did not exceed the 95% confidence interval at any experimental point.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not affected (pH 7.21 in the solvent control versus pH 7.27 at 1260 µg/mL)
- Effects of osmolality: No relevant increase (381 mOsm in the solvent control versus 387 mOsm at 1260 µg/mL)
- Precipitation: at 40.0 µg/mL with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 9.8 µg/mL and 1260 µg/mL were used. The highest concentration was based on the solubility properties of the test item. No relevant toxic effects were observed after 4 hours treatment up to the highest concentration with and without metabolic activation. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 78.8 µg/mL and above with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
- Additional information on cytotoxicity: No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation.
- Genotoxicity results: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"
Conclusions:
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1260 µg/mL) was chosen with the respect to the current OECD guideline limited by the solubility of the test item. The highest concentration in the main experiment was limited by precipitation observed in the pre-experiment. No relevant and dose dependent increase in mutant colony numbers/106cells was observed in the main experiment up to the maximum concentration. No cytotoxic effects indicated by an adjusted cloning efficiency below 50% was observed neither in absence nor presence of metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in mammalian cells, key study


The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (1260 µg/mL) was chosen with the respect to the current OECD guideline limited by the solubility of the test item. The highest concentration in the main experiment was limited by precipitation observed in the pre-experiment. No relevant and dose dependent increase in mutant colony numbers/106cells was observed in the main experiment up to the maximum concentration. No cytotoxic effects indicated by an adjusted cloning efficiency below 50% was observed neither in absence nor presence of metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.


 


In vitro bacterial reverse mutation assay, key study


The purpose of this assay according to OECD guideline 471 was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in tetrahydrofuran and tested at concentrations ranging from 2.81 to 2810 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations of >= 281 μg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.


 


In vitro mammalian chromosome aberration test, key study


The test item suspended in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:


 



































 



Without S9 mix



With S9 mix



 



Exp. I



Exp. II



Exp. I



Exposure period



4 hrs



22 hrs



4 hrs



Recovery



18 hrs



-



18 hrs



Preparation interval



22 hrs



22 hrs



22 hrs



In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (1260 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance OECD Guideline 473. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation. In the absence and presence of S9 mix, no relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In the presence of S9 mix, however, two statistically significant increases in chromosomal aberrations (1.7 and 2.3 % aberrant cells, excluding gaps) were observed after treatment with 14.6 and 25.6 µg/mL. Dose dependency, tested by a trend test, was statistically significant. Since the values are clearly within the range of the laboratory control data (0.4 – 3.2 % aberrant cells, excluding gaps), these findings can be regarded as biologically irrelevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations. 


 


Conclusion


Based on the results of three genetic toxicity studies, the test item is not considered to be mutagenic or clastogenic.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008


The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.