Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Ä uvrB); ampicillin resistance (R factor plasmid). Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
E. coli WP2 uvrA is checked for UV sensitivity. Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/β-naphthoflavone induced rat livers
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2750 and 5500 μg/plate
Vehicle / solvent:
DMSO. Which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
No metabolic activation, Strains: TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
No metabolic activation, Strains: TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
No metabolic activation, Strains: TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
No metabolic activation, Strains: E. coli 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 and for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Standard plate test
- Exposure duration: 48 - 72 hours at 37°C in the dark
Preincubation test
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 - 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, background lawn, determination of titer
Evaluation criteria:
ACCEPTANCE CRITERIA:
Generally, the experiment is considered valid if the following criteria are met: The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain; The sterility controls revealed no indication of bacterial contamination; The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above; Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

ASSESSMENT CRITERIA:
The test substance is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if: The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
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:
depending on the strain and test conditions at 2750 μg/plate only without S9 mix (slight decrease in the number of his+ revertants, slight reduction in the titer)
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
No bacteriotoxic effect (reduced his- or trp- background growth, slight decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed in the standard plate test up to the highest required concentration. In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions at 2750 μg/plate only without S9 mix.

SOLUBILITY
No test substance precipitation was found with and without S9 mix.

DISCUSSION
According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
other: OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test".
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cells (obtained from LMP; Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
See "any other information on materials and methods"
Vehicle / solvent:
The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Griseofulvin
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
DOSE-SELECTION:
With respect to the solubility of the test item, 4000.0 µg/mL of Soybean oil, epoxidized, reaction products with methanol was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 7.8 and 4000.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. No precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with the same top concentration due to the low response of the positive control MMC and designated Experiment IB. Since no clear cytotoxicity and test item precipitation was observed in Experiment IA and IB, the same concentrations were applied in Experiment II.

EXPERIMENTAL PERFORMANCE:
- Culture Medium and Conditions: For seeding and treatment of the cell cultures the culture medium was MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL). All cultures were incubated at 37°C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Seeding of the Cultures: Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37°C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1.0 x 10^5 – 1.5 x 10^5 cells were seeded with regard to the preparation time.

TREATMENT:
- Exposure period 4 hours: The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium was added. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. Then the cells were cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
- Exposure period 24 hours: The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.

PREPARATION OF THE CULTURES:
For the micronucleus analysis, 24 hours after the start of the exposure, the cells were treated on the slides in the chambers of the quadriperm dishes with deionised water for 1 to 1.5 min at 37°C. Afterwards the cells were fixed twice with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25 % (v/v) formaldehyde. After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.

ANALYSIS OF MICRONUCLEI AND CYTOTOXICITY:
Evaluation of the cultures was performed manually using NIKON microscopes with 40x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). Briefly the micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 1000 cells in two parallel cultures were scored for micronuclei, so that at least 2000 cells from clones with 2-8 cells were analysed per test group. The frequency of micronucleated cells was reported as % micronucleated cells. Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3-4 cells (c4), and 5-8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated. Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for a sufficient cell proliferation during treatment and recovery.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronucleated cells carrying one or more micronuclei found in the solvent controls falls within the range of the historical laboratory control data range.
b) The positive control substances produce a significant increase (at least two-fold the respective control value) in the number of micronucleated cells exceeding the historical laboratory solvent control data range.

EVALUATION OF RESULTS:
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical control data and
- either a statistically significant concentration-related increase in three test groups or a significant increase of micronucleated cells in at least one test group is observed.
A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated test groups is in the range of the historical control data and
- no statistically significant concentration-related increase in the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment IA, IB and II in the presence of S9 mix at 125.0 µL/mL and above. In Experiment II in the absence of S9 mix phase separation was observed at 250.0 µg/mL and above. No relevant influence on pH value was observed. The osmolarity was slightly increased at the highest applied concentration in Experiment II. In the absence and presence of metabolic activation no clear cytotoxicity measured as reduced proliferation index was observed. No mutagenicity was observed at the concentrations evaluated. In Experiment IA and IB in the absence and presence of S9 mix two statistically significant increases were observed after treatment with 1000 µg/mL (1.20 and 1.30 %). The values are in the range of the laboratory historical control data (0.15-1.50 % and 0.05-1.70 % micronucleated cells, respectively) and therefore biologically irrelevant. In Experiment II in the presence of S9 mix two statistically significant increases were observed after treatment with 62.5 and 2000.0 µg/mL (1.55 and 1.45 %). The values are in the range of the laboratory historical control data (0.05 - 1.70 % micronucleated cells) and therefore biologically irrelevant. In the presence of S9 mix one single value (1.60 %) exceeded the range of the laboratory historical control data (0.05-1.50 % micronucleated cells) after treatment with 1000.0 µg/mL. The value is not statistically significant and therefore biologically irrelevant. Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium as described in. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
See "any other information on materials and methods"
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO. The final concentration of DMSO in the culture medium was 0.5% v/v.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
PRE-TEST:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

DOSE SELECTION:
The highest concentration used in the pre-test was 4000 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 31.3 µg/mL and 4000 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Based on cytotoxicity and phase separation noted in the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 3 at the low doses (until 500 µg/mL) and 2 at the higher doses in the experiments with S9. In the experiments without S9 the individual concentrations were spaced by a factor of 2. Narrower spacing at high concentrations was used in the absence of metabolic activation to cover the cytotoxic range more closely.

EXPERIMENTAL PERFORMANCE:
- Culture Medium: For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
- Seeding: Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in PBS. The PBS is composed as follows (per litre): NaCl: 8000 mg, KCl: 200 mg, KH2PO4: 200 mg, Na2HPO4: 150 mg. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/L EDTA (ethylene diamine tetraacetic acid). Approximately 1.5x10^6 (single culture) and 5x10^2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.
- Treatment: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS, in the absence of metabolic activation. The "saline G" solution had the following constituents (per litre): NaCl: 8000 mg, KCl: 400 mg, Glucose: 1100 mg, Na2HPO4.2H2O: 192 mg, KH2PO4: 150 mg. The pH was adjusted to 7.2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment. Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm^2 flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm^2 cell culture flasks were seeded with about 3 – 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm^2 flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37°C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
Acceptability of the Assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.
- The data of this study comply with the above mentioned criteria and the historical data.

Evaluation of Results:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies 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. However, both, biological relevance and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 62.5 µg/mL and above in the first experiment (4 hrs), and at 250 µg/mL in the second experiment (24h hrs)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TEST:
Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 250.0 µg/mL and above without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal of the test item. Turbidity was observed at 31.3 - 500 µg/mL after 4 hours treatment with and without metabolic activation. Phase separation was noted from 1000 µg/mL up to the maximum concentration. After 24 hours treatment without metabolic activation turbidity was observed at 62.5 µg/mL and above. There was no relevant shift of the osmolarity and pH value even at the maximum concentration of the test item.

MAIN EXPERIMENTS:
Phase separation was noted in both experiments at 166.5 µg/mL and above with and at 31.3 µg/mL and above without metabolic activation. Turbidity at the end of treatment was observed at 15.6 µg/mL without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The induction factor reached or exceeded the threshold of three times the corresponding solvent control in both cultures of the second experiment with metabolic activation at 55.6 and 1000.0 µg/mL. These effects however, are based on the rather low solvent control of 6.5 and 8.5 mutant colonies/10^6 cells. The actual values of the mutation frequency did not exceed the historical range of solvent controls. There was no dose dependent trend as indicated by the lacking statistical significance. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred without metabolic activation at 62.5 µg/mL and above in the first experiment and at 250 µg/mL in the second experiment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A GLP-compliant Ames test according to OECD guideline 471 was conducted (BASF SE, 2013). The test substance Soybean oil, epoxidized, reaction products with methanol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium (TA 1535, 1537, 100 and 98) and Escherichia coli (WP2 uvrA) in a reverse mutation assay at the following doses: 33, 100, 333, 1000, 2750 and 5500 μg/plate. A standard plate test and pre-incubation test both with and without metabolic activation (liver S9 mix from phenobarbital/ β-naphthoflavone induced rats) were performed. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2750 μg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the pre-incubation test either without S9 mix or after the addition of a metabolizing system. No precipitation of the test substance was found with and without S9 mix. Thus, under the experimental conditions of this study, the test substance Soybean oil, epoxidized, reaction products with methanol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In a GLP-compliant micronucleus test, tested according to OECD guideline 487 Chinese hamster V79 cells were exposed to the test substance, Soybean oil, epoxidized, reaction products with methanol, with and without metabolic activation (Harlan CCR, 2013). Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with metabolic activation. In Experiment IB the exposure period was 4 hours without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. Doses up to 4000 µg/mL were chosen. No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment IA, IB and II in the presence of S9 mix at 125.0 µL/mL and above. In Experiment II in the absence of S9 mix phase separation was observed at 250.0 µg/mL and above. No relevant influence on pH value was observed. The osmolarity was slightly increased at the highest applied concentration in Experiment II. In the absence and presence of metabolic activation no clear cytotoxicity measured as reduced proliferation index was observed. No mutagenicity was observed at the concentrations evaluated. In Experiment IA and IB in the absence and presence of S9 mix two statistically significant increases were observed after treatment with 1000 µg/mL (1.20 and 1.30 %). The values are in the range of the laboratory historical control data (0.15-1.50 % and 0.05-1.70 % micronucleated cells, respectively) and therefore biologically irrelevant. In Experiment II in the presence of S9 mix two statistically significant increases were observed after treatment with 62.5 and 2000.0 µg/mL (1.55 and 1.45 %). The values are in the range of the laboratory historical control data (0.05-1.70 % micronucleated cells) and therefore biologically irrelevant. In the presence of S9 mix one single value (1.60 %) exceeded the range of the laboratory historical control data (0.05-1.50 % micronucleated cells) after treatment with 1000.0 µg/mL. The value is not statistically significant and therefore biologically irrelevant. Mitomycin C (0.1 µg/mL), Griseofulvin (8.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells. In conclusion, it can be stated that under the experimental conditions reported, the test item Soybean oil, epoxidized, reaction products with methanol did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

 

In a GLP-compliant gene mutation assay tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance, Soybean oil, epoxidized, reaction products with methanol, with and without metabolic activation (Harlan CCR, 2013). The highest concentration used in the range finding pre-experiment was 4000 µg/mL of the test item based on the solubility properties of the test item. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects observed in the preliminary test. The test item was dissolved in DMSO. The following doses were tested in the main experiments; Experiment I and II without S9: 15.6, 31.3, 62.5, 125.0, 250.0 and 375.0 µg/mL and in experiment I and II with S9 these doses were tested: 18.5, 55.6, 166.5, 500.0, 1000.0 and 2000.0 µg/mL. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred without metabolic activation at 62.5 µg/mL and above in the first experiment and at 250 µg/mL in the second experiment. No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. 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, Soybean oil, epoxidized, reaction products with methanol is considered to be non-mutagenic in this HPRT assay.


Short description of key information:
The test substance is considered not to be mutagenic based on the results of the in the in vitro micronucleus test, the HPRT locus in V79 and the Ames test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity tests,the test substancedoes not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.