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EC number: 946-410-1 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test material was found to be non-mutagenic in an in vitro Ames test and in vitro mouse lymphoma assay, conducted in accordance with OECD Guideline 471 and 476, respectively, and non-clastogenic in an in vitro chromosome aberration study, conducted in accordance with OECD Guideline 473.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 November 2014 to 19 December 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- OPPTS harmonised guidelines
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Not required
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Non-mammalian study
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Non-mammalian study
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1: Range-finding test: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: Main test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The substance was not misicible in water and DMSO but fully soluble in Acetone - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Concurrent - Acetone
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- 2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Concurrent - Acetone
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- 80 µg/plate for TA1537
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Concurrent - Acetone
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- 0.2 µg/plate for TA98
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Concurrent - Acetone
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- 1, 2, 10 µg/plate for TA100, TA1535&TA1537, WP2uvrA respectively
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Concurrent - Acetone
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 5 µg/plate for TA98
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).
RANGE FINDING
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.5.1.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 °Ci 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
MAIN TEST
Dose selection
The dose range used for the main test was determined by the results of the range-finding test and was 5 to 5000 µg/plate.
Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.
Without Metabolic Activation
The procedure was the same as described previously
With Metabolic Activation
The procedure was the same as described previously
Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A
NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9
NUMBER OF CELLS EVALUATED:
All strains 0.9 to 9 * 10>9
DETERMINATION OF CYTOTOXICITY
- Method: N/A
OTHER EXAMINATIONS:
N/A
OTHER:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60 - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1 . A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.
- Key result
- 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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The genetic toxicity potential of the test item was assessed in accordance with OECD Guideline 471. The test item was considered to be non-mutagenic under the conditions of this test.
- Executive summary:
Introduction
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Methods
Salmonella typhimurium strains TAl53 5, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co—factors). The dose range for the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of the range-findi ng test, and was 5 to 5000 µg/plate.
Seven test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.
Results
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix although substantial reductions in revertant colony frequency were observed to several tester strains (particularly TA1535 and TA1537) at the upper dose levels. Consequently the same maximum dose level was used in the second mutation test.
Results from the second mutation test showed the test item inducing toxicity as a weakening of the bacterial background lawns of all of the tester strains dosed in the absence of S9 at the upper test item dose levels. In the presence of S9-mix, weakened lawns were noted to TA1535 and TA100 at 5000 µg/plate. A test item precipitate (greasy/globular in appearance) was observed under an inverted microscope at 500 µg/plate and by eye from 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.
Conclusion
The test substance was considered to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 December 2014 to 26 March 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted to GLP in accordance with recognised guideline. A minor deficiency was that 200 cells per dose level were evaluated whereas the latest version of the OECD 473 requires that 300 cells be evaluated. This change in the OECD occured in the version that was adopted on 26 september 2014. This minor deviation is considered not to affect the validity of the study or the conlusion of the study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Principles of method if other than guideline:
- N/A
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- N/A
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment 1
0, 45, 90, 180, 360, 540, 720 µg/ml without S9
0, 45, 90, 180, 360, 540, 720 µg/ml with S9
Experiment 2
0, 22.5, 45, 90, 180, 360, 540 µg/ml without S9
0, 45, 90, 180, 360, 450, 540 µg/ml with S9 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 50 mg/ml but was soluble in DMSO at 500 mg/ml. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- 0.4 ug/ml in Expt. 1, 0.2 ug/ml in Expt. 2
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- 5 ug/ml in both experiments
- Details on test system and experimental conditions:
- - Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA
METHOD OF APPLICATION: in medium
With Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.
Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours.
DURATION
- Preincubation period: 48
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 or 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed
SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 ug/ml
STAIN (for cytogenetic assays): 5% Gurrs Giemsa
NUMBER OF REPLICATIONS: Treatments performed in duplicate.
NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Where possible the first 100 consecutive well-spread metaphases from each culture were counted; i.e. 200 cells per dose level.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated - Evaluation criteria:
- A test iten can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated groups is within the range of the historical control data.
2. No toxicologically or statistically significant increase in the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced chromosome aberrations in all evaluated groups is not within the range of historical control data.
and
2. Either a concentration-related or statistically significant increase in the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
- Key result
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml.
The maximum dose was based on the maximum recommended dose level. Precipitate was observed at and above 156.25 µg/ml in the exposure groups without S9-mix, and at and above 625 µg/ml in the presence of S9-mix. Haemolysis was observed following exposure to the test item between 625 and 2500 µg/ml. Haemolysis is an indication of toxicity top erythrocytes but is not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 312.5 ug/mL in both of the 4(20)-hour exposure groups. The selection of the maximum dose level was based on toxicity.
Experiment 1.
A precipitate was noted at 90 to 360 ug/ml in the absence of S9 and at 180 and 360 ug/ml in the presence of S9. Haemolysis was observed in the absence of S9 at and above 360 ug/ml, and at and above 540 ug/ml in the presence of S9. There was a moderate decrease in mitotic index in the 4(20)-hour exposure group dosed in the absence of S9-mix, 27 and 51% mitotic inhibition at 180 and 360 ug/ml respectively. In the presence of S9 the was no dose-related inhibition of mitotic index but there was complete mitotic inhibition above 360 ug/ml. Therefore, the maximum dose level selected for metaphase analysis was based on toxicity.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy.
Experiment 2.
A precipitate was noted at and above 360 ug/ml in both the absence and presence of S9. Haemolysis was observed at 540 ug/ml. There was a moderate decrease in mitotic index in the 4(20)-hour exposure group dosed in the absence of S9-mix, 11 and 31% mitotic inhibition at 45 and 90 ug/ml respectively. In the presence of S9 a plateau of toxicity was observed where 40, 33 and 48% mitotic inhibition was observed at 45, 90 and 180 ug/ml. There was complete mitotic inhibition above 180 ug/ml. Therefore, the maximum dose level selected for metaphase analysis was based on toxicity.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. There was no statistically significant increase in the frequency of polyploid cells in either exposure group. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The genetic toxicity of the test item was assessed in accordance with OECD Guideline 473. The test item did not induce a statistically significant increase in the frequency of cells with aberrations, in either the presence or absence of a liver enzyme metabolising system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
- Executive summary:
Introduction
This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.
Method
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.
Results
All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method operating as expected. The test item was toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that generally induced approximately 50% mitotic inhibition or approaching optimum toxicity levels.
Conclusion
The test item was considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 November 2014 to 20 January 2015
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media) at 37 oC with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 was prepared in-house from the livers of male Wistar Han™ rats weighing -200g. These had each received, orally, three consecutive daily doses of phenobarbitall~-naphthoflavone(80/100 mg per kg per day) prior to S9 preparation on the fourth day.
- Test concentrations with justification for top dose:
- Experiment 1 (ug/ml) without S9: 0, 20, 40, 60, 80, 100, 120, 140, 160 ug/ml
Experiment 1 (ug/ml) with S9: 0, 9.75, 19.5, 39, 78, 156, 208, 260, 312 ug/ml
Experiment 2 (ug/ml) without S9: 0, 18.75, 37.5, 75, 100, 125, 150, 175, 200 ug/ml
Experiment 2 (ug/ml) with S9: 0, 9.38, 18.75, 37.5, 75, 100, 150, 175, 200 ug/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble at 500 mg/ml in DMSO. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- 400 ug/ml in Expt. 1, 150 ug/ml in Expt. 2
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- 2 ug/ml in both experiments
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media). Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimes were as follows:
DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 both with and without S9, and Experiment 2 with S9), or 24 h (without S9 Experiment 2).
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10~14 days (plate scoring for colony formation)
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): MTT vital stain for viable cells
NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: seeded 2000 cells/well for mutant frequency; 2 cells/well for viability.
DETERMINATION OF CYTOTOXICITY
- Method: other: Relative Suspension Growth values (RSG)
OTHER: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value. The experimental mutation frequency data were analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS. - Evaluation criteria:
- For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10'6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the
corresponding vehicle control by the GEF of 126 x 10 " and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered tobe toxicologically significant. Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion. - Statistics:
- The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS statistical package. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
RANGE-FINDING/SCREENING STUDIES:
The dose range of the test item used in the preliminary toxicity test was 19.53 to 5000 ug/mL . In the 4-hour exposures,both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. The toxicity curve in all three exposure groups was quite steep. A precipitate of the test item was observed at and above 156.25 ug/mL in the absence of metabolic activation and at and above 312.5 ug/mL in the presence of metabolic activation. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity. - Remarks on result:
- other: strain/cell type: mouse lymphoma L5178Y cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- The genetic toxicity potential of the test item was assessed in accordance with OECD Guideline 476. The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
- Executive summary:
Introduction The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.
Methods Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.
Results The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. A precipitate of test item was observed (pre-washing) at and above 260 ug/mL in the presence of metabolic activation in Experiment 1 and at 200 ug/mL in the absence of metabolic activation in Experiment 2. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.
Conclusion The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
Referenceopen allclose all
The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible eduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix although substantial reductions in revertant colony frequency were observed to several tester strains (particularly TA1535 and TA1537) at the upper dose levels. Consequently the same maximum dose level was used in the second mutation test.
Results from the second mutation test showed the test item inducing toxicity as a weakening of the bacterial background lawns of all of the tester strains dosed in the absence of S9 at the upper test item dose levels. In the presence of S9-mix, weakened lawns were noted to TAl535 and TA100 at 5000 µg/plate. A test item precipitate (greasy/globular in appearance) was observed under an inverted microscope at 500 µg/plate and by eye from 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
See attached background material.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Bacterial Mutation Assay
Introduction
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.
Methods
Salmonella typhimurium strains TAl53 5, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co—factors). The dose range for the range-finding test was pre-determined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of the range-finding test, and was 5 to 5000 µg/plate.
Seven test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.
Results
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix although substantial reductions in revertant colony frequency were observed to several tester strains (particularly TA1535 and TA1537) at the upper dose levels. Consequently the same maximum dose level was used in the second mutation test.
Results from the second mutation test showed the test item inducing toxicity as a weakening of the bacterial background lawns of all of the tester strains dosed in the absence of S9 at the upper test item dose levels. In the presence of S9-mix, weakened lawns were noted to TA1535 and TA100 at 5000 µg/plate. A test item precipitate (greasy/globular in appearance) was observed under an inverted microscope at 500 µg/plate and by eye from 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.
Conclusion
The test substance was considered to be non-mutagenic under the conditions of this test.
Mammalian Cell Gene Mutation Assay
Introduction
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.
Methods
Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test.ResultsThe maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. A precipitate of test item was observed (pre-washing) at and above 260 ug/mL in the presence of metabolic activation in Experiment 1 and at 200 ug/mL in the absence of metabolic activation in Experiment 2. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.ConclusionThe test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
Mammalian Cell Chromosome Aberration Test
Introduction
This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.
Method
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.
Results
All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method operating as expected. The test item was toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that generally induced approximately 50% mitotic inhibition or approaching optimum toxicity levels.
Conclusion
The test item was considered to be non-clastogenic to human lymphocytes in vitro.
Justification for selection of genetic toxicity endpoint
No mutagenic or genotoxic positive results.
Short description of key information:
A K1 bacterial gene mutation assay (Ames test) performed under GLP
to the OECD 471 test method.
A K1 mammalian cell gene mutation assay (MLA assay) performed under GLP
to the OECD 476 test method.
A K1 mammalian cell chromosome aberration test performed under GLP to
the OECD 473 test method.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
In accordance with the CLP Regulation, no 1272/2008, a substance should be classified as mutagenic if there is evidence that it may induce heritable mutations in the germ cells of humans. The test material was found to be non-mutagenic in an in vitro Ames test and in vitro mouse lymphoma assay and non-clastogenic in an in vitro chromosome aberration study. As such, the test material does not meet the criteria for classification.
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