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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In vitro Ames test (TA1535, TA1537, TA102, TA98, TA 100 and WP2uverA-): Non-mutagenic Mouse lymphoma L5178Y TK+/- Gene Mutation Assay: Non-mutagenic Chromosome aberration study in human lymphocytes: Non-clastogenic Chromosome aberration study in CHO cells: Non-clastogenic In Vivo Mouse micronucleus study: negative
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
Study period:
21/08/1996 to 04/11/1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
Toxicity pre-screen 50, 167, 500, 1670, 5000 ug/plateInitial study for Salmonella and E.coli 16.7, 50, 167, 500, 1670, 5000 ug/plateConfirmatory study for Salmonella and E. coli 16.7, 50, 167, 500, 1670, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: none given
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate-incorporation and preincubationDURATION- Preincubation period: 30 minutes at 37 C- Exposure duration: 48 hoursSELECTION AGENT (mutation assays): absence of histidine or tryptophan in agarNUMBER OF REPLICATIONS: triplicate platesDETERMINATION OF CYTOTOXICITY- Method: growth of background lawn of non-revertant bacteria and size and number of revertant colonies
Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. If the test article does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine- or tryptophan-independent revertants.
Statistics:
Statistical analyses were performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon normal, spontaneous variation observed among replicate negative control cultures, as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test articles judged to be negative in this assay.
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA100, TA102, WP2uvrA-
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:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: precipitation was observed at 1670 ug/plate and above- Other confounding effects:RANGE-FINDING/SCREENING STUDIES: A toxicity screening test using both the plate-incorporation and pre-incubation methods, was perforem in TA1537, TA100 and WP2uvrA-, in the absence of S9 only.COMPARISON WITH HISTORICAL CONTROL DATA: All control values were comparable to historical ranges
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The results for OS#114461 were negative in the Ames/Salmonella-E.coli Liquid reverse mutation assay under the conditions, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.
Executive summary:

Test Guidance

OECD Guideline No. 471

Method

The test material was evaluated in the Ames/Salmonella-E.coli bacterial reverse mutation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA102, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2uvrA-), in the presence and absence of an exogenous metabolic activation system (S9).

Results

Toxicity of the test material was first evaluated in a prescreen by treating duplicate cultures of strains TA1537, TA100 and WP2uvrA- with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9, using both the pre-incubation and plate-incorporation methods. Results of the prescreen indicated the test material was toxic to all three tester strains at 5000 ug/plate in the pre-cubation treatment conditions only. In addition, the test article was found to be incompletely soluble at doses => 500 ug/plate.

Based upon these findings, the test material was evaluated in triplicate cultures in all five strains of Salmonella strain WP2uvrA- at dose of 16.7, 50, 167, 500, 1670 and 5000 ug/plate, in the presence and absence of S9, using the plate-incorporation method. Six dose levels of the test material were evaluated with and without S9 in the event of unacceptable insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors.The test article again precipitated from solution =>167 ug/plate. No toxicity was again observed in any of the tester strains, with or without S9. Revertant frequencies for all doses of the test material in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative control cultures except for strain TA102 in the absence of S9. In this case a statistically significant increase in revertant frequencies, to approximately 1.1- to 1.5 -fold control values, were observed at doses of 50 to 1670 ug/plate. However, these increases were not linear.

The test material was re-evaluated in the confirmatory assay using the same dose levels under pre-incubation treatment conditions and also re-evaluated in TA102 without S9 under plate incorporation conditions. The test article again precipitated from solution at doses =>167 ug/plate. Inhibited growth again was observed in all tester strains at 5000 ug/plate without S9 Revertant frequencies for all dose of the test material in all tester strains with and without S9 approximated or were less than control value. All positive and negative control values in both assays were within acceptable limits.

Conclusion

Therefore, the results for the test material were negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
18 September 1996 to 05 June 1997
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Technical deficiencies that included: no historical data included in the report, mitotic index data missing from evaluated dose levels, absence of dose response relationship for toxicity, some cells with multiple aberrations of chromatid deletion type which are typical of toxicity-related false positive responses, weak effects in first experiement were not reproduced inthe confirmatory experiment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: Human, whole blood cultures
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9
Test concentrations with justification for top dose:
0.167, 0.5, 1.67, 5.0, 13.7, 50, 167, 500, 1670, 5000 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test material was not soluble in water but was soluble in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.25 ug/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
40 ug/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 48 hours- Exposure duration: 5 or =>24 hours- Fixation time (start of exposure up to fixation or harvest of cells): >=24 hoursSPINDLE INHIBITOR (cytogenetic assays): ColcemidSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 100 per duplicateDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cell-cycle kineticsOTHER EXAMINATIONS:- Determination of polyploidy: Yes- Determination of endoreplication: Yes-Osmolarity: Yes-pH: Yes
Evaluation criteria:
Criteria for a valid assay: The results of the assay are valid if: 1) the number of aberrant metaphasesin the solvent controls is <= 9 aberrant metaphases; and 2) both positive controls induce statistically significant increases (p<0.05) in the frequency of aberrations/cell and the proportion of aberrant metaphases (as compared to the concurrent negative controls).Interpretation of results: The test article is considered to have produced a positive result if: 1) it induces a statistically significant, dose-dependent increase in the frequency of aberrations/cell or in the proportion of aberrant metaphases; or 2) it induces a reproducible, statistically significant increase in either endpoint for at least one test article concentration at the same treatment/sampling interval. If the test article does not meet either criterion above, it will be considered negative (non-clastogenic) in this assay.The above criteria will be used as a guide inevaluating the test results. However, the Study Director may take other factors into consideration in evaluating the test results, since biological and statistical significance should be considered in evaluation of assay results.
Statistics:
The five different treatment groups (Schedules I-V) were analysed separately, and gaps were excluded from the statistical analysis. Chi-square analysis was performed to evaluate the proportion of aberrant metaphases, while a one-way analysis of variance was performed to evaluate the frequency of aberrations/cell. Data from the replicate cultures were pooled, where appropriate, prior to analysis, and all comparisons were made relative to the appropriate solvent control. The Dunnett's test subsequently was used to determine if any differences from the controls were significant. In addition, the Cochran-Armitage test fro trend analysis was used to assess any possible dose dependency. Statistical significance for all analyses was established at the p<0.05 level.
Species / strain:
lymphocytes: Human, whole blood cultures
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: None- Effects of osmolality: None- Precipitation: No dataRANGE-FINDING/SCREENING STUDIES: A screening study was performed that included an evaluation of cell-cycle kinetics.COMPARISON WITH HISTORICAL CONTROL DATA: No historical control data reported.ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The conclusion of the study was negative (non-clastogenic). However, there were technical flaws in the study related to missing data and poor reproducibility that make the study result unreliable.
Executive summary:

Study disregarded because of technical flaws, discrepancies and poor reproducibility of toxicity and aberration frequencies.

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:
2012-03-21 to 2012-08-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)
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:
Cell Growth Inhibition Test0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000Experiment 1 4(20) hour without S9 15*, 30*, 60*, 120, 240, 360 µg/ml4(20) hour with S9 15, 30, 60, 90*, 120*, 180* µg/ml* = Dose levels selected for metaphase analysisExperiment 224 hour without S9 7.5, 15, 30, 60*, 90*, 120* µg/ml4(20) hour with S9 30, 60, 90*, 120*, 180*, 240 µg/ml* = Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test substance was insoluble in water but miscible in DMSO at 400 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
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: NDAMETHOD OF APPLICATION: in mediumWith Metabolic ActivtionCultures 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 ActivationCultures 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 hoursDURATION- Preincubation period: 48 hours- Exposure duration: 4 or 24 hours- Expression time (cells in growth medium): 20 or 0 hours - Selection time (if incubation with a selection agent): N/A- 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 fixedSELECTION AGENT (mutation assays): N/ASPINDLE INHIBITOR (cytogenetic assays): demecolcineSTAIN (for cytogenetic assays): 5% Gurrs GiemsaNUMBER 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.DETERMINATION OF CYTOTOXICITY - Method: mitotic indexOTHER 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 positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
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 TestThe dose range for the Preliminary Toxicity Test was 0, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000, 4000 µg/ml. The maximum dose was based on the maximum recommended dose level equivalent to 10 mM. Precipitate was observed at and above 125µg/ml in the 4-hour exposure group in the presence and absence of metabolic activation and at 250µg/ml in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 125 ug/ml in the 4(20)-hour exposure group in the presence of S9 and up to 250 ug/ml in the exposure groups in the absence of S9.The selection of the maximum dose level was based on toxicity.Chromosome Aberration Test - Experiment 14(20) hour without S9 15*, 30*, 60*, 120, 240, 360 µg/ml4(20) hour with S9 15, 30, 60, 90*, 120*, 180* µg/ml* = Dose levels selected for metaphase analysisChromosome Aberration Test - Experiment 224 hour without S9 7.5, 15, 30, 60*, 90*, 120* µg/ml4(20) hour with S9 30, 60, 90*, 120*, 180*, 240 µg/ml* = Dose levels selected for metaphase analysisAll of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials 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 itself was operating as expected.The test item, OS43721AC did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either of two experiments, using a dose range that generally included a dose level that induced or exceeded approximately 50% mitotic inhibition. The test item was therefore concluded to be non-clastogenic to human lymphocytes in vitro.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached background material.

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either of two experiments, using a dose range that generally included a dose level that induced or exceeded approximately 50% mitotic inhibition. The test item was therefore concluded to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Test Guidance

OECD Guidelines for Testing of Chemicals No. 473 " In Vitro Mammalian Chromosome Aberration Test"

Method BIO of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

US EPA OPPTS 870.5375 Guideline.

40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.

Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

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 hours 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 and were in the range of 15 to 360 ug/ml for all exposure groups.

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 indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was non-toxic and 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 was the lowest precipitating dose level.

Conclusion

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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:
26 May 2015 to 21 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The thymidine kinase, TK +/-, 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, 5, 10, 20, 40, 60, 80, 120, 160 ug/mlExperiment 1 (ug/ml) with S9: 0, 10, 20, 40, 60, 80, 100, 120, 160 ug/mlExperiment 2 (ug/ml) without S9: 0, 2.5, 5, 10, 20, 40, 60, 80, 100 ug/mlExperiment 2 (ug/ml) with S9: 0, 20, 40, 80, 90, 100, 110, 120, 130 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test item was insufficiently soluble in water.
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:
1.5 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 cellsNUMBER OF REPLICATIONS: DuplicateNUMBER 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 10E6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than thecorresponding vehicle control by the GEF of 126 x 10E6 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 to be 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: NoneRANGE-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 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. A precipitate of the test item was observed at and above 312.5 ug/mL in all of the exposure group. 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 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 level used in the main test was limited by test item-induced toxicity. 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. A small but statistically significant linear trend increase in mutant frequency was observed in the 4-hour with S9 exposure group of Experiment 2. However, the mutant frequency values were all within the accetable range for a vehicle control and the GEF was not exceeded at any dose level. The reponse observwed was considered to be artefactual and of no toxicological significance. Conclusion The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20 February 2012 to 21 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Japanese Joint Notification Yakusyoku 0331 No.7 and the Notification of Minitry of Labour, No 77 September 1, 1988 and No 67 (revised), June 2, 1997.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine or tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone induced S9
Test concentrations with justification for top dose:
Initial test: 4.88, 19.5, 78.1, 313, 1250 and 5000 ug/plateMutagenicity test: 156, 313, 625, 1250, 2500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO- Justification for choice of solvent/vehicle: The test substance was not soluble in water at 50 mg/ml but was soluble in DMSO at 50 mg/ml.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation, 20 minutes at 37CDURATION- Exposure duration: 48 hours- Expression time (cells in growth medium): 48 hoursNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 1.49 to 7.44 x 10E8 per plateDETERMINATION OF CYTOTOXICITY- Method: growth of background lawn of bacteria and reduction in number of revertant colonies
Evaluation criteria:
The test material should have induced a reproducible and dose-related increase in the revertant count in at least one strain of bacteria.
Statistics:
Not applicable
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
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, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: A precipitate was observed at 5000 ug/plate without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bactyerial strains at any dose level, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Test Guidance

Japanese Joint Notification Yakusyoku 0331 No.7 and the Notification of Minitry of Labour, No 77 September 1, 1988 and No 67 (revised), June 2, 1997.

Method

This study was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, 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 dose determination test was 4.88 to 5000 ug/plate. The experiment was repeated on a separate day using the dose range, 156 to 5000 ug/plate, fresh cultures of bacterial strains and fresh test material formulations.

Results

Cytotoxicity to bacteria by the test material was not observed for TA98, TA100, TA1535, TA1537 and WP2uvrA with or without metabolic activation. Precipitate of the test material was observed at 5000 ug/plate for TA98, TA100, TA1535, TA1537 and WP2uvrA without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Conclusion

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 September 1987 to 19 November 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
High and variable frequency in the number of cells with aberrations in the solvent control group. The number of cells with aberrations in the vehicle control cultures occasionally exceeded the maximum number spcified in the protocol. No conclusion given by the Study Director based on the study results even though the results met the criteria specified in the protocol for a negative result.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Atypical cell harvest time-points
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: no
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50.1, 150, 501, 1503, 5009 ug/ml for the 19-hour non-activated exposure group of the range-fonder experiment0.5, 1.0, 1.5, 3.0, 5.0, 10.1, 15.1, 30.2, 50.3, 101, 151, 302 ug/ml for the 10-hour non-activated exposure group0.5, 1.0, 1.5, 3.0, 5.0, 10, 15, 30, 50, 150 ug/ml for the 20-hour non-activated exposure group0.15, 1.5, 5.1, 15.2, 50.7, 152 ug/ml for the 10-hour with-activation exposure group0.15, 0.5, 1.5, 5.0, 15, 50.1, 150.3, 501, 5010 ug/ml for the 20-hour with-activation exposure group
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;- Justification for choice of solvent/vehicle: The solubility of the test material was shown to be 506 mg/ml in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.3 and 5.0 ug/ml without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
50 ug/ml with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: 24 hours- Exposure duration: 2, 10 or 20 hours- Fixation time (start of exposure up to fixation or harvest of cells): 10 or 20 hoursSPINDLE INHIBITOR (cytogenetic assays): ColcemidSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: DuplicateNUMBER OF CELLS EVALUATED: 100 per duplicateDETERMINATION OF CYTOTOXICITY- Method: mitotic indexOTHER EXAMINATIONS:- Determination of polyploidy: Not determined- Determination of endoreplication: Not determined- Other:
Evaluation criteria:
A negative control frequency of 1-2% cells with chromosomal aberrations is expected (excluding gaps). A test sample is considered negative if less than 9% aberrant cells are observed. To verify a positive response data from the three highest concentrations will be analysed statistically according to the methods outlined in a paper by Margolin et al., Environ. Mutagen. 8:, 1986.
Statistics:
The data were analysed statistically using the method described in Margolin et al (19860, "Statristical Analysis in In Vitro Cytogenetic Assays Using Chinese Hamster Ovary Cells." Environ. Mutagen 8: 183-204.
Species / strain:
Chinese hamster Ovary (CHO)
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:
The test material was tested in two nonactivated assays of 10 and 20 hours. In the 10-hour assay, toxicity was seen at 151 µg/mL since no metaphase cells were found. A reduced mitotic index was observed at 101 µg/mL and these metaphase cells were not suitable for analysis. The cells treatedd with three concentrations spanning a log range from 5.0 to 50.3 µg/mL were analysed. The percentages of aberrant cells excluding gaps in the solvent controls were 4% and 6%. The percentages of aberrant cells were not significantly increased in the cells treated with any of the test sample concentrations. Analysis of the data by including gaps as aberrations did not change the outcome. The background levels of the solvent controls increased to 6% and 7% in the two replicates, but there was no significant increase due to the test chemical.In the 20-hour non-activated assay, the chemical was toxic at 150 µg/mL since no metaphase cells were found. A reduced mitotic index was observed at 50 µg/mL and these metaphase cells were not suitable for analysis. A total of three concentrations spanning a log range between 3.0 and 30.0 µg/mL were analysed. The percentages of aberrant cells excluding gaps in the solvent controls were 1% and 3%. The percentage of aberrant cells was statistically significantly increased in one of the flasks treated with 3 µg/mL (11% aberrant cells), but the replicate flask only had 4% aberrant cells. Although there was no concentration dependent increase, the average of the two values at 3 µg/mL, 7.5%, is statistically significant compared to the negative control. When gaps are added into the calculation, the background levels are 2% and 4% aberrant cells. At 3.0 µg/mL a slight increase in the number of aberrant cells was also observed over these background values.The test material was also tested in two activated assays using S9. The substance was toxic at the highest concentration tested, 152 µg/mL, in the 10-hour assay. The percentages of aberrant cells excluding gaps for the solvent control in the 10-hour assay were 5% and 6%, and there was no statistically significant increase in aberrant cells due to the test substance. Analysis of the data by including aberrations with gaps did not change the result. The background levels were increased to 10% and 13% aberrant cells, and there was no signifcant increase in the cells exposed to the substance.The test substance was also toxic at 150.3 µg/mL to the cells treated in the 20-hour activated assay. The background percentages of aberrant cells excluding gaps for solvent control were 2% and 5%. The cells exposed to the test sample concentrations had similar levels of aberrant cells compared to the negative control values. Analysis of the data by including gaps did change the result. The background levels were 3% and 5% while a statistically significant increase in the percentage of aberrant cells was observed at 150.3µg/mL (14% and 15%).The positive controls of cyclophosphamide, benzo(a)pyrene and mitomycin C were shown to be positive, demonstrating the efficacy of the test system and for S9 in the experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
None of the mean frequencies of cells with aberrations exceeded the criteria for a positive response given in the study protocol. It is considered that the result of the study is negative (non-clastogenic).
Executive summary:

Test Guidance

OECD Guideline No 473

Method

The test material was investigated for its ability to induce chromosomal aberrations in Chinese Hamster ovary (CHO) cells in the presence and the absence of a rat liver homogenate metabolic activation system. Two assay periods, 10 hours and 20 hours, were used which totalled four independent assays. The test concentrations that were used for the study ranged from 0.5 to 302 ug/mL in the non-activated assays and 0.15 to 5010 ug/mL in the activated assays. The data were analysed using two methods. The first used the traditional method inwhich the gaps were not counted as chromosomal aberations while the second counted the gaps as chromosomal aberrations.

Results

In the activated assays, the compound was toxic in the 10 -hour assay at the highest concentration, 152 ug/mL, and in the 20 -hour assay at 150 ug/mL. In the non-activated assays, toxicity was observed at the concentrations above 101 ug/mL in the 10 -hour assay and above 50 ug/mL in the 20 -hour assay. A statistically significant increase in the percent of cells with aberrations (excluding gaps) was found in the 20 -hour non-activated assay at one concentration. In the 20 -hour activated assay, the increase in the percent of cells with chromosomal breaks or aberrations was not statistically significant unless gaps were included in the calculation. The response obtained from the negative and the positive controls demonstrated that the test system was capable of detecting chemicals which caused chromosomal damage.

Conclusion

The results indicate that the test material caused a slight increase in chromosomal aberrations in CHO cells in the 20-hour activated and non-activated assays. The reproducibility of this slight increase was not determined. The small increase seen in the absence of activation was not dose-related (it occured at the low dose level), and the small increase in the with-activation group was only significant when gaps were included and with the exclusion of gaps only one of the duplicate cultures showed an increase. According to the criteria of the protocol the response in both cases is negative.

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

Genetic toxicity in vivo

Description of key information

Test Guidance

OECD Guideline No 474

Method

This study was designed to evaluate the potential of the test material to induce micronuclei in the newly formed polychromatic erythrocytes (PCEs) in mouse bone marrow.

A preliminary toxicity screen was conducted to select the appropriate doses for the definitive micronucleus test (MNT). Eight groups of mice (two/sex/dose level) were administered a single intraperitoneal (ip) injection of the test or negative control articles at a dose volume of 10 mL/kg. The test material was evaluated at doses of 500, 750, 1500, 1750, 2250, 3000 and 5000 mg/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and at 24, 48 and 72 hours, after dosing. Except for one female treated at a dose of 5000 mg/kg, all females treated at dose =>1750 mg/kg, and all males treated at doses =>3000 mg/kg, died within 48 hours after treatment. All surviving mice were sacrificed at the last observation time by cervical dislocation. Bone marrow was harvested from these animals and scored for the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) per 1000 total erythrocytes, as an index of toxicity

Results

The test material did not produce any statistically significant depressions in PCE/NCE ratios at any dose level, as compared to the concurrent negative control. In the definitive MNT, nine groups of mice (five/sex/dose) were administered a single ip injection of the test material at doses of 250, 1250 and 2500 mg/kg (males), or at dose of 150, 750 and 1500 mg/kg (females), for sacrifice at 24, 48 or 72 hours after injection. The negative control, corn oil, was administered concurrently to three groups of mice (five/sex) for sacrifice at 24, 48 or 72 hours, while the positive control, cyclophosphamide (CP; 60 mg/kg), was administered concurrently to a single group of mice (five/sex) for sacrifice 24 hours after treatment. All test and control articles were administered in a volume of 10 mL/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and approximately 24, 48 or 72 hours after dosing. Bone marrow slides were prepared, stained, coded and scored for the number of micronucleated PCEs (MPCEs) in 2000 PCEs/mouse, as well as for the number of micronucleated normochromatic erythrocytes (MNCEs) present in the optical fields containing 2000 PCEs. The PCE/NCE ratio per 1000 erythrocytes also was determined for each mouse as an index of toxicity. MPCE frequencies for all negative control groups were within acceptable ranges, and the CP positive control induced statistically significant increase in MPCE frequency (combined- and by-sex; p<0.01). Analysis of the by- and combined-sex indicated that the test material did not induce any statistically significant or dose-dependent increases in MPCE frequencies, at any harvest time evaluated, as compared to the compared to the concurrent negative controls. In addition, all observed MPCE frequencies in the animals treated with the test material were within historical ranges. Analysis of the PCE/NCE ratios revealed that the test material induced statistically significant depressions in female mice treated at 750 and 1500 mg/kg and harvested at 24 hours (p<0.05), as well as in male mice treated at 2500 mg/kg and harvested at 48 hours (p<0.05). A statistically significant depression in PCE/NCE ratios was also observed in the high dose groups, at the 48 -hour harvest time, when combining sexes (p<0.05; females treated at 1500 mg/kg, males treated at 2500 mg/kg).

Conclusion

The test substance is considered to be negative in the In Vivo Micronucleus Test in Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 1996 to 03 March 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories- Weight at study initiation: males 29-38g and females 20-29g for the preliminary test, males 26-34g and females 20-26g for the main test.- Assigned to test groups randomly: Yes- Fasting period before study: No- Housing: Stainless steel wire mesh caging- Diet (e.g. ad libitum): PMI Certified Rodent Diet ad libitum- Water (e.g. ad libitum): Tap water ad libitum- Acclimation period: 5 to 12 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18-26°C- Humidity (%): >=25%- Photoperiod (hrs dark / hrs light): 12 hour light-12 hour darkIN-LIFE DATES: From: 27 August 1996 To: 03 March 1997
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: OS#114461 was diluted in corn oil.
Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
Single dose
Post exposure period:
24, 48, 72 hours
Remarks:
Doses / Concentrations:500, 750, 1500, 1750, 2250, 3000, 5000 mg/kgBasis:other: actual injected
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide- Justification for choice of positive control(s): It is a known clastogen- Route of administration: Intraperitoneal- Doses / concentrations: 60 mg/kg in sterile water for injection
Tissues and cell types examined:
Bone marrow polychromatic and normochromatic cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Maximum tolerated dose level, and two lower dose levels TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24, 48 and 72 hoursDETAILS OF SLIDE PREPARATION: Bone marrow cell suspension spread onto slide, fixed and stained before coding.METHOD OF ANALYSIS: 1000 erythrocytes (PCE and NCE) were scored for the PCE/NCE ratio. 2000 PCEs were scored for the presence of micronuclei.OTHER:
Evaluation criteria:
A test article is considered to have produced a positive response if it induces a statistically significant increase in the frequency of MPCEs at one or more test article concentration for a single sacrifice time or sex (p<0.05), and the increase in MPCE frequency is dose-dependent (p<0.05). If the test article induces either a statistically significant or dose-dependent increase in MPCE frequency it is considered to have produced an equivocal response. A test article producing neither type of increase is considered to be negative (non-clastogenic) in this assay.The above criteria are used as a guide in evaluating test results. however, the Study Director may take other factors (e.g., historical control values) into consideration in evaluating the test results, since biological and statistical significance should be considered inevaluation of the asay results.
Statistics:
The frequency of MPCEs i each group was compared to its respective negative control group using a one-tailed Student's t-test. In addition, the Cochran-Armitage test was used to determine the statistical significance of any possible dose trends. Statistical significance was evaluated at the p<0.05 probability level. PCE/NCE ratios were analysed as described above. All comparisons were made separately for each sacrifice time.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- Dose range: 500, 750, 1500, 1750, 2250, 3000, 5000 mg/kg- Solubility: Dissolved in corn oil- Clinical signs of toxicity in test animals: Yes, including premature mortalityRESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay):- Ratio of PCE/NCE (for Micronucleus assay): Changed ratio of PCE/NCE in higher dose groups- Statistical evaluation: Performed
Conclusions:
The test material is considered to be negative in the In Vivo Micronucleus Test in the Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.
Executive summary:

Test Guidance

OECD Guideline No 474

Method

This study was designed to evaluate the potential of the test material to induce micronuclei in the newly formed polychromatic erythrocytes (PCEs) in mouse bone marrow.

A preliminary toxicity screen was conducted to select the appropriate doses for the definitive micronucleus test (MNT). Eight groups of mice (two/sex/dose level) were administered a single intraperitoneal (ip) injection of the test or negative control articles at a dose volume of 10 mL/kg. The test material was evaluated at doses of 500, 750, 1500, 1750, 2250, 3000 and 5000 mg/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and at 24, 48 and 72 hours, after dosing. Except for one female treated at a dose of 5000 mg/kg, all females treated at dose =>1750 mg/kg, and all males treated at doses =>3000 mg/kg, died within 48 hours after treatment. All surviving mice were sacrificed at the last observation time by cervical dislocation. Bone marrow was harvested from these animals and scored for the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) per 1000 total erythrocytes, as an index of toxicity

Results

The test material did not produce any statistically significant depressions in PCE/NCE ratios at any dose level, as compared to the concurrent negative control. In the definitive MNT, nine groups of mice (five/sex/dose) were administered a single ip injection of the test material at doses of 250, 1250 and 2500 mg/kg (males), or at dose of 150, 750 and 1500 mg/kg (females), for sacrifice at 24, 48 or 72 hours after injection. The negative control, corn oil, was administered concurrently to three groups of mice (five/sex) for sacrifice at 24, 48 or 72 hours, while the positive control, cyclophosphamide (CP; 60 mg/kg), was administered concurrently to a single group of mice (five/sex) for sacrifice 24 hours after treatment. All test and control articles were administered in a volume of 10 mL/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and approximately 24, 48 or 72 hours after dosing. Bone marrow slides were prepared, stained, coded and scored for the number of micronucleated PCEs (MPCEs) in 2000 PCEs/mouse, as well as for the number of micronucleated normochromatic erythrocytes (MNCEs) present in the optical fields containing 2000 PCEs. The PCE/NCE ratio per 1000 erythrocytes also was determined for each mouse as an index of toxicity. MPCE frequencies for all negative control groups were within acceptable ranges, and the CP positive control induced statistically significant increase in MPCE frequency (combined- and by-sex; p<0.01). Analysis of the by- and combined-sex indicated that the test material did not induce any statistically significant or dose-dependent increases in MPCE frequencies, at any harvest time evaluated, as compared to the compared to the concurrent negative controls. In addition, all observed MPCE frequencies in the animals treated with the test material were within historical ranges. Analysis of the PCE/NCE ratios revealed that the test material induced statistically significant depressions in female mice treated at 750 and 1500 mg/kg and harvested at 24 hours (p<0.05), as well as in male mice treated at 2500 mg/kg and harvested at 48 hours (p<0.05). A statistically significant depression in PCE/NCE ratios was also observed in the high dose groups, at the 48 -hour harvest time, when combining sexes (p<0.05; females treated at 1500 mg/kg, males treated at 2500 mg/kg).

Conclusion

The test substance is considered to be negative in the In Vivo Micronucleus Test in Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.

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

Additional information

Additional information from genetic toxicity in vitro:

In Vitro

Bacterial Mutagenicity Test (Ames)

In a key study performed according to OECD Guideline No. 471, the test material was evaluated in the Ames/Salmonella-E.coli bacterial reverse mutation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA102, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2uvrA-), in the presence and absence of an exogenous metabolic activation system (S9).

Toxicity of the test material was first evaluated in a prescreen by treating duplicate cultures of strains TA1537, TA100 and WP2uvrA- with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9, using both the pre-incubation and plate-incorporation methods. Results of the prescreen indicated the test material was toxic to all three tester strains at 5000 ug/plate in the pre-cubation treatment conditions only. In addition, the test article was found to be incompletely soluble at doses => 500 ug/plate.

Based upon these findings, the test material was evaluated in triplicate cultures in all five strains of Salmonella strain WP2uvrA- at dose of 16.7, 50, 167, 500, 1670 and 5000 ug/plate, in the presence and absence of S9, using the plate-incorporation method. Six dose levels of the test material were evaluated with and without S9 in the event of unacceptable insolubility at the highest dose levels evaluated in the mutation assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors.The test article again precipitated from solution =>167 ug/plate. No toxicity was again observed in any of the tester strains, with or without S9. Revertant frequencies for all doses of the test material in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative control cultures except for strain TA102 in the absence of S9. In this case a statistically significant increase in revertant frequencies, to approximately 1.1- to 1.5 -fold control values, were observed at doses of 50 to 1670 ug/plate. However, these increases were not linear.

The test material was re-evaluated in the confirmatory assay using the same dose levels under pre-incubation treatment conditions and also re-evaluated in TA102 without S9 under plate incorporation conditions. The test article again precipitated from solution at doses =>167 ug/plate. Inhibited growth again was observed in all tester strains at 5000 ug/plate without S9 Revertant frequencies for all dose of the test material in all tester strains with and without S9 approximated or were less than control value. All positive and negative control values in both assays were within acceptable limits.

Therefore, the results for the test material were negative in the Ames/Salmonella-E.coli Reverse Mutation Assay, using liquid pre-incubation and plate incorporation treatments, under the conditions, and according to the criteria, of the test protocol.

In a supporting study performed to Japanese Joint Notification Yakusyoku 0331 No.7 and the Notification of Minitry of Labour, No 77 September 1, 1988 and No 67 (revised), June 2, 1997, the mutagenic potential of the test material was assessed using a bacterial/microsome test system. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, 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 dose determination test was 4.88 to 5000 ug/plate. The experiment was repeated on a separate day using the dose range, 156 to 5000 ug/plate, fresh cultures of bacterial strains and fresh test material formulations.

Cytotoxicity to bacteria by the test material was not observed for TA98, TA100, TA1535, TA1537 and WP2uvrA with or without metabolic activation. Precipitate of the test material was observed at 5000 ug/plate for TA98, TA100, TA1535, TA1537 and WP2uvrA without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

In Vitro

Chromosome Aberration

The key study was performed in accordance with the following guidelines:

OECD Guidelines for Testing of Chemicals No. 473 " In Vitro Mammalian Chromosome Aberration Test"

Method BIO of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

US EPA OPPTS 870.5375 Guideline.

40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.

Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

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 hours 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 and were in the range of 15 to 360 ug/ml for all exposure groups.

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 indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was non-toxic and 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 was the lowest precipitating dose level.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

In a supporting study performed to OECD Guideline No 473, the test material was investigated for its ability to induce chromosomal aberrations in Chinese Hamster ovary (CHO) cells in the presence and the absence of a rat liver homogenate metabolic activation system. Two assay periods, 10 hours and 20 hours, were used which totalled four independent assays. The test concentrations that were used for the study ranged from 0.5 to 302 ug/mL in the non-activated assays and 0.15 to 5010 ug/mL in the activated assays. The data were analysed using two methods. The first used the traditional method inwhich the gaps were not counted as chromosomal aberations while the second counted the gaps as chromosomal aberrations.

In the activated assays, the compound was toxic in the 10 -hour assay at the highest concentration, 152 ug/mL, and in the 20 -hour assay at 150 ug/mL. In the non-activated assays, toxicity was observed at the concentrations above 101 ug/mL in the 10 -hour assay and above 50 ug/mL in the 20 -hour assay. A statistically significant increase in the percent of cells with aberrations (excluding gaps) was found in the 20 -hour non-activated assay at one concentration. In the 20 -hour activated assay, the increase in the percent of cells with chromosomal breaks or aberrations was not statistically significant unless gaps were included in the calculation. The response obtained from the negative and the positive controls demonstrated that the test system was capable of detecting chemicals which caused chromosomal damage.

The results indicate that the test material caused a slight increase in chromosomal aberrations in CHO cells in the 20-hour activated and non-activated assays. The reproducibility of this slight increase was not determined. The small increase seen in the absence of activation was not dose-related (it occured at the low dose level), and the small increase in the with-activation group was only significant when gaps were included and with the exclusion of gaps only one of the duplicate cultures showed an increase. According to the criteria of the protocol the response in both cases is negative.

In Vitro

Mammalian Cell Gene Mutation Assay

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.

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.

The maximum dose level used in the main test was limited by test item-induced toxicity. 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. A small but statistically significant linear trend increase in mutant frequency was observed in the 4-hour with-S9 exposure group of Experiment 2. However, the mutant frequency values were all within the accetable range for a vehicle control and the GEF was not exceeded at any dose level. The reponse observwed was considered to be artefactual and of no toxicological significance.

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.

In Vivo

A key study performed to OECD Guideline No 474 was designed to evaluate the potential of the test material to induce micronuclei in the newly formed polychromatic erythrocytes (PCEs) in mouse bone marrow.

A preliminary toxicity screen was conducted to select the appropriate doses for the definitive micronucleus test (MNT). Eight groups of mice (two/sex/dose level) were administered a single intraperitoneal (ip) injection of the test or negative control articles at a dose volume of 10 mL/kg. The test material was evaluated at doses of 500, 750, 1500, 1750, 2250, 3000 and 5000 mg/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and at 24, 48 and 72 hours, after dosing. Except for one female treated at a dose of 5000 mg/kg, all females treated at dose =>1750 mg/kg, and all males treated at doses =>3000 mg/kg, died within 48 hours after treatment. All surviving mice were sacrificed at the last observation time by cervical dislocation. Bone marrow was harvested from these animals and scored for the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) per 1000 total erythrocytes, as an index of toxicity.

The test material did not produce any statistically significant depressions in PCE/NCE ratios at any dose level, as compared to the concurrent negative control. In the definitive MNT, nine groups of mice (five/sex/dose) were administered a single ip injection of the test material at doses of 250, 1250 and 2500 mg/kg (males), or at dose of 150, 750 and 1500 mg/kg (females), for sacrifice at 24, 48 or 72 hours after injection. The negative control, corn oil, was administered concurrently to three groups of mice (five/sex) for sacrifice at 24, 48 or 72 hours, while the positive control, cyclophosphamide (CP; 60 mg/kg), was administered concurrently to a single group of mice (five/sex) for sacrifice 24 hours after treatment. All test and control articles were administered in a volume of 10 mL/kg. Mice were observed for mortality and pharmacotoxic signs immediately (0 -2 hours), and approximately 24, 48 or 72 hours after dosing. Bone marrow slides were prepared, stained, coded and scored for the number of micronucleated PCEs (MPCEs) in 2000 PCEs/mouse, as well as for the number of micronucleated normochromatic erythrocytes (MNCEs) present in the optical fields containing 2000 PCEs. The PCE/NCE ratio per 1000 erythrocytes also was determined for each mouse as an index of toxicity. MPCE frequencies for all negative control groups were within acceptable ranges, and the CP positive control induced statistically significant increase in MPCE frequency (combined- and by-sex; p<0.01). Analysis of the by- and combined-sex indicated that the test material did not induce any statistically significant or dose-dependent increases in MPCE frequencies, at any harvest time evaluated, as compared to the compared to the concurrent negative controls. In addition, all observed MPCE frequencies in the animals treated with the test material were within historical ranges. Analysis of the PCE/NCE ratios revealed that the test material induced statistically significant depressions in female mice treated at 750 and 1500 mg/kg and harvested at 24 hours (p<0.05), as well as in male mice treated at 2500 mg/kg and harvested at 48 hours (p<0.05). A statistically significant depression in PCE/NCE ratios was also observed in the high dose groups, at the 48 -hour harvest time, when combining sexes (p<0.05; females treated at 1500 mg/kg, males treated at 2500 mg/kg).

The test substance is considered to be negative in the In Vivo Micronucleus Test in Mouse Bone Marrow Erythropoetic Cells, under the conditions, and according to the criteria, of the test protocol.


Justification for selection of genetic toxicity endpoint
Key study

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 an in vitro mammalian cell gene mutation assay, and non-clastogenic in an in vitro chromosome aberration study both with and without metabolic activation. The test material was also negative in an in vivo mouse micronucleus study. As such, the test item does not meet the classification criteria.