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Registration Dossier
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EC number: 835-272-7 | CAS number: 256374-76-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames assay
SH-0850 does not have mutagenic potential in the absence and presence of the metabolic activation system (S9 mix) using the Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and the Escherichia coli strain WP2uvrA.
Chromosome Aberration Test
SH-0850 does not induce numerical chromosome aberration but induces structural chromosome aberration in Chinese hamster lung fibroblast cells (CHL/IU cells).
Gene mutation assay - mammalian cell assay
No mutagenic effect of DAIGUARD-850 was observed either in the presence or in the absence of metabolic activation system in the Mouse Lymphoma Assay.
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:
- November 22, 2012 to December 3, 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- “OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test” (Adopted: July 21, 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- “Bacterial Reverse Mutation Test” of “Mutagenicity Test” stipulated in the “Test Methods of the New Chemical Substances etc.” (March 31, 2011, Yakushokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No. 5, Kanpokihatsu No. 110331009, partly amended by Yakushokuhatsu 0402 No. 1, Heisei 24.03.28 Seikyoku No. 2, Kanpokihatsu No. 120402001 on April 2, 2012)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- “Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” (Public Notice No. 77, the Ministry of Labour, September 1, 1988, amended by Public Notice No. 67, the Ministry of Labour, June 2, 1997 and Public Notice No. 120, the Ministry of Labour, December 25, 2000) and “Specific Test Methods and Test Result Evaluation Methods for Microbial Mutagenicity Test” (Memorandum, Manager of Chemical Substances Investigation Division, Industrial Safety and Health Department, Labour Standards Bureau, the Ministry of Labour, February 8, 1999)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- histidine and tryptophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Species and reason for selection
Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 were used as the tester strains in this study. Salmonella typhimurium strains were generously provided by Dr. Taijiro Matsushima, Japan Bioassay Research Center on March 13, 2003 and September 20, 2003, respectively. The use of these tester strains in the microbial mutagenicity test and bacterial reverse mutation test is recommended in “Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” and “Test Methods of the New Chemical Substances etc.”, and “OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test”.
Storage
The tester strains were preliminarily checked for the amino acid requirement, ultraviolet sensitivity, rfa wall mutation, presence or absence of plasmid pKM101 and for the negative and positive control value. It was confirmed that these tester strains had the appropriate properties.
Dimethyl sulfoxide was mixed into the culture medium of the tester strains at a volume ratio of 10:0.9, and the mixtures were dispensed and stored as working stocks at -80 °C or below in an ultra-deep freezer 3 in Ames test room 1. The working stocks were freshly thawed before use. - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Species and reason for selection
Escherichia coli strain WP2uvrA was used as the tester strain in this study. Escherichia coli strain was generously provided by Dr. Taijiro Matsushima, Japan Bioassay Research Center on March 13, 2003 and September 20, 2003, respectively. The use of the tester strain in the microbial mutagenicity test and bacterial reverse mutation test is recommended in “Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” and “Test Methods of the New Chemical Substances etc.”, and “OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test”.
Storage
The tester strain was preliminarily checked for the amino acid requirement, ultraviolet sensitivity, rfa wall mutation, presence or absence of plasmid pKM101 and for the negative and positive control value. It was confirmed that the tester strain had the appropriate properties. Dimethyl sulfoxide was mixed into the culture medium of the tester strains at a volume ratio of 10:0.9, and the mixtures were dispensed and stored as working stocks at -80 °C or below in an ultra-deep freezer 3 in Ames test room 1. The working stocks were freshly thawed before use. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
1) Rat liver S9
S9 (lot number 12090706, manufactured on September 7, 2012, Oriental Yeast Co., Ltd.,) prepared from the livers of 7-week old male SD rats (body weight: 208.5 ± 8.8 g) treated (intraperitoneal administration) with the combination of phenobarbital (single dose of 30 mg/kg and three doses of 60 mg/kg) and 5,6-benzoflavone (single dose of 80 mg/kg) was used. S9 was stored at -80 °C or below frozen in an ultra-deep freezer 3 in Ames test room 1 until use. The frozen S9 was freshly thawed just before use.
2) Composition of S9 mix
S9 mix was freshly prepared before use in the test. 1 mL of S9 mix contained 8 μM of MgCl2, 33 μM of KCl, 5 μM of glucose-6-phosphate, 4 μM of NADPH, 4 μM of NADH, 100 μM of sodium phosphate buffer (pH 7.4) and 0.1 mL of S9. - Test concentrations with justification for top dose:
- Based on the result of range-finding test, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313μg/plate in the absence of S9 mix of any tester strains for Main test-1. In the presence of S9 mix, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313μg/plate of any tester strains for Main test-1.
Main test-2
As the result of Main test-1, no increase to 2-fold or more over the negative control value in the number of revertant colonies was noted for all tester strains in the presence and absence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the presence and absence of S9 mix. Therefore, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313 μg/plate of any tester strains for Main test-2. - Vehicle / solvent:
- Negative control substance (vehicle)
Name: DMSO
Manufacturer, lot number, purity and grade
Manufacturer: Dojindo Laboratories
Lot number: DB136
Purity: 99.9 %
Grade: Pure solvent for ultraviolet absorption spectrum
Reason for vehicle selection
The test substance was not soluble in distilled water (50.0 mg/mL) and acetone (100 mg/mL) but suspended in DMSO (50.0 mg/mL). Heat generation, bubbling and change of color tone were not observed for 50.0 mg/mL suspension using DMSO up to 2 hours after preparation at room temperature. Therefore, it was determined to be stable and DMSO was selected as vehicle.
Storage conditions
The vehicle was frozen (allowable range: -30 - -10 oC) and stored in Freezer 5 in Ames experiment Room 1. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracence (2AA); ICR-191
- Details on test system and experimental conditions:
- Experimental method
The tests were performed by the pre-incubation method in the absence and presence of S9 mix. Three plates were used for the negative control and 2 plates per dose level were used for each positive control and the test substance treatment groups in the dose-range finding test. In the main studies 3 plates per dose level were used for each negative control, positive control and test substance treatment groups. The study code number, the name of bacterial strain, presence or absence of S9 and the dose level were written on each plate for identification.
Operational procedure
In each operation, a mixture consisting of 0.1 mL of the test substance solution, the solvent or the positive control solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix and 0.1 mL of bacterial culture solution were added in a test tube and shook at 37 ± 0.5 °C for 20 minutes. 2 mL of the soft agar was then added to the test tube and the mixture was overlaid onto the surface of the plate. After the plate was incubated at 37 ± 0.5 °C for 48 hours, the number of revertant colonies formed on the plate was counted.
Sterility test
After 2 mL of soft agar was added to each of the test substance solution of the highest dose level (0.1 mL) and S9 mix (0.5 mL), these mixtures were overlaid onto the surface of the plates. The plates were incubated at 37 ± 0.5 °C for 48 hours and the presence or absence of contamination was determined for each plate.
Observation and counting
Observation
At the end of culture period, the presence or absence of precipitation of the test substance was determined by visual examination and the presence or absence of growth inhibition was determined using a stereoscopic microscope.
Counting
The number of colonies was counted using a colony analyzer (CA-11D, System Science Co., Ltd.,) for all plates. When using the colony analyzer, the colony count was corrected for the area and count loss and used as the number of revertant colony. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- The test result was considered to be positive when the number of revertant colonies was increased to 2-fold or more over the negative control value and the mode of increase was dose dependent or reproducible, and all other test results were considered to be negative.
- Statistics:
- No statistical procedure was applied.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Dose-range finding test
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for TA100 in the absence of S9 mix. No growth inhibition was observed. Precipitation of the test substance was not observed.
Main test-1
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for all tester strains in the absence and presence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the absence and presence of S9 mix.
Main test-2
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for all tester strains in the absence and presence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the absence and presence of S9 mix.
Discussion
As the result of the tests, the number of revertant colonies was below 2-fold over the negative control value for all tester strains in the absence and presence of S9 mix. The mutagenicity of the test substance was judged negative.
The number of revertant colonies for positive control was 2-fold or more value over the negative control, and the numbers of revertant colonies for the negative control and positive controls were within the range of the historical data. Furthermore, the test system was found to be free of contamination. Therefore, it was judged that the study was performed properly. - Conclusions:
- It was concluded that SH-0850 is not mutagenic under the condition of the study.
- Executive summary:
The mutagenic potential of SH-0850 was assessed by the pre-incubation method in the absence and presence of the metabolic activation system (S9 mix) using the Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and the Escherichia coli strain WP2uvrA.
The test result showed that the number of revertant colonies for all tester strains was less than two-fold of the negative control and the mutagenicity was considered to be negative.
Therefore, it was concluded that SH-0850 does not have mutagenic potential.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- March 28, 2013 to June 7, 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- "OECD Guidelines for the Testing of Chemicals, No.473, In Vitro mammalian Chromosome Aberration Test" (Adopted: July 21, 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- "Chromosomal aberration test in cultured mammalian cells" of "Mutagenicity Test" stipulated in the "Test Methods of the New Chemical Substances etc." (March 31, 2011, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, partly amended by Yakushokuhatsu 0402 No.1, Heisei 24.03.28 Seikyoku No.2, Kanpokihatsu No. 120402001 on April 2, 2012)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- clastogenic potential using Chinese hamster lung fibroblast cells (CHL/IU cells).
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- Type and reason for selection
Cells used: Cells of fibroblast cell line isolated from Chinese hamster lung (CHL/IU cells)
Obtained from: Health Science Research Resources Bank, Japan Health Sciences Foundation
Received on: April 17, 2002
Mode chromosome number: 25
Doubling time: Approximately 15 hours
Mycoplasma contamination: Negative
Spontaneous appearance frequency of cells with structural or numerical chromosome aberration: < 5%
Reason for selection: Recommendation in the applied guideline.
Storage
Cells were suspended in the culture medium containing 10 vol % DMSO [Eagle MEM (Nissui Pharmaceutical Co., Ltd.,) + 10 vol % heat-inactivated newborn calf serum (NBCS, Sanko Junyaku Co., Ltd.,)] and stored frozen in liquid nitrogen.
Culture conditions (nominal)
Culture incubator: Cell culture CO2 incubator (MCO-18AIC, SANYO Electric Co., Ltd.)
Temperature: 37°C
Humidity: Under humidified conditions
CO2 concentration: 5%.
Passage
Vessel: 90-mm plastic dish (Nunc)
Frequency: Twice weekly
Passage number: Cell growth inhibition test: passage 5
Chromosome aberration test: passage 10 - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Culture medium and S9 mix
Culture medium
The basal medium (MEM) was prepared by adding L-glutamine (final concentration: 0.292 g/L) and sodium hydrogen carbonate (final concentration: approximately 1.95 g/L) to Eagle MEM (Lot No. 686211, Nissui Pharmaceutical Co., Ltd.). The medium for cell culture was prepared by adding 10 vol % heat-inactivated NBCS (Lot No.: AWJ22312, HyClone Laboratories) to MEM.
S9 mix
Rat liver S9
Manufacturer: Oriental Yeast Co., Ltd.
Date of manufacture: December 21, 2012
Lot number: 12122110
S9 protein amount: 23.7 mg/mL
Animal used:
Species, strain: SD strain rat
Sex: Male
Age: 7 weeks old
Body weight: 207.9 +/- 9.2 g
Inducing substance
Name: Phenobarbital (PB) and 5,6-benzoflavone (5,6-BF)
Administration method: Intraperitonearl
Administration period and dose level (mg/kg):
PB 30 mg/kg single dose; 60 mg/kg 3 doses
5,6-BF 80 mg/kg single dose
Storage place: Ultra-deep freezer 4 in Cell Experiment Room 2
Storage temperature: -80 °C or less (allowable range)
Usage: Freshly thawed before use. - Test concentrations with justification for top dose:
- Cell growth inhibition test: Nominal dose level of the test substance: 55.6, 111, 223, 445, 890, 1780 and 3560 μg/mL
Chromosomal aberration test
Short-term treatment; -S9 mix: 445, 890, 1780 and 3560 µg/mL
Short-term treatment; +S9 mix: 890, 1780 and 3560 µg/mL
24-hour continuous treatment: 315, 445, 629, 890, 1260 and 1780 µg/mL
The highest dose level: 3560 μg/mL, equivalent to 10 mmol/L specified in the guideline as the upper limit in the absence of cytotoxicity. - Vehicle / solvent:
- Negative control substance (vehicle)
Name: DMSO
Manufacturer, lot number, purity and grade
Manufacturer: Dojindo Laboratories
Lot number; JT012
Purity: 99.9 %
Grade: Pure solvent for ultraviolet absorption spectrum
Reason for vehicle selection
The test substance was not soluble in water at 35.6 mg/mL but suspended in DMSO at 356 mg/mL. Heat generation, bubbling and change of color tone were not observed for 356 mg/mL test substance solution using DMSO up to 2 hours after preparation at room temperature. Therefore, it was determined to be stable and DMSO was selected as vehicle. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Preparation of the test substance solution and positive control substance solution
Preparation of test substance solution
Method of preparation
DMSO was added to the weighed test substance. The solution was stirred (by tube mixer) and ultrasonically treated (ultrasonic cleaner) to prepare stock dose suspension of the test substance. The stock dose suspension was further diluted with the same vehicle to prepare the test substance dose formulation at a concentration of 100-fold dose level of the test substance in the culture medium. The test substance solution was prepared without purity correction because the purity of the test substance was 95% or more.
Timing of preparation
The test substance solution was freshly prepared before use, stored at room temperature, and used within 2 hours.
Preparation of positive control substance solution
Method of preparation and storage
Previously MMC and CPA were dissolved in distilled water (lot No.: K1E86, for injection, Otsuka Pharmaceutical Factory, Inc.,) to prepare 0.01 mg/mL and 1.00 mg/mL solutions, respectively, and stored frozen in Ultra-deep freezer 4 (allowable range: -80 °C or below) in Cell Experiment Room 2.
Timing of preparation
The frozen solutions were freshly thawed before use, stored at room temperature and used within 2 hours.
Experimental method
Cell growth inhibition test
Operational procedure
Treatment
One culture vessel was used per dose level for the test.
In the short-term treatment in the absence of S9 mix (-S9 mix), the culture medium was removed from pre-culture and a well-mixed mixture of 30 μL of the test substance dose formulation or solvent and 3 mL of fresh culture medium was added to cells.
In the short-term treatment in the presence of S9 mix (+ S9 mix), the culture medium was removed from pre-culture and a well-mixed mixture of 30 μL of the test substance dose formulation or solvent, 2.5 mL of fresh culture medium, and 0.5 mL of S9 mix was added to cells.
In each case of - S9 mix and + S9 mix, the culture medium was removed after treatment for 6 hours, cells were washed 3 times with 2 mL of Dulbecco's phosphate buffered saline solution without Ca2+ and Mg2+, 5 mL of fresh culture medium was added, and the cells were cultured for 18 hours.
In the continuous treatment, the culture medium was removed from pre-culture, a well-mixed mixture of 50 μL of the test substance dose formulation or solvent and 5 mL of fresh culture medium was added to cells, and the cells were treated for 24 hours.
A total of 50 μL of 10 μg/mL demecolcine solution was added per culture vessel at 2 hours before the completion of cell culture.
The presence or absence of precipitation of the test substance, discoloration of the culture medium, and corrosion of the culture vessel was visually confirmed at the start of treatment, at the completion of treatment, and at the completion of cell culture.
After the completion of cell culture, cells were detached by treatment with 2 mL of 0.25 w/v% trypsin to prepare a cell suspension. A 200 μL-aliquot of the cell suspension was sampled and diluted with 10 mL of Cell Pack (Sysmex Corporation), the number of cells was counted with Microcell Counter (CDA-500, Sysmex Corporation), and the cell growth rate and 50% inhibitory concentration (IC50) were determined. The value of IC50 was calculated from the straight line obtained from the cell growth rate of the lowest concentration at which the cell growth rate was lowered to less than 50% and the cell growth rate of the next lowest concentration.
The following procedure was not performed for negative control and test substance dose levels exceeding the lowest concentration at which the calculated cell growth rate was below 20% and not exceeding the highest concentration at which the calculated cell growth rate was more than 90%. The cell growth rate was 90.1% at 445 μg/mL in 24-hour continuous treatment, but less than 90% at the dose levels of 223 μg/mL and below.
Therefore, the following procedure was performed.
The remainder of the suspension was centrifuged at 1000 rpm for 5 minutes to collect cells, and the collected cells were hypotonically treated with 3 mL of 0.075 mol/L KCl at 37°C for 15 minutes. The hypotonically treated cells were semi-fixed with approximately 0.3 mL of fixative solution (methanol : acetic acid = 3 : 1) and then fixed with 3 mL of the fixative solution with 2 changes. A cell suspension of appropriate concentration was then prepared with the fixative solution and dropped onto a glass slide. One chromosomal specimen was prepared for each dose level.
Each chromosomal specimen was air-dried and stained with 2 vol % Giemsa solution prepared by dilution with 1/15 mol/L phosphate buffer (pH 6.8) for approximately 15 minutes.
Observation of specimen
The presence or absence of dividing cells was examined and 50 metaphase cells were observed to determine the appearance frequency of cells with chromosome aberration for the dose levels that provide useful information on dose level setting in the chromosomal aberration test.
Structural aberration
The number of cells with structural aberration except gap described below was recorded.
-Gap was defined as an achromatic region narrower than the chromatid width.
-Chromatid breaks
-Chromatid exchanges
-Chromosome breaks
-Chromosome exchanges (dicentric, circular chromosome, translocation)
-Fragmentation
Numerical aberration
The number of cells showing polyploidy (at least triploidy) and endoreduplication was recorded.
Chromosomal aberration test
Procedure
Using the positive controls shown below, the chromosomal aberration test was performed. Two culture vessels were used per dose level and four chromosomal specimens were prepared for each dose level (2 specimens per culture vessel). Procedure after hypotonical treatment was also performed for negative control, positive controls and all dose levels of the test substance.
Dose level
The dose levels of the test substance were determined based on the results of the cell growth inhibition test. The established dose levels and reasons for selection are described below.
Since IC50 value was obtained for -S9 mix and for the 24-hour continuous treatment, dose level exceeding IC50 was set as the highest dose level of the test substance. Dose levels were determined by serial dilutions at common ratio 2 and √2 for -S9 mix and 24-hour continuous treatment, respectively.
For +S9 mix, IC50 value exceeded 3560 μg/mL and 3560 μg/mL was set as the highest dose level.
Observation of specimen
Dose levels for observation
All negative control specimens and positive control specimens were observed.
Dose levels shown in the following table were selected as the dose levels of the test substance for specimen observation because IC50 value exceeded 3560 μg/mL for -S9 mix and +S9 mix.
Dose levels shown in the following table were selected as the dose levels of the test substance for specimen observation because IC50 value was obtained for 24-hour continuous treatment.
All specimens to be observed were identified by randomly assigned slide numbers and observed under blind method.
As a result of specimen observation in 24-hour continuous treatment, the appearance frequency of cells with structural aberration was 15.5% at the highest, but obvious dose dependency was not confirmed. Therefore additional observation of 445 μg/mL was performed to confirm dose dependency of the appearance frequency of cells with chromosome aberration. Slide number was assigned to the specimens for observation.
Structural aberration
For each dose level, 200 (50 for each specimen) metaphase cells having 25 ± 2 chromosomes (centromeres) were observed. The total number of cells with structural aberration was recorded and, at the same time, the number of cells and number of aberrations by the type of structural aberration were recorded.
Numerical aberration
For each dose level, 200 (50 for each specimen) metaphase cells were observed and polyploid cells having 38 or more chromosomes and endoreduplication cells were recorded respectively.
D20 value
D20 value (the concentration at which aberration is noted in 20 % of cells) was calculated when 5 % or more cells with chromosome aberration are noted except the result of the treatment is negative. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- Evaluation criteria of the results
The test result was considered to be positive when the appearance frequency of cells with structural aberration or numerical aberration was 10% or higher and the appearance was dose dependent; or when 5% or higher appearance frequency was reproducible in chromosomal aberration test and confirmatory test. The other test results were considered to be negative. No statistical procedures were applied.
Conditions for valid test
a) The appearance frequency of cells with chromosome aberration for the negative control is less than 5%, and
b) The appearance frequency of cells with structural aberration for the positive control is 20% or higher. - Statistics:
- Not specified
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Cell growth inhibition test
IC50 value was calculated to be 3400 μg/mL and 860 μg/mL for -S9 mix and 24-hour continuous treatment, respectively. IC50 value exceeded 3560μg/mL for +S9 mix.
Precipitation of the test substance was observed at dose levels of 111 μg/mL and higher at the start of treatment, at dose levels of 445 μg/mL and higher at the completion of treatment, and at dose levels of 1780 μg/mL and higher at the completion of cell culture for -S9 mix. For +S9 mix precipitation of the test substance was observed at dose levels of 111 μg/mL and higher at the start of treatment, at dose levels of 445 μg/mL and higher at the completion of treatment. Precipitation of the test substance was observed at dose levels of 111 μg/mL and higher at the start of treatment, at dose levels of 890 μg/mL and higher at the completion of treatment for 24-hour continuous treatment.
No discoloration of culture medium or corrosion of culture vessel was observed at any of the treatment.
Chromosomal aberration test
Short-term treatment
-S9 mix
IC50 value: >3560 μg/mL
Precipitation of the test substance, discoloration of culture medium and corrosion of culture vessel: Precipitation of the test substance was observed at all dose levels at the start and the completion of treatment, at dose levels of 1780 μg/mL and higher at the completion of cell culture. No discoloration of culture medium or corrosion of culture vessel was observed.
Appearance frequency of cells with structural aberration
Negative control: <5%
Positive control: >=20%
Test substance: less than 5%, therefore, judged to be negative
Appearance frequency of cells with numerical aberration: The appearance frequency of cells with numerical aberration was less than 5% in all groups, and therefore the test was judged to be negative.
+S9 mix
IC50 value: > 3560 μg/mL
Precipitation of the test substance, discoloration of culture medium and corrosion of culture vessel: Precipitation of the test substance was observed at all dose levels of the test substance at the start and the completion of treatment. No discoloration of culture medium or corrosion of culture vessel was observed.
Appearance frequency of cells with structural aberration
Negative control: <5%
Positive control: >=20%
Test substance: less than 5%, therefore, judged to be negative
Appearance frequency of cells with numerical aberration: The appearance frequency of cells with numerical aberration was less than 5% in all groups, and therefore the test was judged to be negative.
24-hour continuous treatment
IC50 value: 1200 μg/mL
Precipitation of the test substance, discoloration of culture medium and corrosion of culture vessel: Precipitation of the test substance was observed at all dose levels at the start of treatment, at dose levels of 629 μg/mL and higher at the completion of treatment. No discoloration of culture medium or corrosion of culture vessel was observed.
Appearance frequency of cells with structural aberration
Negative control: <5%
Positive control: >=20%
Test substance: 10% or more and dose dependency on the appearance was noted, therefore, the test was judged to be positive.
Appearance frequency of cells with numerical aberration: The appearance frequency of cells with numerical aberration was less than 5% in all groups, and therefore the test was judged to be negative.
D20 value: Structural aberration: 1.5 μg/mL
Discussion
The appearance frequency of cells with chromosome aberration for the negative control was less than 5%, and the appearance frequency of cells with structural aberration except gap for the positive control was 20% or higher in any treatment. Therefore, it was judged that the test was performed properly.
The appearance frequency of cells with numerous aberration was below 5 % in any observed dose level of the test substance in any treatment, therefore it was determined to be negative for numerous aberration. On the other hand, the appearance frequency of cells with structural aberration was below 5 % in any observed dose level of the test substance for -S9 mix and +S9 mix, but in 24-hour continuous treatment, it was 15.5 % at the highest and dose dependency was noted in the appearance. Therefore, it was determined to be positive for structural aberration. - Conclusions:
- It was concluded that SH-0850 does not induce numerical chromosome aberration but induces structural chromosome aberration under the condition of this study.
- Executive summary:
The clastogenic potential of SH-0850 was assessed using Chinese hamster lung fibroblast cells (CHL/IU cells).
Specimen were prepared and observed at 890, 1780 and 3560 μg/mL in the absence and presence of S9 mix in short-term treatment, 445, 629, 890 and 1260 μg/mL in 24-hour continuous treatment.
The appearance frequency of cells with numerous aberration was below 5 % in any observed dose level of the test substance in any treatment. Therefore, it was determined to be negative for numerous aberration. The appearance frequency of cells with structural aberration was below 5 % in any observed dose level of the test substance in the absence and presence of S9 mix in short-term treatment. In 24-hour continuous treatment, it was 15.5 % at the highest and the dose dependency was noted in the appearance. Therefore, it was determined to be positive for structural aberration.
Based on above results, it was concluded that SH-0850 does not induce numerical chromosome aberration but induces structural chromosome aberration under the condition of this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 December 2015 to 03 May 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, " In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene ", 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- genotoxicity (point mutations and/or gross chromosomal changes) at the thymidine kinase (tk)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Cell Line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 1661603
Supplier: American Type Culture Collection
(Manassas,Virginia, USA)
Date of receipt: 22 January 2004
Date of working lot: 18 October 2013 / 21 January 2016
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). - Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- The post-mitochondrial fraction (S9 fraction) was prepared from rat liver by the Microbiological Laboratory of CiToxLAB Hungary Ltd. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived annually.
Induction of Liver Enzymes:
Male Wistar rats (365-453 g, animals were 9 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage (for both inducers) for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 13 October 2014.
Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. Sterility of the preparation was confirmed.
The protein concentration was determined by colorimetric test by chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 30.9 g/L. The date of preparation of S9 fraction for this study was 16 October 2014 (CiToxLAB code: E11960).
The biological activity in the Salmonella assay of each batch of S9 was characterized using the 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study was found active under the test conditions.
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath. - Test concentrations with justification for top dose:
- A preliminary toxicity test was performed to select dose levels for the main assays.
Assay 1, 2 and 3
-/+S9 mix: 10, 500, 250, 125, 62.5, 31.25 & 15.625 μg/mL
The tested concentration range in the study was considered to be adequate as the highest examined concentration was the highest achievable concentration due to the limited solubility of the test item. Lower test concentrations were spaced by a factor of two. - Vehicle / solvent:
- Dimethyl sulfoxide was used for vehicle of the test item as well as vehicle (solvent) of the positive control chemicals in the study.
Data of the chemical used in the study are shown below:
Name: Dimethyl sulfoxide
Abbreviation: DMSO
Supplier: Sigma-Aldrich Co.
Lot No.: SZBE2790V / SZBF3070V
Expiry date: 20 September 2017 / 18 October 2018
Storage conditions: Room temperature, under N2 - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- Preliminary Toxicity Test
A preliminary toxicity test was performed to select dose levels for the main assays.
During the preliminary test, a 3 hour treatment in the presence and absence of S9-mix and a 24 hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity.
The test used single cultures and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer. Visual examination for any kind of insolubility in the final culture medium was conducted at the beginning and end of treatment. Measurement of pH and osmolality was performed at the end of the treatment period. To check if late phase cytotoxicity exists, cells were transferred for the expression period for two extra days and repeated cell counting was performed.
Main Mutation Assays
In Assay 1, cells were treated for 3-hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 3-hours in the presence of S9 mix and for 24-hours in the absence of S9 mix. In Assay 3, cells were treated for 3-hours in the presence of S9 mix, and for 3 and 24 hours in the absence of S9 mix.
A suitable volume (0.2 mL) of RPMI-5 medium, vehicle, test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks.
For the 24-hour treatment, 6x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37°C ± 1°C (approximately 5% CO2 in air). Gentle shaking was used during the 3- hour treatments. Measurement of pH and osmolality was also performed after the treatment period. Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer.
Where sufficient cells survived, cell density was adjusted to a concentration of 2x105 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.
Plating for Survival
Cultures of cell density 2x105 cells/mL, were further diluted to 8 cells/mL.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for about two weeks. Wells containing viable clones were identified by eye using background illumination and counted.
Expression Period
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days.
During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2x105 cells/mL and transferred to flasks for further growth.
On completion of the expression period, six test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.
Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1x104 cells/mL with RPMI-20 for plating for a viability test.
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.
Plating for -trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1x104 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2x103 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase is concentration-related (p < 0.05) as indicated by the linear trend analysis.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis and verified by an additional experiment documented by an amendment. Similarly, positive responses seen only at high levels of cytotoxicity might require careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).
The test item will be considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, but it does not exceed the GEF. The test item is then considered unable to induce mutations in this test system. - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY EXPERIMENT
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment was 500 μg/mL (the highest achievable concentration based on the limited solubility of the test item).
Minor insolubility and no cytotoxicity were observed in the preliminary experiment.
Concentrations up to the highest available concentrations were selected for the main experiments according to the instructions of the relevant OECD guideline regarding non-cytotoxic test items with limited solubility. Six concentrations were selected for the main experiments in each assay.
MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel subcultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.
Assay 1
In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
In Assay 1, no insolubility or large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment.
In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
Assay 2
In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
In Assay 2, no insolubility or large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment.
In the presence of S9-mix (3-hour treatment), similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (24-hour treatment), no cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.
Although there was clearly no indication of mutagenicity after the treatments in Assays 1-2, but as the results of the first two main tests did not fully meet the acceptance criteria (the mutation frequency of the untreated and vehicle control were slightly higher than the upper limit of the acceptable range in some cases), thus an additional experiment (Assay 3) was performed to provide fully valid datasets for interpretation.
Assay 3
In Assay 3, a 3-hour treatment with and without metabolic activation (in the presence and absence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL in all cases.
In Assay 3, insolubility (minimal amount of precipitate) was observed at the highest examined concentration of 500 μg/mL in all cases. There were no large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment.
In the presence of S9-mix (3-hour treatment), similarly to the previous tests, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (24-hour treatment), no cytotoxicity was observed (survival results are shown in Table 7 of Appendix 5). An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant doseresponse to the treatment was indicated by the linear trend analysis.
Some minor increases in the mutation frequency were observed sporadically in Assays 1, 2 and 3; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they was considered as biologically not relevant increases, just showing the biological variability of the test system.
VALIDITY OF THE MUTATION ASSAYS
Untreated, negative (vehicle) and positive controls were run concurrently in the study.
In Assay 1 with and without metabolic activation as well as in Assay 2 without metabolic activation, the spontaneous mutation frequency of the negative (vehicle) and/or untreated control was slightly higher* than the upper limit of the recommended range (50-170 mutants per 106 viable cells). In Assay 2 with metabolic activation, the observed values were in the acceptable range. However, in order to provide fully valid datasets, an additional experiment (Assay 3) was performed. In Assay 3, all the spontaneous mutation frequency of the negative (vehicle) and untreated controls were in the recommended range, thus this criterion was considered to be fulfilled.
*Note: Mutation frequency values of 206.4, 198.9 and 154.6 were recorded for negative (vehicle) control in Assay 1 with and without metabolic activation and in Assay 2 without metabolic activation: while Mutation frequency values of 198.5, 206.7 and 179.1 were recorded for untreated control in Assay 1 with and without metabolic activation and in Assay 2 without metabolic activation.
The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD criteria.
The plating efficiencies for the negative (vehicle) control of the test item at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays.
The number of test concentrations evaluated was six in each case, which met the acceptance criteria of at least four evaluated concentrations.
The tested concentration range in the study was considered to be adequate as the highest examined concentration was the highest achievable concentration due to the limited solubility of the test item*. Lower test concentrations were spaced by a factor of two.
*Note: Based on the limited solubility (maximum achievable concentration in DMSO was 50 mg/mL) and according to the recommendation of the OECD 490 guideline (organic solvent concentration should not exceed 1% (v/v) in the final treatment medium), the highest achievable concentration was 500 μg/mL.
The overall study was considered to be valid. - Conclusions:
- The Mouse Lymphoma Assay with DAIGUARD-850 on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.
Treatment with the test item did not result in a statistically significant or biologically relevant increase in mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. Therefore, no mutagenic activity of the test item was observed in the performed experiments.
In conclusion, no mutagenic effect of DAIGUARD-850 was observed either in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay. - Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of DAIGUARD-850 to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).
Dimethyl sulfoxide was used as vehicle of the test item in this study. The test item was examined up to 500 μg/mL (highest achievable concentration based on the limited solubility of the test item) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with and without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL,
Assay 2, 3-hour treatment with metabolic activation and 24-hour treatment without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL,
Assay 3, 3-hour treatment with and without metabolic activation and 24-hour treatment without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
In Assays 1-3, there were no large changes in pH or osmolality after treatment. No insolubility was detected in the final treatment medium at the end of the treatment in Assays 1-2. In assay 3, minimal amount of precipitate was detected at the highest examined concentration of 500 μg/mL in all cases.
In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
Although there was clearly no indication of mutagenicity after the treatments in Assays 1-2, but as the results of the first two main tests did not fully meet the acceptance criteria (the mutation frequency of the untreated and vehicle control were slightly higher than the upper limit of the acceptable range in some cases), thus an additional experiment (Assay 3) was performed to provide fully valid datasets for interpretation.
In Assay 3, following a 3-hour treatment with metabolic activation), similarly to the previous tests, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 3, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 3, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. In overall, the criterion spontaneous mutation frequency of the negative (vehicle) controls was considered to be fulfilled. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate based on the limited solubility of the test item. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
In conclusion, no mutagenic effect of DAIGUARD-850 was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Referenceopen allclose all
Table of the test result (Main test-1)
Name of test substance: SH-0850
Test period |
From November 27, 2012 to November 30, 2012 |
|||||||||||
Presence or absence of metabolic activation |
Dose level of test substance (µg/plate) |
Number of reverse mutations (number of colonies) |
||||||||||
Base pair substitution type |
Frame shift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
-S9 mix |
Negative control |
117 119 123 |
(120+/-3) |
17 11 11 |
(13+/-3) |
31 33 33 |
(32+/-1) |
24 19 19 |
(21+/-3) |
8 14 14 |
(12+/-3) |
|
313 |
136 130 146 |
(137+/-8) |
11 11 6 |
(9+/-3) |
32 31 28 |
(30+/-2) |
26 25 19 |
(23+/-4) |
6 19 13 |
(13+/-7) |
||
625 |
130 100 128 |
(119+/-17) |
6 12 10 |
(9+/-3) |
34 27 29 |
(30+/-4) |
15 15 20 |
(17+/-3) |
12 13 13 |
(13+/-1) |
||
1250 |
163 136 146 |
(148+/-26) |
11 13 12 |
(12+/-1) |
35 22 19 |
(25+/-9) |
19 29 24 |
(24+/-5) |
11 18 19 |
(16+/-4) |
||
2500 |
178 132 134 |
(148+/-26) |
11 13 12 |
(12+/-1) |
35 22 19 |
(25+/-9) |
19 29 24 |
(24+/-5) |
11 18 19 |
(16+/-4) |
||
5000 |
177 166 146 |
(163+/-16) |
12 6 7 |
(8+/-3) |
28 39 32 |
(33+/-6) |
28 21 19 |
(23+/-5) |
14 14 10 |
(13+/-2) |
||
+S9 mix |
Negative control |
122 105 119 |
(115+/-9) |
12 10 6 |
(9+/-3) |
39 33 27 |
(33+/-6) |
27 27 30 |
(25+/-4) |
20 17 11 |
(16+/-5) |
|
313 |
109 113 113 |
(112+/-2) |
14 6 12 |
(11+/-4) |
29 18 31 |
(26+/-7) |
39 31 35 |
(35+/-4) |
20 13 15 |
(16+/-4) |
||
625 |
132 140 115 |
(129+/-13) |
12 14 12 |
(13+/-1) |
34 20 24 |
(26+/-7) |
33 24 29 |
(29+/-5) |
18 22 15 |
(18+/-4) |
||
1250 |
118 108 132 |
(119+/-13) |
15 14 11 |
(13+/-2) |
29 19 33 |
(27+/-7) |
27 22 32 |
(27+/-5) |
14 11 15 |
(13+/-2) |
||
2500 |
111 130 129 |
(123+/-11) |
10 8 7 |
(8+/-2) |
34 34 22 |
(30+/-7) |
39 25 28 |
(31+/-7) |
15 13 21 |
(16+/-4) |
||
5000 |
115 147 144 |
(135+/-18) |
14 13 8 |
(12+/-3) |
32 29 45 |
(35+/-9) |
28 34 20 |
(27+/-7) |
21 13 12 |
(15+/-5) |
||
Positive control |
Positive control groups for which S9 mix is not necessary |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
|||||
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|||||||
(Number of colonies/plate) |
747 717 696 |
(720+/-26) |
311 289 307 |
(302+/-12) |
300 362 299 |
(320+/-36) |
473 513 513 |
(500+/-23) |
1810 1698 1769 |
(1759+/-57) |
||
Positive control groups for which S9 mix is necessary |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
||||||
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|||||||
(Number of colonies/plate) |
693 701 732 |
(709+/-21) |
170 144 193 |
(169+/-25) |
772 682 689 |
(714+/-50) |
201 217 252 |
(223+/-26) |
119 103 107 |
(110+/-8) |
||
[Notes]
( ): Mean colony number +/- standard deviation (n=3)
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: Sodium azide
2AA: 2-Aminoanthracene
Table of the test result (Main test-2)
Name of test substance: SH-0850
Test period |
From November 30, 2012 to December 3, 2012 |
|||||||||||
Presence or absence of metabolic activation |
Dose level of test substance (µg/plate) |
Number of reverse mutations (number of colonies) |
||||||||||
Base pair substitution type |
Frame shift type |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
-S9 mix |
Negative control |
144 141 141 |
(142+/-2) |
11 12 18 |
(14+/-4) |
26 33 46 |
(35+/-10) |
27 15 21 |
(21+/-6) |
20 19 15 |
(18+/-3) |
|
313 |
144 146 164 |
(151+/-11) |
17 12 11 |
(13+/-3) |
46 32 41 |
(40+/-7) |
20 26 28 |
(25+/-4) |
8 17 14 |
(13+/-5) |
||
625 |
139 158 151 |
(149+/-10) |
18 14 18 |
(17+/-2) |
32 35 32 |
(33+/-2) |
26 19 32 |
(26+/-7) |
20 13 20 |
(18+/-4) |
||
1250 |
152 128 150 |
(143+/-13) |
11 10 14 |
(12+/-2) |
35 35 33 |
(34+/-1) |
21 20 18 |
(20+/-2) |
17 19 19 |
(18+/-1) |
||
2500 |
153 179 162 |
(165+/-13) |
21 11 15 |
(16+/-5)
|
40 34 26 |
(33+/-7) |
29 32 22 |
(28+/-5) |
10 21 14 |
(15+/-6) |
||
5000 |
210 217 178 |
(202+/-21) |
13 18 19 |
(17+/-3) |
34 32 38 |
(35+/-3) |
33 24 27 |
(28+/-5) |
20 11 14 |
(15+/-5) |
||
+S9 mix |
Negative control |
168 153 129 |
(150+/-20) |
14 17 18 |
(16+/-2) |
27 33 25 |
(28+/-4) |
36 40 26 |
(34+/-7) |
25 29 17 |
(24+/-6) |
|
313 |
123 135 144 |
(134+/-11) |
13 7 7 |
(9+/-3) |
45 41 48 |
(45+/-4) |
34 33 33 |
(33+/-1) |
21 29 29 |
(26+/-5) |
||
625 |
142 145 135 |
(140+/-6) |
13 6 18 |
(12+/-6) |
33 41 35 |
(36+/-4) |
33 34 31 |
(33+/-2) |
15 21 26 |
(21+/-6) |
||
1250 |
132 138 128 |
(132+/-6) |
13 8 14 |
(12+/-3) |
35 29 20 |
(28+/-8) |
40 41 21 |
(34+/-11) |
19 20 17 |
(19+/-2) |
||
2500 |
139 133 156 |
(143+/-12) |
7 12 14 |
(11+/-4) |
42 35 31 |
(36+/-6) |
21 35 28 |
(28+/-7) |
12 14 19 |
(15+/-4) |
||
5000 |
142 148 145 |
(145+/-3) |
10 11 14 |
(12+/-2) |
28 34 52 |
(38+/-12) |
25 38 34 |
(32+/-7) |
17 24 11 |
(17+/-7) |
||
Positive control |
Positive control groups for which S9 mix is not necessary |
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
|||||
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
|||||||
(Number of colonies/plate) |
786 797 727 |
(770+/-38) |
398 430 403 |
(410+/-17) |
343 401 397 |
(380+/-32) |
431 442 435 |
(436+/-6) |
2084 2138 2149 |
(2124+/-35) |
||
Positive control groups for which S9 mix is necessary |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
||||||
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|||||||
(Number of colonies/plate) |
899 873 771 |
(848+/-68) |
215 218 214 |
(216+/-2) |
772 739 781 |
(764+/-22) |
255 250 235 |
(247+/-10) |
100 110 99 |
(103+/-6) |
||
[Notes]
( ): Mean colony number +/- standard deviation (n=3)
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: Sodium azide
2AA: 2-Aminoanthracene
Historical data
Negative control (mean +/- 3S.D.)
|
-S9 mix |
+S9 mix |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Mean
Standard deviation |
107
15 |
10
4 |
25
7 |
20
6 |
10
4 |
112
16 |
10
4 |
28
6 |
28
7 |
14
4 |
Highest count
Lowest count |
152
62 |
22
1 |
46
4 |
38
2 |
22
1 |
160
64 |
22
1 |
46
10 |
49
7 |
26
2 |
Positive control (mean +/- 3S.D.)
|
-S9 mix |
+S9 mix |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|
Name |
AF-2 |
NaN3 |
AF-2 |
AF-2 |
ICR-191 |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose level (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
0.5 |
1 |
2 |
10 |
0.5 |
2 |
Mean
Standard deviation |
737
85 |
255
65 |
315
44 |
522
80 |
1462
243 |
835
115 |
213
34 |
712
125 |
271
40 |
161
24 |
Highest count
Lowest count |
992
482 |
450
60 |
447
183 |
762
282 |
2191
733 |
1180
490 |
315
111 |
1087
337 |
391
151 |
233
89 |
[Notes]
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3: Sodium azide
2AA: 2-Aminoanthracene
Test period: From May, 2012 to October, 2012
Test media: AN medium was used.
Results of the cell growth inhibition test of SH-0850
Substance |
Dose level (µg/mL) |
Treatment recovery time (h) |
S9 mix |
Cell growth rate (%) |
Precipitate of test substance in culture mediuma) |
Appearance frequency of dividing cell (%) |
Appearance frequency of abnormal cell (%)b) |
|||
At the start of treatment |
At the completion of treatment |
At the completion of cell culture |
Structural aberration |
Numerical aberration |
||||||
DMSO |
0 |
6-18 |
- |
100 |
- |
- |
- |
n.s. |
n.s. |
n.s. |
SH-0850 |
55.6 111 223 445 890 1780 3560 |
6-18 6-18 6-18 6-18 6-18 6-18 6-18 |
- - - - - - - |
91.9 101.3 100.8 89.0 85.2 66.1 48.3 |
- + + + + + + |
- - - + + + + |
- - - - - + + |
n.s. n.s. n.s. abundant abundant abundant abundant |
n.s. n.s. n.s. n.o. n.o. 0.0 4.0 |
n.s. n.s. n.s. n.o. n.o. 0.0 4.0 |
IC50: 3400 µg/mL |
||||||||||
DMSO |
0 |
6-18 |
+ |
100 |
- |
- |
- |
n.s. |
n.s. |
n.s. |
SH-0850 |
55.6 111 223 445 890 1780 3560 |
6-18 6-18 6-18 6-18 6-18 6-18 6-18 |
+ + + + + + + |
101.3 99.1 104.4 96.0 97.8 85.8 89.8 |
- + + + + + + |
- - - + + + + |
- - - - - - - |
n.s. n.s. n.s. n.s. n.s. abundant abundant |
n.s. n.s. n.s. n.s. n.s. 0.0 2.0 |
n.s. n.s. n.s. n.s. n.s. 0.0 0.0 |
IC50: >3560 µg/mL |
||||||||||
DMSO |
0 |
24-0 |
- |
100 |
- |
- |
|
n.s. |
n.s. |
n.s. |
SH-0850 |
55.6 111 223 445 890 1780 3560 |
24-0 24-0 24-0 24-0 24-0 24-0 24-0 |
- - - - - - - |
86.7 88.4 85.0 90.1 47.6 17.2 5.3 |
- + + + + + + |
- - - - + + + |
|
abundant abundant abundant abundant abundant n.s. n.s. |
n.o. n.o. n.o. 2.0 6.0 n.s. n.s. |
n.o. n.o. n.o. 0.0 4.0 n.s. n.s. |
IC50: 860 µg/mL |
||||||||||
DMSO: dimethylsulfoxide
n.s.: Specimen was not made, n.o.: Not observed, abundant: dividing cell appeared sufficiently.
a) Presence of precipitate of the test substance is shown by ‘+’, absence is shown by ‘-‘.
b) Fifty cells were observed per dose to determine appearance frequency of chromosome aberration.
Note) 3560 µg/mL, that corresponds to 10 mmol/L of the upper limit without toxicity in the guideline, was set as the highest dose and other dose levels were set as described be serial dilution at common ratio 2.
Results of chromosomal aberration test (short-term treatment)
Name of test substance: SH-0850
Treatment time (h) |
S9 mix |
Test material dose level (µg/ml) |
Number of cells with chromosome structural aberration (appearance frequency %) |
Number of gaps (appearance frequency %) |
Cell growth rate (%) |
Number of cells with chromosome numerical aberration (appearance frequency %) |
|||||||||
Number of cells observed |
Chromatid breaks |
Chromatid exchanges |
Chromosome breaks |
Chromosome exchanges |
Others |
Total number of cells with aberration |
Number of cells observed |
Polyploid cells |
Others |
Total cell number with aberrations |
|||||
6-18 |
- |
Negative control (DMSO) 0 |
100 |
2 |
2 |
0 |
0 |
0 |
4 |
0 |
100 |
100 |
0 |
0 |
0 |
100 |
3 |
0 |
0 |
0 |
0 |
3 |
1 |
100 |
0 |
0 |
0 |
||||
200 |
5(2.5) |
2(1.0) |
0(0.0) |
0(0.0) |
0(0.0) |
7(3.5) |
1(0.5) |
200 |
0(0.0) |
0(0.0) |
0 0.0) |
||||
- |
445† |
0 |
|
93.5 |
0 |
|
|||||||||
0 |
104.0 |
0 |
|||||||||||||
0 |
(98.8) |
0 |
|||||||||||||
- |
890† |
100 |
2 |
0 |
0 |
0 |
0 |
2 |
0 |
92.1 |
100 |
1 |
0 |
1 |
|
100 |
4 |
0 |
0 |
0 |
0 |
4 |
0 |
97.5 |
100 |
0 |
0 |
0 |
|||
200 |
6(3.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
6(3.0) |
0(0.0) |
(94.8) |
200 |
1(0.5) |
0(0.0) |
1(0.5) |
|||
- |
1780† |
100 |
2 |
0 |
0 |
0 |
0 |
2 |
1 |
70.4 |
100 |
1 |
0 |
1 |
|
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
63.5 |
100 |
1 |
0 |
1 |
|||
200 |
2(1.0) |
1(0.5) |
0(0.0) |
0(0.0) |
0(0.0) |
3(1.5) |
1(0.5) |
(67.0) |
200 |
2(1.0) |
0(0.0) |
2(1.0) |
|||
- |
3560† |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
52.0 |
100 |
2 |
0 |
2 |
|
100 |
4 |
0 |
0 |
0 |
0 |
4 |
0 |
49.8 |
100 |
2 |
0 |
2 |
|||
200 |
4(2.0) |
1(0.5) |
0(0.0) |
0(0.0) |
0(0.0) |
5(2.5) |
0(0.0) |
(50.9) |
200 |
4(2.0) |
0(0.0) |
4(2.0) |
|||
- |
Positive control (MMC) 0.1 |
100 |
60 |
45 |
0 |
0 |
0 |
75 |
0 |
|
100 |
0 |
0 |
0 |
|
100 |
54 |
67 |
0 |
0 |
0 |
80 |
1 |
100 |
0 |
0 |
0 |
||||
200 |
114(57.0) |
112(56.0) |
0(0.0) |
0(0.0) |
0(0.0) |
155(77.5) |
1(0.05) |
200 |
0(0.0) |
0(0.0) |
0(0.0) |
||||
+ |
Negative control (DMSO) 0 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
100 |
0 |
0 |
0 |
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
0 |
0 |
0 |
||||
200 |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
200 |
0(0.0) |
0(0.0) |
0(0.0) |
||||
+ |
890† |
100 |
0 |
0 |
1 |
0 |
0 |
1 |
0 |
101.1 |
100 |
0 |
0 |
0 |
|
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
106.2 |
100 |
0 |
0 |
0 |
|||
200 |
1(0.5) |
0(0.0) |
1(0.05) |
0(0.0) |
0(0.0) |
2(1.0) |
0(0.0) |
(103.7) |
200 |
0(0.0) |
0(0.0) |
0(0.0) |
|||
+ |
1780† |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
98.9 |
100 |
0 |
0 |
0 |
|
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
101.1 |
100 |
1 |
0 |
1 |
|||
200 |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
(100.0) |
200 |
1(0.5) |
0(0.0) |
1(0.05) |
|||
+ |
3560† |
100 |
1 |
1 |
0 |
0 |
0 |
2 |
0 |
101.4 |
100 |
0 |
0 |
0 |
|
100 |
1 |
1 |
0 |
0 |
0 |
2 |
0 |
98.3 |
100 |
1 |
0 |
1 |
|||
200 |
2(1.0) |
2(1.0) |
0(0.0) |
0(0.0) |
0(0.0) |
4(2.0) |
0(0.0) |
(99.9) |
200 |
1(0.5) |
0(0.0) |
1(0.5) |
|||
+ |
Positive control (CPA) 6 |
100 |
7 |
17 |
0 |
0 |
0 |
23 |
0 |
|
100 |
0 |
0 |
0 |
|
100 |
15 |
20 |
0 |
0 |
0 |
34 |
2 |
100 |
0 |
0 |
0 |
||||
200 |
22(11.0) |
37(18.5) |
0(0.0) |
(0.0) |
0(0.0) |
57(28.5) |
2(1.0) |
200 |
0(0.0) |
0(0.0) |
0(0.0) |
||||
Treatment time indicates treatment time – recovery time.
Regarding number of cells with aberration, data per each plate of each group is shown in the first and second line, and its total is shown in the third line.
Regarding cell growth rate, value per each plate of each test substance is shown in the first and second line, and its mean is shown in the third line.
DMSO: dimethylsulfoxide, MMC: mitomycin C, CPA: cyclophosphamide hydrate
†: Precipitate of the test substance was observed at all dose range at the start and completion of the treatment and at and above 1780 µg/ml at the completion of culture without S9 mix in short-term treatment.
Precipitate of the test substance was observed at all dose range at the start and completion of the treatment with S9 mix in the short-term treatment.
Specimen was not observed at 445 µg/ml without S9 mix in short-term treatment.
Results of chromosomal aberration test (continuous treatment)
Name of test substance: SH-0850
Treatment time (h) |
Test material dose level (µg/ml) |
Number of cells with chromosome structural aberration (appearance frequency %) |
Number of gaps (appearance frequency %) |
Cell growth rate (%) |
Number of cells with chromosome numerical aberration (appearance frequency %) |
|||||||||
Number of cells observed |
Chromatid breaks |
Chromatid exchanges |
Chromosome breaks |
Chromosome exchanges |
Others |
Total number of cells with aberration |
Number of cells observed |
Polyploid cells |
Others |
Total cell number with aberrations |
||||
24-0 |
Negative control (DMSO) 0 |
100 |
3 |
0 |
0 |
1 |
0 |
4 |
1 |
100 |
100 |
0 |
0 |
0 |
100 |
3 |
0 |
0 |
0 |
0 |
3 |
0 |
100 |
1 |
0 |
1 |
|||
200 |
6(3.0) |
0(0.0) |
0(0.0) |
1(0.5) |
0(0.0) |
7(3.5) |
1(0.5) |
200 |
1(0.5) |
0(0.0) |
1(0.5) |
|||
3155† |
0 |
|
105.9 |
0 |
|
|||||||||
0 |
101.5 |
0 |
||||||||||||
0 |
(103.7) |
0 |
||||||||||||
445† |
100 |
3 |
0 |
0 |
0 |
0 |
3 |
0 |
98.5 |
100 |
0 |
0 |
0 |
|
100 |
4 |
0 |
0 |
0 |
0 |
4 |
0 |
98.8 |
100 |
1 |
0 |
1 |
||
200 |
7(3.5) |
0(0.0) |
0(0.0) |
0(0.0) |
0(0.0) |
7(3.5) |
0(0.0) |
(98.7) |
200 |
1(0.5) |
0(0.0) |
1(0.5) |
||
629† |
100 |
7 |
1 |
0 |
0 |
0 |
8 |
0 |
87.9 |
100 |
0 |
0 |
0 |
|
100 |
7 |
1 |
0 |
0 |
0 |
8 |
0 |
88.3 |
100 |
2 |
0 |
2 |
||
200 |
14(7.0) |
2(1.0) |
0(0.0) |
0(0.0) |
0(0.0) |
16(8.0) |
0(0.0) |
(88.1) |
200 |
2(1.0) |
0(0.0) |
2(1.0) |
||
890† |
100 |
10 |
5 |
0 |
0 |
0 |
15 |
0 |
74.4 |
100 |
2 |
0 |
2 |
|
100 |
14 |
2 |
0 |
0 |
0 |
16 |
0 |
73.7 |
100 |
0 |
0 |
0 |
||
200 |
24(12.0) |
7(3.5) |
0(0.0) |
0(0.0) |
0(0.0) |
31(15.5) |
0(0.0) |
(74.1) |
200 |
2(1.0) |
0(0.0) |
2(1.0) |
||
1260† |
100 |
7 |
1 |
0 |
0 |
0 |
8 |
0 |
54.7 |
100 |
0 |
0 |
0 |
|
100 |
5 |
3 |
0 |
0 |
0 |
8 |
0 |
43.8 |
100 |
2 |
0 |
2 |
||
200 |
12(6.0) |
4(2.0) |
0(0.0) |
0(0.0) |
0(0.0) |
16(8.0) |
0(0.0) |
(49.3) |
200 |
2(1.0) |
0(0.0) |
2(1.0) |
||
1780† |
0 |
|
28.7 |
0 |
|
|||||||||
0 |
26.8 |
0 |
||||||||||||
0 |
(27.8) |
0 |
||||||||||||
Positive control (MMC) 0.05 |
100 |
48 |
51 |
0 |
0 |
0 |
75 |
0 |
|
100 |
0 |
0 |
0 |
|
100 |
53 |
43 |
0 |
0 |
0 |
75 |
0 |
100 |
0 |
0 |
0 |
|||
200 |
101(50.5) |
94(47.0) |
0(0.0) |
0(0.0) |
0(0.0) |
150(75.0) |
0(0.0) |
200 |
0(0.0) |
0(0.0) |
0(0.0) |
|||
Treatment time indicates treatment time – recovery time.
Regarding number of cells with aberration, data per each plate of each group is shown in the first and second line, and its total is shown in the third line.
Regarding cell growth rate, value per each plate of each test substance is shown in the first and second line, and its mean is shown in the third line.
DMSO: dimethylsulfoxide, MMC: mitomycin C
†: Precipitate of the test substance was observed at all dose range at the start and completion of the treatment and at 629 µg/ml at the completion of treatment.
Specimen was not observed at 315 and 1780 µg/ml without S9 mix in short-term treatment.
Background data at Hita Laboratory
Negative Control
Treatment method |
Appearance frequency of the aberration cell (%, average ± standard deviation) |
||
Structural aberration |
Numerical aberration |
||
Short term treatment |
-S9 mix |
1.2 ± 0.89 |
0.3 ± 0.30 |
+S9 mix |
0.9 ± 0.67 |
0.1 ± 0.28 |
|
24 hours continuous treatment method |
1.8 ± 1.18 |
0.1 ± 0.28 |
|
Treatment method |
Appearance frequency range of the aberration cell (%, average ± 3 standard deviations) |
||
Structural aberration |
Numerical aberration |
||
Short term treatment |
-S9 mix |
0.0 - 3.9 |
0.0 - 1.2 |
+S9 mix |
0.0 - 2.9 |
0.0 - 0.9 |
|
24 hours continuous treatment method |
0.0 - 5.3 |
0.0 - 0.9 |
|
If lower limit of the range is less than 0, 0.0 is stated.
Positive control
Treatment method |
Material name |
Dose (µg/ml) |
Appearance frequency of the aberration cell (%, average ± standard deviation) |
||
Structural aberration |
Numerical aberration |
||||
Short term treatment |
-S9 mix |
MMC |
0.1 |
76.6 ± 4.54 |
0.2 ± 0.24 |
+S9 mix |
CPA |
6 |
26.8 ± 6.02 |
0.1 ± 0.24 |
|
24 hours continuous treatment method |
MMC |
0.05 |
72.7 ± 4.62 |
0.1 ± 0.22 |
|
Treatment method |
Appearance frequency range of the aberration cell (%, average ± 2 standard deviations) |
||
Structural aberration |
Numerical aberration |
||
Short term treatment |
-S9 mix |
67.5 – 85.7 |
0.0 – 0.7 |
+S9 mix |
14.8 – 38.8 |
0.0 – 0.6 |
|
24 hours continuous treatment method |
63.5 – 81.9 |
0.0 – 0.5 |
|
If lower limit of the range is less than 0, 0.0 is stated.
All of them are the latest 20 experimental data that were finalized before March 21, 2013.
Results of the Preliminary Toxicity Test
(3-hour treatment in the presence of metabolic activation)
Test item (or solvent) concentration |
Cell number, cells/mL (Relative Survival*, %) after treatment Day 0 |
Cell number, cells/mL (Relative Survival*, %) on Day 1 |
Cell number, cells/mL (Relative Survival*, %) on Day 2 |
Observation at the beginning / after treatment ** |
Untreated control |
2.70E±05 (91) |
4.40E±05 (92) |
6.90E±05 (101) |
B: normal A: normal (pH: 7.2, osmolality: 301 mmol/kg) |
Negative (vehicle) control (1% DMSO) |
2.98E±05 (100) |
4.80E±04 (100) |
6.80E±05 (100) |
B: normal A: normal (pH: 7.2, osmolality: 451 mmol/kg) |
500 µg/mL |
2.50E±05 (84) |
4.75E±05 (99) |
6.00E±05 (88) |
B: normal A: precipitate#(pH: 7.2, osmolality: 451 mmol/kg) |
250 µg/mL |
2.48E±05 (83) |
4.60E±05 (96) |
7.85E±05 (115) |
B: normal A: precipitate#(pH: 7.2, osmolality: 453 mmol/kg) |
125 µg/mL |
2.73E±05 (92) |
4.20E±04 (88) |
7.20E±05 (106) |
B: normal A: normal (pH: 7.2, osmolality: 455 mmol/kg) |
62.5 µg/mL |
2.75E±05 (92) |
3.75E±05 (78) |
6.95E±05 (102) |
B: normal A: normal (pH: 7.2, osmolality: 455 mmol/kg) |
31.25 µg/mL |
2.53E±05 (85) |
5.05E±05 (105) |
6.25E±05 (92) |
B: normal A: normal (pH: 7.2, osmolality: 455 mmol/kg) |
15.625 µg/mL |
2.83E±05 (95) |
5.05E±05 (105) |
6.65E±05 (98) |
B: normal A: normal (pH: 7.2, osmolality: 453 mmol/kg) |
7.813 µg/mL |
2.53E±05 (85) |
5.10E±05 (106) |
5.40E±05 (79) |
B: normal A: normal (pH: 7.2, osmolality: 458 mmol/kg) |
3.906 µg/mL |
2.75E±05 (92) |
4.90E±05 (102) |
6.40E±05 (94) |
B: normal A: normal (pH: 7.2, osmolality: 456 mmol/kg) |
1.953 µg/mL |
2.98E± 05 (100) |
4.85E±05 (101) |
6.35E±05 (93) |
B: normal A: normal (pH: 7.2, osmolality: 453 mmol/kg) |
0.977 µg/mL |
2.48E±05 (83) |
3.80E±05 (79) |
6.35E±05 (93) |
B: normal A: normal (pH: 7.2, osmolality: 457 mmol/kg) |
*: compared to the relevant negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Results of the Preliminary Toxicity Test
(3-hour treatment in the absence of metabolic activation)
Test item (or solvent) concentration |
Cell number, cells/mL (Relative Survival*, %) after treatment Day 0 |
Cell number, cells/mL (Relative Survival*, %) on Day 1 |
Cell number, cells/mL (Relative Survival*, %) on Day 2 |
Observation at the beginning / after treatment ** |
Untreated control |
3.85E±05 (121) |
4.50E±05 (101) |
6.60E±05 (95) |
B: normal A: normal (pH: 7.2, osmolality: 274 mmol/kg) |
Negative (vehicle) control (1% DMSO) |
3.18E±05 (100) |
4.45E±05 (100) |
6.95E±05 (100) |
B: normal A: normal (pH: 7.2, osmolality: 434 mmol/kg) |
500 µg/mL |
3.55E±05 (112) |
5.30E±05 (119) |
7.65E±05 (11) |
B: normal A: precipitate#(pH: 7.2, osmolality: 429 mmol/kg) |
250 µg/mL |
3.68E±05 (116) |
4.30E±05 (97) |
8.10E±05 (117) |
B: normal A: normal (pH: 7.2, osmolality: 426 mmol/kg) |
125 µg/mL |
3.98E±05 (125) |
5.35E±05 (120) |
7.00E±05 (101) |
B: normal A: normal (pH: 7.2, osmolality: 432 mmol/kg) |
62.5 µg/mL |
4.58E±05 (144) |
3.35E±05 (75) |
8.00E±05 (115) |
B: normal A: normal (pH: 7.2, osmolality: 434 mmol/kg) |
31.25 µg/mL |
3.28E±05 (103) |
4.15E±05 (93) |
7.50E±05 (108) |
B: normal A: normal (pH: 7.2, osmolality: 430 mmol/kg) |
15.625 µg/mL |
3.38E±05 (106) |
5.15E±05 (116) |
8.40E±05 (121) |
B: normal A: normal (pH: 7.2, osmolality: 430 mmol/kg) |
7.813 µg/mL |
3.45E±05 (109) |
4.90E±05 (110) |
6.85E±05 (99) |
B: normal A: normal (pH: 7.2, osmolality: 434 mmol/kg) |
3.906 µg/mL |
3.75E±05 (118) |
4.10E±05 (92) |
7.30E±05 (105) |
B: normal A: normal (pH: 7.2, osmolality: 431 mmol/kg) |
1.953 µg/mL |
3.83E±05 (120) |
4.70E±05 (106) |
6.15E±05 (88) |
B: normal A: normal (pH: 7.2, osmolality: 433 mmol/kg) |
0.977 µg/mL |
4.15E±05 (131) |
3.45E±05 (78) |
8.20E±05 (118) |
B: normal A: normal (pH: 7.2, osmolality: 432 mmol/kg) |
*: compared to the relevant negative (vehicle) control (1% (v/v) DMSO) #: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Summary Tables of Survival Data
Survival Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
+ |
3 |
A1 |
500 µg/mL |
59 |
1.171 |
97 |
116 |
250 µg/mL |
49 |
1.287 |
101 |
96 |
|||
125 µg/mL |
58 |
1.181 |
108 |
107 |
|||
62.5 µg/mL |
38 |
1.446 |
98 |
112 |
|||
31.25 µg/mL |
38 |
1.446 |
110 |
119 |
|||
15.625 µg/mL |
36 |
1.479 |
105 |
121 |
|||
Vehicle control |
55 |
1.215 |
100 |
100 |
|||
Untreated control |
53 |
1.238 |
114 |
111 |
|||
Positive control (CP) |
157 |
0.559 |
34 |
19 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
- |
3 |
A1 |
500 µg/mL |
67 |
1.091 |
89 |
91 |
250 µg/mL |
67 |
1.091 |
90 |
92 |
|||
125 µg/mL |
90 |
0.907 |
88 |
90 |
|||
62.5 µg/mL |
100 |
0.841 |
78 |
98 |
|||
31.25 µg/mL |
76 |
1.012 |
86 |
84 |
|||
15.625 µg/mL |
81 |
0.973 |
89 |
99 |
|||
Vehicle control |
52 |
1.250 |
100 |
100 |
|||
Untreated control |
62 |
1.140 |
96 |
114 |
|||
Positive control (NQO) |
158 |
0.555 |
47 |
35 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
+ |
3 |
A2 |
500 µg/mL |
87 |
0.928 |
92 |
110 |
250 µg/mL |
53 |
1.238 |
126 |
128 |
|||
125 µg/mL |
72 |
1.046 |
107 |
121 |
|||
62.5 µg/mL |
73 |
1.038 |
126 |
105 |
|||
31.25 µg/mL |
72 |
1.046 |
119 |
99 |
|||
15.625 µg/mL |
58 |
1.181 |
129 |
102 |
|||
Vehicle control |
71 |
1.055 |
100 |
100 |
|||
Untreated control |
63 |
1.130 |
114 |
163 |
|||
Positive control (CP) |
336 |
0.083 |
9 |
6 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 2 (II.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
- |
24 |
A2 |
500 µg/mL |
143 |
0.617 |
101 |
111 |
250 µg/mL |
153 |
0.575 |
92 |
88 |
|||
125 µg/mL |
151 |
0.583 |
87 |
90 |
|||
62.5 µg/mL |
143 |
0.617 |
95 |
115 |
|||
31.25 µg/mL |
172 |
0.502 |
83 |
131 |
|||
15.625 µg/mL |
162 |
0.539 |
90 |
95 |
|||
Vehicle control |
132 |
0.667 |
100 |
100 |
|||
Untreated control |
129 |
0.682 |
108 |
114 |
|||
Positive control (NQO) |
216 |
0.360 |
47 |
51 |
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 3 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
+ |
3 |
A3 |
500 µg/mL |
82 |
0.965 |
80 |
102 |
250 µg/mL |
101 |
0.835 |
61 |
103 |
|||
125 µg/mL |
96 |
0.866 |
69 |
93 |
|||
62.5 µg/mL |
72 |
1.046 |
82 |
94 |
|||
31.25 µg/mL |
89 |
0.914 |
69 |
111 |
|||
15.625 µg/mL |
65 |
1.110 |
85 |
124 |
|||
Vehicle control |
60 |
1.160 |
100 |
100 |
|||
Untreated control |
63 |
1.130 |
102 |
141 |
|||
Positive control (CP) |
246 |
0.278 |
21 |
20 |
A3 = Assay 3
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 3 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
- |
3 |
A3 |
500 µg/mL |
103 |
0.822 |
91 |
108 |
250 µg/mL |
97 |
0.860 |
92 |
85 |
|||
125 µg/mL |
112 |
0.770 |
81 |
113 |
|||
62.5 µg/mL |
89 |
0.914 |
98 |
92 |
|||
31.25 µg/mL |
101 |
0.835 |
93 |
91 |
|||
15.625 µg/mL |
111 |
0.776 |
86 |
94 |
|||
Vehicle control |
82 |
0.965 |
100 |
100 |
|||
Untreated control |
97 |
0.860 |
93 |
114 |
|||
Positive control (NQO) |
160 |
0.547 |
63 |
56 |
A3 = Assay 3
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Survival Results of Assay 3 (iII.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
Relative survival§(%RS) |
Relative Total Growth (%RTG) |
- |
24 |
A3 |
500 µg/mL |
152 |
0.579 |
84 |
91 |
250 µg/mL |
132 |
0.667 |
100 |
100 |
|||
125 µg/mL |
148 |
0.596 |
93 |
107 |
|||
62.5 µg/mL |
143 |
0.617 |
89 |
91 |
|||
31.25 µg/mL |
152 |
0.579 |
91 |
100 |
|||
15.625 µg/mL |
139 |
0.635 |
94 |
87 |
|||
Vehicle control |
128 |
0.687 |
100 |
100 |
|||
Untreated control |
128 |
0.687 |
101 |
145 |
|||
Positive control (NQO) |
195 |
0.424 |
46 |
46 |
A3 = Assay 3
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
§ = Relative survival values (%) corrected with the post treatment cell concentrations.
Summary Tables of Viability Data
Viability Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
+ |
3 |
A1 |
500 µg/mL |
81 |
0.973 |
250 µg/mL |
79 |
0.988 |
|||
125 µg/mL |
72 |
1.046 |
|||
62.5 µg/mL |
83 |
0.957 |
|||
31.25 µg/mL |
86 |
0.935 |
|||
15.625 µg/mL |
93 |
0.886 |
|||
Vehicle control |
86 |
0.935 |
|||
Untreated control |
81 |
0.973 |
|||
Positive control (CP) |
218 |
0.354 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
Viability Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
- |
3 |
A1 |
500 µg/mL |
109 |
0.787 |
250 µg/mL |
102 |
0.829 |
|||
125 µg/mL |
113 |
0.765 |
|||
62.5 µg/mL |
95 |
0.873 |
|||
31.25 µg/mL |
115 |
0.754 |
|||
15.625 µg/mL |
88 |
0.921 |
|||
Vehicle control |
107 |
0.799 |
|||
Untreated control |
104 |
0.816 |
|||
Positive control (NQO) |
150 |
0.588 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 µg/mL)
Viability Results of Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
+ |
3 |
A2 |
500 µg/mL |
113 |
0.765 |
250 µg/mL |
90 |
0.907 |
|||
125 µg/mL |
113 |
0.765 |
|||
62.5 µg/mL |
99 |
0.847 |
|||
31.25 µg/mL |
108 |
0.793 |
|||
15.625 µg/mL |
118 |
0.737 |
|||
Vehicle control |
121 |
0.722 |
|||
Untreated control |
77 |
1.004 |
|||
Positive control (CP) |
308 |
0.138 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
Viability Results of Assay 2 (II.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
- |
24 |
A2 |
500 µg/mL |
106 |
0.805 |
250 µg/mL |
99 |
0.847 |
|||
125 µg/mL |
103 |
0.822 |
|||
62.5 µg/mL |
101 |
0.835 |
|||
31.25 µg/mL |
67 |
1.091 |
|||
15.625 µg/mL |
106 |
0.805 |
|||
Vehicle control |
89 |
0.914 |
|||
Untreated control |
121 |
0.722 |
|||
Positive control (NQO) |
121 |
0.722 |
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
Viability Results of Assay 3 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
+ |
3 |
A3 |
500 µg/mL |
101 |
0.835 |
250 µg/mL |
87 |
0.928 |
|||
125 µg/mL |
111 |
0.776 |
|||
62.5 µg/mL |
102 |
0.829 |
|||
31.25 µg/mL |
101 |
0.835 |
|||
15.625 µg/mL |
68 |
1.082 |
|||
Vehicle control |
125 |
0.701 |
|||
Untreated control |
92 |
0.893 |
|||
Positive control (CP) |
188 |
0.446 |
A3 = Assay 3
+ = in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
Viability Results of Assay 3 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
- |
3 |
A3 |
500 µg/mL |
90 |
0.907 |
250 µg/mL |
112 |
0.770 |
|||
125 µg/mL |
76 |
1.012 |
|||
62.5 µg/mL |
104 |
0.816 |
|||
31.25 µg/mL |
129 |
0.682 |
|||
15.625 µg/mL |
130 |
0.677 |
|||
Vehicle control |
99 |
0.847 |
|||
Untreated control |
92 |
0.893 |
|||
Positive control (NQO) |
141 |
0.626 |
A3 = Assay 3
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 µg/mL)
Viability Results of Assay 3 (III.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating efficiency (PE) |
- |
24 |
A3 |
500 µg/mL |
125 |
0.701 |
250 µg/mL |
122 |
0.717 |
|||
125 µg/mL |
112 |
0.770 |
|||
62.5 µg/mL |
129 |
0.682 |
|||
31.25 µg/mL |
115 |
0.754 |
|||
15.625 µg/mL |
113 |
0.765 |
|||
Vehicle control |
121 |
0.722 |
|||
Untreated control |
115 |
0.754 |
|||
Positive control (NQO) |
166 |
0.524 |
A3 = Assay 3
- = in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
Summary Tables of Mutagenicity Data
Mutagenicity Results of Assay 1 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
+ |
3 |
500 µg/mL |
535/768 |
195/768 |
38/768 |
0.286 |
185.8 |
250 µg/mL |
488/768 |
233/768 |
47/768 |
0.305 |
229.4 |
||
125 µg/mL |
524/768 |
200/768 |
44/768 |
0.392 |
182.7 |
||
62.5 µg/mL |
531/768 |
189/768 |
48/768 |
0.123 |
192.7 |
||
31.25 µg/mL |
510/768 |
217/768 |
41/768 |
0.091 |
218.9 |
||
15.625 µg/mL |
554/768 |
170/768 |
44/768 |
0.326 |
184.3 |
||
Vehicle control |
522/768 |
205/768 |
41/768 |
-- |
206.4 |
||
Untreated control |
522/768 |
202/768 |
44/768 |
-- |
198.5 |
||
Positive control (CP: 4 µg/mL) |
243/768 |
197/768 |
328/768 |
◊◊ 3.40E-12 |
1626.0* |
In linear trend analysisβ2/var (β) = 0.04, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
+= in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 1 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
- |
3 |
500 µg/mL |
537/768 |
189/768 |
42/768 |
0.394 |
227.3 |
250 µg/mL |
564/768 |
174/768 |
30/768 |
0.091 |
186.3 |
||
125 µg/mL |
541/768 |
179/768 |
48/768 |
0.440 |
229.1 |
||
62.5 µg/mL |
546/768 |
184/768 |
38/768 |
0.007 |
195.4 |
||
31.25 µg/mL |
524/768 |
191/768 |
53/768 |
1.324 |
253.7 |
||
15.625 µg/mL |
539/768 |
193/768 |
36/768 |
0.025 |
192.3 |
||
Vehicle control |
559/768 |
175/768 |
34/768 |
-- |
198.9 |
||
Untreated control |
548/768 |
180/768 |
40/768 |
-- |
206.7 |
||
Positive control (NQO: 0.15 µg/mL) |
279/768 |
232/768 |
257/768 |
◊◊ 3.63E-10 |
861.8* |
In linear trend analysisβ2/var (β) = 0.10, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
-= in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
+ |
3 |
500 µg/mL |
615/768 |
108/768 |
45/768 |
0.352 |
145.3 |
250 µg/mL |
609/768 |
99/768 |
60/768 |
1.350 |
127.9 |
||
125 µg/mL |
630/768 |
86/768 |
52/768 |
1.157 |
129.5 |
||
62.5 µg/mL |
615/768 |
95/768 |
58/768 |
1.091 |
131.1 |
||
31.25 µg/mL |
634/768 |
96/768 |
38/768 |
1.861 |
120.9 |
||
15.625 µg/mL |
629/768 |
85/768 |
54/768 |
0.787 |
135.4 |
||
Vehicle control |
604/768 |
106/768 |
58/768 |
-- |
166.4 |
||
Untreated control |
605/768 |
92/768 |
71/768 |
-- |
118.8 |
||
Positive control (CP: 4 µg/mL) |
400/768 |
134/768 |
234/768 |
◊◊ 1.71E-08 |
2366.3* |
In linear trend analysisβ2/var (β) = 0.02, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
+= in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (II.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
- |
24 |
500 µg/mL |
571/768 |
124/768 |
73/768 |
0.719 |
184.2 |
250 µg/mL |
584/768 |
118/768 |
66/768 |
0.046 |
161.6 |
||
125 µg/mL |
563/768 |
126/768 |
79/768 |
0.945 |
188.8 |
||
62.5 µg/Ml |
586/768 |
121/768 |
61/768 |
0.050 |
162.0 |
||
31.25 µg/mL |
554/768 |
137/768 |
77/768 |
0.025 |
149.7 |
||
15.625 µg/mL |
581/768 |
120/768 |
67/768 |
0.304 |
173.4 |
||
Vehicle control |
579/768 |
122/768 |
67/768 |
-- |
154.6 |
||
Untreated control |
593/768 |
104/768 |
71/768 |
-- |
179/1 |
||
Positive control (NQO: 0.1 µg/mL) |
231/768 |
252/768 |
285/768 |
◊◊ 7.70E-11 |
832.2* |
In linear trend analysisβ2/var (β) = 0.65, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
-= in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 3 (I.)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
+ |
3 |
500 µg/mL |
651/768 |
58/768 |
59/768 |
1.709 |
99.0 |
250 µg/mL |
626/768 |
86/768 |
56/768 |
0.823 |
110.2 |
||
125 µg/mL |
642/768 |
64/768 |
62/768 |
0.478 |
115.5 |
||
62.5 µg/mL |
647/768 |
65/768 |
56/768 |
1.291 |
103.5 |
||
31.25 µg/mL |
594/768 |
97/768 |
77/768 |
0.265 |
153.9 |
||
15.625 µg/mL |
625/768 |
81/768 |
62/768 |
2.316 |
95.2 |
||
Vehicle control |
634/768 |
74/768 |
60/768 |
-- |
136.7 |
||
Untreated control |
614/768 |
80/768 |
74/768 |
-- |
125.3 |
||
Positive control (CP: 4 µg/mL) |
245/768 |
195/768 |
328/768 |
◊◊ 1.38E-08 |
1279.8* |
In linear trend analysisβ2/var (β) = 0.65, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
+= in the presence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 3 (II.)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
- |
3 |
500 µg/mL |
636/768 |
83/768 |
49/768 |
0.192 |
104.0 |
250 µg/mL |
658/768 |
57/768 |
53/768 |
0.312 |
100.4 |
||
125 µg/mL |
643/768 |
72/768 |
53/768 |
1.233 |
87.7 |
||
62.5 µg/mL |
662/768 |
64/768 |
42/768 |
0.871 |
91.0 |
||
31.25 µg/mL |
654/768 |
58/768 |
56/768 |
0.004 |
117.8 |
||
15.625 µg/mL |
614/768 |
89/768 |
65/768 |
2.124 |
165.3 |
||
Vehicle control |
631/768 |
65/768 |
72/768 |
-- |
116.0 |
||
Untreated control |
645/768 |
69/768 |
54/768 |
-- |
97.7 |
||
Positive control (NQO: 0.15 µg/mL) |
398/768 |
167/768 |
203/768 |
◊◊ 1.73E-09 |
524.9* |
In linear trend analysisβ2/var (β) = 0.38, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
-= in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 3 (III.)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item or control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ◊ |
Mutation frequency |
- |
24 |
500 µg/mL |
604/768 |
118/768 |
46/768 |
0.972 |
171.2 |
250 µg/mL |
623/768 |
102/768 |
43/768 |
0.073 |
146.0 |
||
125 µg/mL |
618/768 |
96/768 |
54/768 |
0.015 |
141.1 |
||
62.5 µg/mL |
642/768 |
89/768 |
37/768 |
0.034 |
131.4 |
||
31.25 µg/mL |
602/768 |
126/768 |
40/768 |
0.534 |
161.6 |
||
15.625 µg/mL |
620/768 |
102/768 |
46/768 |
0.008 |
140.0 |
||
Vehicle control |
630/768 |
107/768 |
31/768 |
-- |
137.2 |
||
Untreated control |
636/768 |
92/768 |
40/768 |
-- |
125.1 |
||
Positive control (NQO: 0.1 µg/mL) |
291/768 |
303/768 |
174/768 |
◊◊ 6.88E-12 |
925.7* |
In linear trend analysisβ2/var (β) = 0.89, not significant
*= Statistically significant
◊= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.24(at p<0.05).
◊◊= Evaluated by T-test for independent samples. Significant at p<0.05.
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn β = slope of the curve var(β) = variance of the slope
-= in the absence of S9-mix
Negative (vehicle) control = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Historical Control Data
Mutation Frequency of the Negative Controls |
||||||
|
Culture Medium |
Distilled water |
||||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
3h,S9+ |
3h,S9- |
24h,S9- |
Average SD Min. Max. n |
83.5 27.1 39.3 164.4 34 |
91.7 24.5 52.6 147.2 17 |
92.6 33.7 41.7 170.4 18 |
72.7 29.4 33.4 136.8 12 |
95.9 39.0 55.1 161.4 6 |
70.8 22.7 43.2 104.9 6 |
|
Dimethyl sulfoxide (DMSO) |
|
|
|
||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
|
|
|
Average SD Min. Max. n |
82.1 24.4 44.2 142.2 54 |
84.2 25.3 33.7 138.0 32 |
80.4 24.8 47.1 159.4 26 |
|
|
|
Mutation Frequency of the Positive Controls |
||||||
|
Cyclophosphamide |
4-Nitroquonoline-N-oxide |
||||
Treatments |
3h,S9+ |
|
|
|
3h,S9- |
24h,S9- |
Average SD Min. Max. n |
954.5 530.9 196.1 2642.5 74 |
|
|
|
612.2 396.2 207.0 1687.3 39 |
679.4 300.3 245.0 1431.0 35 |
h = hour
SD = Standard Deviation
S9+ = experiment with metabolic activation
S9- = experiment without metabolic activation
n = number of cases
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus assay
SH-0850 does not induce micronuclei.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July 19, 2013 to October 2, 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- "OECD Guidelines for the Testing of Chemicals, No.474, Mammalian Erythrocyte Micronucleus Test " (Adopted: July 21, 1997)
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- "Micronucleus test in rodents" of "Mutagenicity Test" stipulated in the "Test Methods of the New Chemical Substances etc." (March 31, 2011, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, partly amended by Yakushokuhatsu 0402 No.1, Heisei 24.03.28 Seikyoku No.2, Kanpokihatsu No. 120402001 on April 2, 2012)
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- No further details specified in the study report.
- Species:
- mouse
- Strain:
- other: Crlj:CD1(ICR), SPF,
- Details on species / strain selection:
- Mouse is commonly used for micronucleus test because it is easy to observe micronuclei and plenty of background data are available.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Species, strain, sex and supplier: Mouse, Crlj:CD1(ICR), SPF, male and female, Charles River Laboratories Japan, Inc. (Hino Breeding Center)
Weeks of age on arrival and number of animals purchased
Preliminary test: 6 weeks old, 24 males, 24 females
Micronucleus test: 6 weeks old, 35 males
Weeks of age and body weight range at administration
Preliminary test: 7 weeks old, body weight range of within +/-20% of mean value
Micronucleus test: 7 weeks old, body weight range of within +/-20% of mean value
Quarantine, acclimatization method and period
Appearance of the animals was observed on receipt and animals which were judged to be healthy were subjected to quarantine and acclimatization for more than 5 days. During this period, clinical condition was observed once daily. Body weight was measured on receipt and at the end of quarantine.
Allocation
Animals which grew steadily during quarantine and acclimatization period and were judged to be healthy by the administrator of experimental animals were allocated to the groups by random sampling based on body weight level on the day before the start of administration. The animals were allocated to 7 groups of 3 males and females, respectively, in the preliminary test, and to 5 groups of 6 animals in the micronucleus test.
The animals were administered at 1 day after group allocation. Excessive animals which were not used for test were excluded from the study after allocation and euthanized by cervical dislocation under ether anesthesia.
Identification method
Animal
On receipt, animal receipt numbers were assigned randomly. 5 or less animals were housed in each cage in ascending order of the number. However, for animals No. 21 - 24 used in the preliminary test 4 males and 4 females were housed respectively in each cage. 4 animals which were assigned with a smaller number in each cage were marked on their tails by oil ink (the 5th animals had no marking) for identification.
After allocation, the new animal numbers were assigned to the animals and 3 or less animals were housed in each cage (preliminary test: 1 or 2 cages/dose, micronucleus test: 2 cages/dose). The animals were identified on dorsal hair by marking of the number 1 - 3 in the preliminary test and 1 - 6 in the nucleus test in ascending order of the animal number for each dose level. For marking after allocation, water based ink (AnimalMarker FG-2000, Fuchigami-Kikai) was used and different colors were used for each dose level.
Cage
Cages were identified by labels which showed the study number, sex, animal receipt number, cage number, receipt date, weeks of age at receipt, species, strain and name of the study director before allocation, and by labels which showed the study number, cage number, rearing period (the date of allocation to the end date of rearing), strain, species, sex, group, dose, animal number and the name of the study director after allocation.
Weeks of age of the animals at the start of administration, number of animals used in the test and body weight at the initial administration
Preliminary test: 7 weeks old, 21 males, body weight at the initial administration: 27.8 - 33.0 g, mean: 31.1 g; 7 weeks old, 21 females, body weight at the initial administration: 23.1 - 28.7 g, mean: 25.7 g
Micronucleus test: 7 weeks old, 30 males, body weight at the initial administration: 32.0 – 37.4 g, mean: 35.3 g (positive control group is excluded from the values of body weight at the initial administration and mean.)
Husbandry
Environmental conditions
Animal room: During quarantine period: Quarantine room 2; After the end of quarantine: Animal room 4
After allocation for the preliminary test, the animals were maintained in Safety Clean Lack NCR-05 (Shibata Scientific Technology Ltd.,) which was located in Animal room.
Temperature: 21 - 25ºC (measured value: 22.8 - 24.3ºC)
Relative humidity: 40 - 70% (measured value: 47.9 - 60.9%)
Frequency of ventilation: 10 - 15 times/hour (55 - 72 times/hour when Safety Clean Rack was used.)
Lighting cycle: 12 hours light (7:00 – 19:00) / 12 hours dark (19:00 – 7:00)
Type and size of cage: Polycarbonate flat floor cage (210 x 320 x 130 mm, W x D x H)
Number of animals: 5 or less animals/cage
Frequency for change of housing equipment: Cage, cage lid, bedding, enrichment and watering bottle were changed at the end of quarantine. Cage and bedding were also changed on transfer to the necropsy room.
Feeding
Food and feeding method: Autoclaved Pelleted Food MF for experimental animal was given ad libitum by feeder of stainless cage.
Manufacturer and lot number
Manufacturer: Oriental Yeast Co., Ltd.
Lot number: 130515
Confirmation for quality: Based on data of analysis of contaminants at Eurofin which was provided by the manufacturer and “Limit for Contaminants in Feed and Medium” under Toxic Substances Control Act by EPA, US (1979), feed which was confirmed to satisfy the standard of Hita Laboratory was used.
Drinking water
Water and supplying method: Tap water of Hita city to which sodium hypochlorite (Purelox) was added to adjust chlorine concentration between 3 and 5 ppm was given ad libitum by watering bottle.
Confirmation for quality: Water quality was inspected in accordance with “Ministerial Ordinance for Water Quality Standard” (Ordinance by Ministry of Health, Labour and Welfare, No. 101) by the Ministry of Health, Labour and Welfare twice a year. It was confirmed that the latest result of the inspection which had been acquired before animal receipt fulfilled the standard of the Ministry of Health, Labour and Welfare.
Bedding and enrichment
Type: Bedding: Sunflake; Enrichment: Woodbite
Manufacturer and lot number
Manufacturer: Charles River Laboratories Japan, Inc.
Lot number: 130212 (Sunflake); 130219 (Woodbite)
Confirmation for quality: Based on data of analysis for contaminants at Eurofin which was provided by manufacturer and “Limit for Contaminants in Feed and Medium” in Toxic Substances Control Act by EPA (1979), bedding and enrichment which were confirmed to be within standard values of Hita Laboratory were used. - Route of administration:
- oral: gavage
- Vehicle:
- Negative control substance (vehicle)
Name: Olive oil
Manufacturer, lot number and grade
Manufacturer: Nikko Pharmaceutical Co., Ltd.
Lot number: 242509
Grade: Japanese pharmacopeia grade
Reason for vehicle selection
Olive oil was used as the vehicle in “28-day repeated oral dose toxicity study of SH-0850 in the rats” (study number B11-1025). Therefore, olive oil was also selected as a vehicle in this study. As a result of the solubility check, the test substance formed a homogeneous suspension at the concentration of 200 mg/mL in olive oil. Change of color tone, heat generation and bubbling were not observed for the 200 mg/mL test substance suspension prepared with olive oil up to 4 hours after preparation at a room temperature. Therefore, olive oil was determined to be stable and was selected as vehicle. - Details on exposure:
- Preparation of the test substance solution
Preparation method: In the preliminary test, 2.00 g of weighed test substance was added to olive oil, suspended by stirring (with a tube mixer) and by ultrasonication (with an ultrasonic cleaner) to prepare a total volume of 10 mL of the test substance solution at 200 mg/mL. The solution was serially diluted with olive oil by stirring with a magnetic stirrer to prepare the test substance solutions at 100, 50.0, 25.0, 12.5 and 6.25 mg/mL. In the micronucleus test, 2.00 g of weighed test substance was added to olive oil, suspended by stirring (with a tube mixer) and by ultrasonication (with an ultrasonic cleaner) to prepare a total volume of 10 mL of the test substance solution at 200 mg/mL. The solution was serially diluted with olive oil by stirring with a magnetic stirrer to prepare the test substance solutions at 100 and 50.0 mg/mL. The solution was prepared in the safety cabinet.
Preparation timing and storage: The solution was prepared on each administration day and used within 4 hours after preparation. The solution was stored at room temperature until use.
Preparation of positive control solution
Preparation method: Water for injection (lot No.: K2J85, Otsuka Pharmaceutical Factory, Inc.,) was added to a bottle containing 2 mg of MCC. MMC was dissolved in water for injection and a total volume of 10 mL of the solution was prepared at 0.2 mg/mL. The positive control substance solution was handled in the safety cabinet.
Preparation timing and storage: The solution was prepared immediately before use and used within 4 hours after preparation. The solution was stored at a room temperature until use. - Duration of treatment / exposure:
- 24 hours
- Frequency of treatment:
- 2 serial oral gavage administrations were given to the animals with an interval of 24 hours.
- Post exposure period:
- 1 day
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Preliminary test: 42 animals (21 male/21 female)
Micronucleus test: 30 males - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Positive control substance: Mitomycin C (MMC)
Reason for selection: MMC is stated as a positive control substance in the guideline listed in the section of Study method and its background data are available at Hita Laboratory.
Storage conditions: MMC was stored at a room temperature (allowable temperature range: 10-30 °C) in Cabinet 2 in the test material storage room. - Tissues and cell types examined:
- bone marrow cells
- Details of tissue and slide preparation:
- Preparation timing
At 24 hours after the final administration.
Method
After animals were euthanized by cervical dislocation, femur was removed and bone marrow cells were washed out to a centrifuge tube with approximately 0.8 mL of inactivated fetal bovine serum. It was centrifuged at 1000 rpm (210 xg) for 5 minutes and sediment of collected cells was smeared onto a slide glass. After air dried, it was fixed with methanol. Slide was stained with 3 vol% Giemsa solution (Sφerenzen buffer, prepared at pH 6.8) and then was stained differentially with 0.004 w/v% citric acid solution.
Two specimens were prepared from an animal which survived at the specimen preparation phase.
Observation of specimen
Specimens from negative control, positive control and 3 dose levels of test substance treated groups were observed. For each control and test substance groups, specimens from 5 animals in ascending order of the animal number were observed.
Specimens for observation were assigned with random slide numbers and were observed with blinded method using a biological microscope (x1000).
Frequency of micronucleus
For 1 animal, 2000 (1000 per slide) of polychromatic erythrocyte (PCE) was observed to determine the frequency of micronucleated polychromatic erythrocyte (MNPCE).
Growth inhibition for bone marrow cell
For 1 animal, 200 (100 per slide) of total erythrocyte (TE) was observed to determine ratio of PCE (PCE/TE). - Evaluation criteria:
- Criteria for the valid test
Mean value of MNPCE/PCE of both negative and positive control should be within the background data (negative: mean +/- 3 S.D., positive: mean +/- 3 S.D.) from the latest 20 tests in Hita Laboratory.
At specimen observation, there should be 3 or more dose levels at which their PCE/TE is greater than 20% of the PCE/TE of the negative control. - Statistics:
- Determination of MNPCE/PCE
For MNPCE/PCE of each test substance groups and of positive control groups, one-tailed 1% and 5% significant tests were conducted by the conditional binominal test (Kastenbaum and Bowman) compared to the negative control. Cochran-Armitage Trend Test was planned to check the dose-dependency if the test substance groups showed the significant differences, but no significant difference was noted and no trend test was conducted. The result was considered to be positive when significant increase of MNPCE/PCE was noted for the test substance group compared with the negative control and also dose-dependency was noted in the increase.
Determination of PCE/TE
Homogeneity of variances of each group (except the positive control) was examined by Bartlett Test. As a result, the homogeneity of variances was noted and therefore Williams test was conducted. If the result of Williams test did not show any significant difference, Dunnett test was planned to check the difference of mean values between the negative control and each test substance groups, but the significant difference was noted and Dunnett test was not conducted.
For comparison between the negative and positive control, homogeneity of variances between the 2 groups was examined by F test. As a result, variance was judged to be homogeneous and the difference of mean value was examined by Student’s t-test.
A statistical tool, StatLight, was used for the tests. The significance level of each test were 5% for Bartlett test, 5% (two-tailed) for Williams test, 5% for F test, and 5% and 1% (two-tailed) for other tests. - Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary test
Clinical signs
No change in clinical conditions and death were noted in the negative control and all test substance groups up to day 1 after second administration.
Body weight
No significant body weight change was noted in all test substance groups up to day 1 after second administration compared with the negative control group.
Micronucleus test
Clinical signs
No change in clinical conditions and death were noted in the negative control and all test substance groups up to day 1 after second administration and in the positive control up to day 1 after single administration.
Body weight
No significant body weight change was noted in all test substance groups up to day 1 after second administration and in the positive control up to day 1 after single administration compared with the negative control group.
MNPCE/PCE/PCE and PCE/TE
Mean values of MNPCE/PCE of the negative and positive control are 0.09 and 5.39%, respectively. The mean values are 0.06, 0.08 and 0.10% at 500, 1000 and 2000 mg/kg/day of the test substance groups, respectively, and no statistically significant difference was noted for all test substance groups compared with the negative control. For the positive control, significant increase was noted at 1% level.
Mean values of PCE/TE of the negative and positive control are 47.0 and 39.2%, respectively. The mean values are 51.1, 55.6 and 60.9% at 500, 1000 and 2000 mg/kg/day of the test substance groups, respectively. Statistically significant difference was noted at 5% level (two-tailed) for the test substance groups of 1000 and 2000 mg/kg/day compared with the negative control.
Background data of Hita Laboratory
MNPCE/PCE for the negative control is 0.10 +/- 0.021% (mean +/- S.D.) and 6.91 +/- 1.030% (mean +/- S.D.) for the positive control in the background data from the latest 20 tests at Hita Laboratory. Based on this, the following range was determined as appropriate.
Negative control: 0.04 – 0.16% (mean +/- 3 S.D.)
Positive control: 3.82 – 10.00% (mean +/- 3 S.D.)
Discussion
Mean values of MNPCE/PCE of both the negative and positive control were within the background data (from the latest 20 tests) of Hita Laboratory and PCE/TE of 3 dose levels of the test substance exceeded 20% of PCE/TE of the negative control for all test substance groups. Therefore, it was concluded that the criteria of the valid test was satisfied.
Statistically significant differences were not noted in values of MNPCE/PCE of all test substance groups by specimen observation compared with the negative control group.
Therefore, it was concluded that the test substance administration did not increase the frequency of micronucleated polychromatic erythrocyte.
Statistically significant differences were noted in values of PCE/TE at 1000 and 2000 mg/kg of the test substance groups at 5% level (two- tailed). Exposure of bone marrow to the test substance that is indicated by a decrease of PCE/TE could not be verified because these significant differences were due to their higher values compared with the negative control in PCE/TE. However, it was concluded that the result of this study was proper for evaluation of the potential of micronucleus induction because the dose levels were set up to 2000 mg/kg/day which is stipulated in the guideline as the upper limit when no toxicity is noted by continuous administration. - Conclusions:
- It was concluded that SH-0850 does not induce micronuclei under the condition of this study.
- Executive summary:
Potential of micronucleus induction of SH-0850 was assessed using male Crlj:CD1(ICR) mouse of 7-weeks of age. 2 serial oral gavage administrations were given to the animals with an interval of 24 hours.
In the preliminary test at the dose levels of 62.5-2000 mg/kg/day, maximum tolerated dose was estimated to be 2000 mg/kg/day or greater for both male and female when death of animal was served as an index. Therefore, 3 dose levels of 2000 mg/kg/day that is the highest dose level, 1000 and 500 mg/kg/day by dilution with the common ratio of 2 were selected for the micronucleus test.
Male animals only were used because it was concluded that the toxicity level was not different between male and female. A negative and a positive control groups were set as control groups and 2 serial oral gavage administrations of vehicle (olive oil) was given to the negative control group at the dose level of 10 mL/kg. For the positive control, Mitomycin C was administered by single intraperitoneal administration at 2 mg/kg/day.
No death was observed in any groups treated with the test substance at any dose levels up to the highest dose level of 2000 mg/kg/day at the time of specimen preparation which was at 24 hours after the second administration. Therefore, 3 dose levels were supplied for specimen observation and the frequency of micronucleated polychromatic erythrocyte (MNPCE/PCE) and ratio between polychromatic and total erythrocyte (PCE/TE) were examined.
No statistically significant difference was noted in MNPCE/PCE compared to the negative control at any dose levels in test substance groups in specimen observation. It was concluded that the test substance administration did not increase the frequency of micronucleated polychromatic erythrocyte.
Based on above, it was concluded that SH-0850 does not induce micronuclei under the condition of this study.
Reference
Clinical symptoms of males in the preliminary test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours
Route of administration: Oral gavage
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
First administration |
Second administration |
||
Up to 1 hour after administration |
Before administration |
Up to 1 hour after administration |
At day 1 after administration |
|||
Negative control [olive oil] |
0 |
1 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
2 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
3 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Test substance [SH-0850] |
62.5 |
4 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
5 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
6 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
125 |
7 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
8 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
9 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
250 |
10 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
11 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
12 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
500 |
13 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
14 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
15 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
1000 |
16 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
17 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
18 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
2000 |
19 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
20 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
21 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Clinical symptoms of females in the preliminary test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours
Route of administration: Oral gavage
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
First administration |
Second administration |
||
Up to 1 hour after administration |
Before administration |
Up to 1 hour after administration |
At day 1 after administration |
|||
Negative control [olive oil] |
0 |
22 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
23 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
24 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Test substance [SH-0850] |
62.5 |
25 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
26 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
27 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
125 |
28 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
29 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
30 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
250 |
31 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
32 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
33 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
500 |
34 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
35 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
36 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
1000 |
37 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
38 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
39 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
2000 |
40 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
41 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
42 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Body weight of males in the preliminary test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours
Route of administration: Oral gavage
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
Body weight (g) |
||
Before the first administration |
Before second administration |
At 1 day after second administration |
|||
Negative control [olive oil] |
0 |
1 2 3 |
31.3 32.2 33.0 |
31.6 32.6 33.4 |
32.0 33.0 33.9 |
Mean +/- S.D. |
32.2+/-0.85 |
32.5+/-0.90 |
33.0+/-0.95 |
||
Test substance [SH-0850] |
62.5 |
4 5 6 |
30.7 30.5 32.5 |
30.3 31.0 32.4 |
30.7 31.5 32.8 |
Mean +/- S.D. |
31.2+/-1.10 |
31.2+/-1.07 |
31.7+/-1.06 |
||
125 |
7 8 9 |
30.0 31.0 32.1 |
29.4 31.2 32.2 |
29.9 31.7 32.8 |
|
Mean +/- S.D. |
31.0+/-1.05 |
30.9+/-1.42 |
31.5+/-1.46 |
||
250 |
10 11 12 |
29.3 27.8 31.2 |
29.2 28.3 31.3 |
29.2 28.5 32.2 |
|
Mean +/- S.D. |
29.4+/-1.70 |
29.6+/-1.54 |
30.0+/-1.97 |
||
500 |
13 14 15 |
31.3 31.0 32.4 |
30.8 31.8 31.7 |
31.5 32.4 31.8 |
|
Mean +/- S.D. |
31.6+/-0.74 |
31.4+/-0.55 |
31.9+/-0.46 |
||
1000 |
16 17 18 |
30.0 31.6 32.0 |
30.7 32.0 32.0 |
31.0 32.6 31.7 |
|
Mean +/- S.D. |
31.2+/-1.06 |
31.6+/-0.75 |
31.8+/-0.80 |
||
2000 |
19 20 21 |
29.6 32.7 31.7 |
29.5 32.4 31.9 |
29.7 32.1 33.0 |
|
Mean +/- S.D. |
31.3+/-1.58 |
31.3+/-1.55 |
31.6+/-1.71 |
||
Body weight of females in the preliminary test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours
Route of administration: Oral gavage
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
Body weight (g) |
||
Before the first administration |
Before second administration |
At 1 day after second administration |
|||
Negative control [olive oil] |
0 |
22 23 24 |
23.1 24.9 27.0 |
22.4 25.9 26.5 |
23.7 25.3 26.3 |
Mean +/- S.D. |
25.0+/-1.95 |
24.9+/-2.21 |
25.1+/-1.31 |
||
Test substance [SH-0850] |
62.5 |
25 26 27 |
23.5 25.8 27.5 |
23.4 25.6 27.6 |
23.9 25.4 27.5 |
Mean +/- S.D. |
25.6+/-2.01 |
25.5+/-2.10 |
25.6+/-1.81 |
||
125 |
28 29 30 |
24.9 25.9 27.8 |
25.0 25.0 28.0 |
24.3 25.2 27.9 |
|
Mean +/- S.D. |
26.2+/-1.47 |
26.0+/-1.73 |
25.8+/-1.87 |
||
250 |
31 32 33 |
24.5 25.3 26.2 |
24.3 25.4 25.9 |
24.2 24.7 26.3 |
|
Mean +/- S.D. |
25.3+/-0.85 |
25.2+/-0.82 |
25.1+/-1.10 |
||
500 |
34 35 36 |
25.5 25.3 26.5 |
24.9 25.3 26.1 |
24.8 25.3 26.1 |
|
Mean +/- S.D. |
25.8+/-0.64 |
25.4+/-0.61 |
25.4+/-0.66 |
||
1000 |
37 38 39 |
23.7 26.6 28.7 |
24.2 25.9 28.9 |
24.0 25.6 29.7 |
|
Mean +/- S.D. |
26.3+/-2.51 |
26.3+/-2.38 |
26.4+/-2.94 |
||
2000 |
40 41 42 |
25.1 25.7 26.4 |
25.3 26.1 27.0 |
25.4 26.3 27.5 |
|
Mean +/- S.D. |
25.7+/-0.65 |
26.1+/-0.85 |
26.4+/-1.05 |
||
Clinical symptoms in the micronucleus test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours (single
administration for the positive control)
Route of administration: Oral gavage (intraperitoneal administration for the positive control)
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
First administration |
Second administration |
||
Up to 1 hour after administration |
Before administration |
Up to 1 hour after administration |
At day 1 after administration |
|||
Negative control [olive oil] |
0 |
101 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
102 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
103 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
104 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
105 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
106 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Test substance [SH-0850] |
500 |
107 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
108 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
109 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
110 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
111 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
112 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
1000 |
113 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
114 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
115 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
116 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
117 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
118 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
2000 |
119 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
|
120 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
121 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
122 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
123 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
124 |
No abnormality |
No abnormality |
No abnormality |
No abnormality |
||
Positive control [MMC] |
2 |
125 |
|
No abnormality |
No abnormality |
No abnormality |
126 |
No abnormality |
No abnormality |
No abnormality |
|||
127 |
No abnormality |
No abnormality |
No abnormality |
|||
128 |
No abnormality |
No abnormality |
No abnormality |
|||
129 |
No abnormality |
No abnormality |
No abnormality |
|||
130 |
No abnormality |
No abnormality |
No abnormality |
|||
MMC: Mitomycin C
Body weight in the micronucleus test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours (single
administration for the positive control)
Route of administration: Oral gavage (intraperitoneal administration for the positive control)
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Animal number |
Body weight (g) |
||
Before the first administration |
Before second administration |
At 1 day after second administration |
|||
Negative control [olive oil] |
0 |
101 102 103 104 105 106 |
34.6 35.1 35.0 35.9 36.4 36.5 |
34.7 35.2 35.3 36.3 36.7 36.9 |
35.3 36.1 35.2 36.9 37.3 37.0 |
Mean +/- S.D. |
35.6+/-0.79 |
35.9+/-0.90 |
36.3+/-0.91 |
||
Test substance [SH-0850] |
500 |
107 108 109 110 111 112 |
33.1 35.5 34.8 35.4 35.9 37.4 |
33.7 35.5 35.2 35.6 35.7 37.3 |
33.6 35.9 35.4 35.9 36.2 37.9 |
Mean +/- S.D. |
35.4+/-1.41 |
35.5+/-1.15 |
35.8+/-1.38 |
||
1000 |
113 114 115 116 117 118 |
32.0 34.7 35.4 36.7 35.6 36.8 |
32.4 35.4 36.3 36.3 36.0 36.8 |
32.5 35.6 36.5 37.1 36.3 37.1 |
|
Mean +/- S.D. |
35.2+/-1.76 |
35.5+/-1.60 |
35.9+/-1.73 |
||
2000 |
119 120 121 122 123 124 |
33.1 34.4 34.3 36.1 36.2 35.7 |
33.2 34.3 35.1 36.7 36.1 35.6 |
33.6 34.7 35.4 36.0 36.6 36.0 |
|
Mean +/- S.D. |
35.0+/-1.23 |
35.2+/-1.27 |
35.4+/-1.09 |
||
Positive control [MMC] |
2 |
125 126 127 128 129 130 |
|
32.6 35.0 35.8 33.4 36.0 37.9 |
32.2 35.3 35.9 34.1 35.8 38.8 |
Mean +/- S.D. |
|
35.1+/-1.91 |
35.4+/-2.19 |
||
MMC: Mitomycin C
Specimen observation in the micronucleus test
Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old
Number of administrations: 2 serial administrations with an interval of 24 hours (single
administration for the positive control)
Route of administration: Oral gavage (intraperitoneal administration for the positive control)
Dose level: 10 mL/kg
Test group |
Dose level (mg/kg/day) |
Specimen preparation period after final administration (hours) |
Animal number |
Ratio of polychromatic erythrocyte to total erythrocytea) (%) |
Frequency of micronucleated polychromatic erythrocyteb) (%) |
Negative control [olive oil] |
0 |
24 |
101 102 103 104 105 |
50.0 52.0 37.0 43.0 53.0 |
0.05 0.10 0.10 0.10 0.10 |
Mean +/- S.D. |
47.0+/-6.82 |
0.09+/-0.022 |
|||
Maximum/Minimum |
53.0/37.0 |
0.10/0.05 |
|||
Test substance [SH-0850] |
500 |
24 |
107 108 109 110 111 |
47.0 43.5 52.5 62.5 50.0 |
0.05 0.05 0.00 0.05 0.15 |
Mean +/- S.D. |
51.1+/-7.21 |
0.06+/-0.055 |
|||
Maximum/Minimum |
62.5/43.5 |
0.15/0.00 |
|||
1000 |
24 |
113 114 115 116 117 |
52.0 53.0 56.0 60.5 56.5 |
0.15 0.05 0.10 0.10 0.00 |
|
Mean +/- S.D. |
55.6+/-3.334# |
0.08+/-0.057 |
|||
Maximum/Minimum |
60.5/52.0 |
0.15/0.00 |
|||
2000 |
24 |
119 120 121 122 123 |
61.0 56.0 58.5 64.0 65.0 |
0.05 0.10 0.10 0.15 0.10 |
|
Mean +/- S.D. |
60.9+/-3.75# |
0.10+/-0.035 |
|||
Maximum/Minimum |
65.0/56.0 |
0.15/0.05 |
|||
Positive control [MMC] |
2 |
24 |
125 126 127 128 129 |
36.0 38.5 37.0 38.5 46.0 |
4.75 3.95 7.50 3.85 6.90 |
Mean +/- S.D. |
39.2+/-3.95 |
5.39+/-1.702** |
|||
Maximum/Minimum |
46.0/36.0 |
7.50/3.85 |
|||
MMC: Mitomycin C
a) 200 RBC/animal were observed
b) 2000 PCEs/animal were observed
#: Significant difference was noted compared with the negative control (p<0.05, Williams test)
**: Significant difference was noted compared with the negative control (p<0.01, conditioned binominal test (Kastenbaum and Bowmann))
Back group data of the micronucleus test in the mouse
Type on control |
Ratio of polychromatic erythrocyte to total erythrocyte |
Frequency of micronucleated polychromatic erythrocyte |
Number of experiment |
|
Mean +/- S.D. (%) |
Mean +/- S.D. (%) |
Range (%) |
||
Negative |
51.5+/-5.48 |
0.10+/-0.021 |
0.04 – 0.16 (mean+/-3 S.D.) |
20 |
Positive |
41.6+/-4.77 |
6.91+/-1.030 |
3.82 – 10.00 (mean+/-3 S.D.) |
20 |
Data from the latest 20 test completed before July 19, 2013.
For positive control, MMC was administered by single intraperitoneal administration at 2 mg/kg/day.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro - Ames Assay
The mutagenic potential of SH-0850 was assessed by the pre-incubation method in the absence and presence of the metabolic activation system (S9 mix) using the Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and the Escherichia coli strain WP2uvrA.
The test result showed that the number of revertant colonies for all tester strains was less than two-fold of the negative control and the mutagenicity was considered to be negative.
Therefore, it was concluded that SH-0850 does not have mutagenic potential.
Genetic toxicity in vitro - Chromosome aberration test
The clastogenic potential of SH-0850 was assessed using Chinese hamster lung fibroblast cells (CHL/IU cells).
Specimen were prepared and observed at 890, 1780 and 3560 μg/mL in the absence and presence of S9 mix in short-term treatment, 445, 629, 890 and 1260 μg/mL in 24-hour continuous treatment.
The appearance frequency of cells with numerous aberration was below 5 % in any observed dose level of the test substance in any treatment. Therefore, it was determined to be negative for numerous aberration. The appearance frequency of cells with structural aberration was below 5 % in any observed dose level of the test substance in the absence and presence of S9 mix in short-term treatment. In 24-hour continuous treatment, it was 15.5 % at the highest and the dose dependency was noted in the appearance. Therefore, it was determined to be positive for structural aberration.
Based on above results, it was concluded that SH-0850 does not induce numerical chromosome aberration but induces structural chromosome aberration under the condition of this study.
Genetic toxicity in vitro - Mammalian cell gene mutation assay
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of DAIGUARD-850 to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).
Dimethyl sulfoxide was used as vehicle of the test item in this study. The test item was examined up to 500 μg/mL (highest achievable concentration based on the limited solubility of the test item) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with and without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL,
Assay 2, 3-hour treatment with metabolic activation and 24-hour treatment without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL,
Assay 3, 3-hour treatment with and without metabolic activation and 24-hour treatment without metabolic activation: 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
Although there was clearly no indication of mutagenicity after the treatments in Assays 1-2, but as the results of the first two main tests did not fully meet the acceptance criteria (the mutation frequency of the untreated and vehicle control were slightly higher than the upper limit of the acceptable range in some cases), thus an additional experiment (Assay 3) was performed to provide fully valid datasets for interpretation.
In Assay 3, following a 3-hour treatment with metabolic activation), similarly to the previous tests, no cytotoxicity of the test item was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 3, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In Assay 3, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.
In conclusion, no mutagenic effect of DAIGUARD-850 was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Genetic toxicity in vivo - Micronucleus assay
Potential of micronucleus induction of SH-0850 was assessed using male Crlj:CD1(ICR) mouse of 7-weeks of age. 2 serial oral gavage administrations were given to the animals with an interval of 24 hours.
In the preliminary test at the dose levels of 62.5-2000 mg/kg/day, maximum tolerated dose was estimated to be 2000 mg/kg/day or greater for both male and female when death of animal was served as an index. Therefore, 3 dose levels of 2000 mg/kg/day that is the highest dose level, 1000 and 500 mg/kg/day by dilution with the common ratio of 2 were selected for the micronucleus test.
Male animals only were used because it was concluded that the toxicity level was not different between male and female. A negative and a positive control groups were set as control groups and 2 serial oral gavage administrations of vehicle (olive oil) was given to the negative control group at the dose level of 10 mL/kg. For the positive control, Mitomycin C was administered by single intraperitoneal administration at 2 mg/kg/day.
No death was observed in any groups treated with the test substance at any dose levels up to the highest dose level of 2000 mg/kg/day at the time of specimen preparation which was at 24 hours after the second administration. Therefore, 3 dose levels were supplied for specimen observation and the frequency of micronucleated polychromatic erythrocyte (MNPCE/PCE) and ratio between polychromatic and total erythrocyte (PCE/TE) were examined.
No statistically significant difference was noted in MNPCE/PCE compared to the negative control at any dose levels in test substance groups in specimen observation. It was concluded that the test substance administration did not increase the frequency of micronucleated polychromatic erythrocyte.
Based on above, it was concluded that SH-0850 does not induce micronuclei under the condition of this study.
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