Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In Vitro Genetic Toxicity
AMES bacterial reverse mutation assay: Negative
In vitro chromosome aberration assay: Negative
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:
- 1990-01-30 to 1990-06-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Escherichia coli strain not tested
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Purity: 90-95%
Lot no: DEEUGEA NBP#4321285
EHL Test sample T900009
Stated expiration date: January 1991
Storage conditions: Room Temperature - Species / strain / cell type:
- S. typhimurium TA 1535
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA1535 were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California). - Additional strain / cell type characteristics:
- other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.
- Species / strain / cell type:
- S. typhimurium TA 1537
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA1537 were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California). - Additional strain / cell type characteristics:
- other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.
- Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA98, were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California). - Additional strain / cell type characteristics:
- other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.
- Species / strain / cell type:
- S. typhimurium TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells:
Salmonella typhimurium strains TA100, were obtained from the Laboratory of Dr. B. N. Ames (Berkeley, California). - Additional strain / cell type characteristics:
- other: Proper phenotype of each culture was verified for cyrstal violet sensitivity, ampicillin resistance, requirement for histidine and biotin, and spontaneous reversion frequency.
- Metabolic activation:
- with and without
- Metabolic activation system:
- - source of S9
: S9 preparation was purchased from Molecular Toxicology Inc. (College Park, Maryland)
- method of preparation of S9 mix : Prepared from livers of Aroclor-1254 induced male Sprague-Dawley rats (Hilltop Laboratories, Scottsdale, PA). Prepared using procedure described by Ames et al. 1975.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 was tested for metabolic activation capability in a matrix experiment in which both percent S9 in S9 mix and the amount of positive standard per plate were varied. - Test concentrations with justification for top dose:
- 0.03, 0.10, 0.30, 1.00, and 3.00 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: anhydrous acetone
- Justification for choice of solvent/vehicle: Not specified
- Justification for percentage of solvent in the final culture medium: Not specified - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- benzo(a)pyrene
- other: Sodium Nitrite (-S9: TA1535); 2-aminoanthracene (+S9: TA1535 and TA1537)
- Details on test system and experimental conditions:
- General procedures were basically those described by Ames et al.
Doses used per plate with/without S-9 were 0.1, 0.3, 1.0, 3.0, and 5.0 mg (Toxicity Screen). Based on the toxicity and insolubilty observed in the toxicity screen, the maximum dose level for mutagenicity testing was selected as 3 mg/plate.
Plate incorporation tests were performed by mixing 0.1 ml of bacterial culture, and. if appropriate. 0.5 ml of S-9 mix with 2 ml of histidine-biotin top agar (0.5% (w/v) NaCl, 0.6% (w/v) Difco agar, 0.05 mM L-histidine-HCl, 0.05 mM biotin) maintained at 44-48 degrees C. THe mixture was poured onto minimal glucose agar plates (Vogel-Bonner medium E with 2% glucose and 1.5% Difco agar). Toxicity tests employed the same procedures as those used in the plate incorporation test. Single plates were prepared for each strain/S-9/dose level combination for the toxicity test. 3 replicate plates were prepared for each strain/S-9/combination for the plate incorporation tests. Concurrent positive and negative controls were conducted for plate incorporation tests to demonstrate strain sensitivity and metabolic activation system capability. Plates were examined after at least 48 hours at 37 degrees Cl. A screen for suitable solvents, for the possibility of pH or osmolality effects, and for reaction with plastic petri dishes was performed prior to the plate incorporation tests.
Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting revertant colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor related the total plate area to the area of the counted fields. Revertant colonies measured in this manner are calculated to not more than three significant figures. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (<10 revertants/plate). - Evaluation criteria:
- Results were considered to be clearly positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (p<0.01) at three treatment levels, and there was a statistically significant dose response (p<0.01).
- Statistics:
- Statistical analysis was performed on plate incorporation assay results after transforming revertants/plate values as log10(revertants/plate). Analysis included Bartlett's test for homogeneity of variance and comparison of treatrments with controls using within-levels pooled variance and a one-sided t-test. Grant's test was performed to determine if outliers were present. Statistical significance of dose response was evaluated by regression analysis for log 10 transformed values and revertants plate.
A critical level of p<0.01 was used to determine statistical significance. Results with P<0.05 were also indicated to assist in interpretation of results. - Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test sample, XP 2563, was concluded not to be mutagenic towards any of the Salmonella typhimurium test strains used (TA98, TA100, TA1535, and TA1537) in the presence or absence of an Aroclor 1254-induced rat liver homogenate metabolic activation system (S-9 Mix)
- Executive summary:
The test material, XP 2563, was tested in Ames/Salmonella plate incorporation assays using test strains TA98, TAlOO, TA1535 and TA1537 in the presence and absence of an Aroclor 1254-induced rat liver homogenate (S-9) . In the toxicity screen, toxicity and insolubility was observed at levels of 3 and 5 mg/plate with and without activation. The maximum dose level for mutagenicity testing was selected to be 3 mg/plate. No significant mutagenicity was observed in both the initial assays and the subsequent confirmation assays. Results therefore suggest that XP 2563 is not a mutagen in Salmonella typhimurium under our experimental conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999-09-29 to 2000-01-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- yes
- Remarks:
- Deviation was determined to have no impact on the outcome of the study.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Solutia Inc. (10300 Olive Boulevard, PO Box 66760, Louis, Missouri, 63166-6760, USA) Lot # 1999001
- Expiration date of the lot/batch: Not provided by the Sponsor
- Purity test date: Not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in solvent DMSO at a concentration of 500 mg/mL.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : clear colourless liquid
Purity: 100% - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Chinese Hamster Ovary (CHO-K1) cells (repository number CCL 61) were sourced from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. The freeze lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20.
- Metabolic activation:
- with and without
- Metabolic activation system:
- - source of S9
: S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/Kg, five days prior to sacrifice.
- method of preparation of S9 mix: The S9 batch was prepared and stored at ≤-70°C until used. Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(α)anthracene to forms mutagenic to S. typhimuirum TA100. Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotineamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 µg Streptomycin/mL, and 2 mM L-glutamine). - Test concentrations with justification for top dose:
- Preliminary Toxicity Assay:
-S9 (4-hour treatment; 16 hour recovery): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
+S9 (4-hour treatment; 16 hour recovery): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
-S9 (20-hour continuous treatment;): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
Concurrent Toxicity Test
-S9 (4-hour treatment; 16 hour recovery): 19, 37.5, 75, 150, 200, 250, 300, 350, 400 µg/mL
Chromosome Aberration Assay:
-S9 (4-hour treatment; 16 hour recovery): 19, 37.5, 75, 150, 200, 250, 300, 350, 400 µg/mL
-S9 (20-hour treatment; 0 hour recovery): 12.5, 25, 50, 75, 100, 125, 150, 200 µg/mL
+S9 (4-hour treatment; 16 hour recovery): 12.5, 25, 50, 75, 100, 150, 200, 250 µg/mL - Vehicle / solvent:
- -Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide was determined to be the solvent of choice based on the solubility of the test material and compatibility with the target cells. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- Mitomycin C- in non-activated study at final concentrations of 0.1 and 0.2 μg/mL. Cyclophosphamide- in S9-activated study at final concentratons of 10 and 20 μg/mL
- Details on test system and experimental conditions:
- Preliminary Toxicity Assay
CHO cells were seeded for each treatment condition at approximately 5 x 105 cells/25 cm2 flask and were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 16-24 hours. Treatment was carried out by refeeding the flasks with 5 mL complete medium for the non-activated study or S9 reaction mixture for the activated study, to which was added 50 µL dosing solution of the test material in solvent or solvent alone. The osmolality of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after the initiation of treatment the cells were harvested by trypsinization and counted using a coulter counter. The presence of test material precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cells counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
Chromosome Aberration Assay
The chromosome aberration assay performed using standard procedures by exposing duplicate cultures of CHO cells to the test material as well as positive and solvent controls. CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask and were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 5 mL complete medium for the non-activated study or 5 mL S9 reaction mixture for the S9 activated study, to which was added 50 µL of dosing solution of the test material in solvent or solvent alone. The osmolarity of the highest concentration of dosing solution in the treatment medium was measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.
In the non-activated study, the cells were exposed to the test material for 4 hours or continuously for 20 hours up to the cell harvest at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. In the 4 hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to the duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks returned to the incubator until cell collection.
In the S9 activated study, the cells were exposed for 4 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. After the exposure period the treatment medium was removed, cells washed with CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid® was added to the duplicate flasks for each treatment condition at final concentration of 0.1 µg/mL and the flasks were returned to the incubator until cell collection.
A concurrent toxicity test was conducted in both the non-activated and S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a coulter counter. The presence of test material precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
Collection of Metaphase Cells
Two hours after the addition of Colcemid®, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy’s fixative. The cells were stored overnight or longer in fixative at approximately 2-8°C.
Slide Preparation
Fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant aspirated, and 1 mL fresh fixative added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension dropped onto the center of a glass slide and allowed to air dry. Slides were identified by the study number, date prepared, and treatment condition. The dried slides were stained with 5% Giesma, air dried and permanently mounted.
Evaluation of Metaphase Cells
Slides were coded using random numbers. To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) were determined for each treatment group. Metaphase cells for 20±2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Initially, the non-activated and S9 activate 4 hour exposure groups were evaluated for chromosome aberrations and if a positive result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was not evaluated for chromosome aberrations. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored chromatid-type and chromosome-type aberrations. The number of metaphase spreads examined and scored per duplicate flask would be reduced if the percentage of aberrant cells reached a statistically significant level before 100 cells were scored. Chromatid-type aberrations included chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations included chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (≥10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY co-ordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polypoid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
Controls
Mitomycin C was used as the positive control in the non-activated study at final concentrations of 0.1 and 0.2 µg/mL. Cyclophosphamide was used as the positive control in the S9 activated study at final concentrations of 10 and 20 µg/mL. For both positive controls, one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The solvent vehicle for the test material was used as the solvent control at the same concentration as that found in the test material-treated groups.
Evaluation of Test Results
The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gaps were not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. - Evaluation criteria:
- The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fischer's exact test) relative to the solvent control.
The test material was considered to induce a positive response when the percentage of cells with aberrations increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). Test materials not demonstrating a statistically significant increase in aberrations were considered to be negative. - Statistics:
- Statistical analysis of % aberrant cells was performed using the Fisher’s exact test. Fisher’s test was used to compare pairwise the % aberrant cells of each treatment group with the solvent control. If a fisher’s test was positive the Cochran-Armitage test was used to measure dose-responsiveness.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Assay
CHO cells were exposed to the solvent alone and to nine concentrations of of the test material ranging from 0.5 µg/mL to 5000 µg/mL in the absence and presence of an S9 reaction mixture. Visible precipitate was observed in the treatment medium at dose levels of ≥150 µg/mL. Dose levels of ≤50 µg/mL were soluble in the treatment medium. The osmolality in the treatment medium of the highest concentration tested, 5000 µg/mL was 370 mmol/Kg. The osmolality of the solvent (DMSO) in the treatment medium was 478 mmol/Kg. The pH of the highest concentration of the test material in treatment medium was approximately 7.5. Cell growth inhibition relative to the solvent control was 80% and 77% at 5000 µg/mL, the highest concentration tested in both the non-activated and S9 activated 4 hour exposure groups, respectively. Cell growth inhibition relative to the solvent control was 100% at 5000 µg/mL, the highest concentration tested in the non-activated 20 hour continuous exposure group. Based on the results of the toxicity study, the dose levels selected for testing the chromosome aberration assay
-S9 (4-hour treatment; 16 hour recovery): 19, 37.5, 75, 150, 200, 250, 300, 350, 400 µg/mL
-S9 (20-hour treatment; 0 hour recovery): 12.5, 25, 50, 75, 100, 125, 150, 200 µg/mL
+S9 (4-hour treatment; 16 hour recovery): 12.5, 25, 50, 75, 100, 150, 200, 250 µg/mL
Chromosome Aberration Assay
Visible precipitate was observed in treatment medium at dose levels of ≥ 100 µg/mL Dose levels of ≤ 75 µg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest concentration tested, 400 µg/mL, was 410 mmol/Kg. The osmolality of the solvent (DMSO) in treatment medium was 411 mmol/Kg. The pH of the highest concentration of the test material in treatment medium was approximately 7.5.
Toxicity of the test material in CHO cells when treated for 4 hours in the absence of S9 activation was 66% at 150 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 150 µg/mL, was 25% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 37.5, 75, and 150 µg/mL. The percentage of cells with structural aberrations in the test material treated groups was statistically increased above that of the solvent control only at dose level 150 µg/mL (p≤0.01, Fischer’s exact test). The Cochran-Armitage test was also positive for a dose response (p≤0.05). The percentage of cells with numerical aberrations in the test material treated groups was not significantly increased above that of the solvent control (p>0.05, Fischer’s exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (11.5%).
Toxicity of the test material in CHO cells when treated for 4 hours in the presence of S9 activation was 64% at 50 µg/mL, the highest concentration evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 50 µg/mL, was not reduced relative to the solvent control. The dose levels selected for microscopic analysis were 12.5, 25, and 50 µg/mL. The percentage of cells with structural and numerical aberrations in the test material treated groups was statistically increased above that of the solvent control at dose level 50 µg/mL (p≤0.05, Fischer’s exact test). The Cochran-Armitage test was also positive for a dose response for structural aberrations (p≤0.05), and negative for a dose response for numerical aberrations (p>0.05). The statistically significant increases in the percentage of structurally and numerically aberrant cells at dose level 50 µg/mL in the S9 activated historical solvent control (0.0-6.5% and 0.0-13.5%, respectively). Therefore, the statistically significant in structurally and numerically aberrant cells observed in the S9 activated 4 hour exposure group were considered not biologically significant. The percentage of structurally damaged cells in the Cyclophosphamide (CP) group was found to be statistically significant (71.4%).
Slides from the non-activated 20-hour exposure group were evaluated for chromosome aberrations. Toxicity of the test material in CHO cells was 76% at 75 µg/mL, the highest concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure group. The mitotic index at the highest dose level evaluated for chromosome aberrations, 75 µg/mL, was 53% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 25, 50, and 75 µg/mL. The percentage of cells with structural aberrations in the test material treated groups was statistically increased above that of the solvent control at dose level 50 µg/mL (p≤0.05, Fischer’s exact test). The Cochran-Armitage test was negative for a dose-response (p>0.05). The statistically significant increase in the percent of structurally aberrant cells at dose level 50 µg/mL in the non-activated 20 hour exposure group (5.0%) was within the range of the percentage of structurally aberrant cells observed with the non-activated historical control (0.0-6.0%). Therefore, the statistically significant increase in structurally aberrant cells observed in the non-activated 20 hour exposure group was considered not biologically significant. The percentage of cells with numerical aberrations in the test material treated groups was not significantly increased above that of the solvent control (p>0.05, Fischer’s exact test). The percentage of structurally damaged cells in the MMC group was found to be statistically significant (12.0%).
The positive and solvent controls fulfilled the requirements for a valid test. - Conclusions:
- The positive and solvent controls fulfilled the requirements for a valid test. Statsitically significant increase in % structurally aberrant cells at 150 μg/mL in non-activated 4 hour exposure group (9.0%) was outside historical controls and therefore considered significant.
Santicizer 2148 was concluded to be positive for the induction of structural chromosome aberrations in absence of S9 activation, and negative for induction of structural chromosome aberrations in presence of S9 activation in Chinese hamster ovary (CHO) cells. Santicizer 2148 was concluded to be negative for the induction of numberical chromosome aberrations in absence and presence of S9 activation in Chinese hamster ovary (CHO) cells. - Executive summary:
The test article, Santicizer 2148, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9-activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article.
Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of 500 mg/mL, the maximum concentration used.
In the preliminary toxicity assay, the maximum dose tested was 5000μg/mL. Visible precipitate was observed in treatment medium at dose levels of > 150μg/L. Dose levels of < 50μg/mL were soluble in treatment medium. Selection of doe levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity, i.e., at least 50% cell growth inhibition, was observed at dose levels of >500 and > 150μg/mL in the non-activated and S9-activateed 4 hour exposure groups respectively, and at dose levels > 150μg/mL in the non-activated 20 hour continuous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 19 to 400μg/mL for the non-activated 4 hour exposure group, from 12.5 go 250μg/mL for the S9-activated 4 hour exposure group, and from 12.5 to 200μg/mL for the non-activated 20 hour continuous exposure group.
In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system, and all cells were harvested at 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at concentrations of > 100μg/mL. Concentrations of < 75μg/mL were soluble in the treatment medium. Toxicity (cell growth inhibition) was 66% and 76% at the highest dose level s evaluated for chromosome aberrations, 150 and 75μg/mL, in the non-activated 4 hour and 20 hour exposure groups respectively. Toxicity (cell growth inhibition) was approximately 64% at the highest dose level evaluated for chromosome aberrations, 50μg/mL, in the S9-activated study. Initially, the non-activated and S9-activated 4 hour exposure groups were scored for structural and numerical chromosome aberrations. Statistically significant increases in structural chromosome aberrations were observed in the non-activated and S(-activated 4 hour exposure groups relative to the solvent control group, at dose levels 150μg/mL in the non-activated 4 hour exposure group (p<0.01, Fisher’s exact test), and at dose level 50μg/mL in the S9 activated 4 hour exposure group (p<0.05, Fisher’s exact test). The Cochran-Armitage test was also positive for a dose response in the non-activated and the S9-activated test systems (p<0.05). No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 4 hour exposure group relative to the solvent control group, at dose level 50μg/mL (p<0.05, Fisher’s exact test). The Cochran-Armitage test was negative for a dose response in the S9 activated test system (p>0.05). The non-activated 20 hour exposure group was then evaluated for chromosome aberrations. A statistically significant increase in structural chromosome aberrations was observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, at dose level 50μg/mL (p<0.05, Fisher’s exact test). The Cochran-Armitage was negative for a dose response (p>0.05). No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, regardless of dose level (p>0.05, Fisher’s exact test).
The statistically significant increases in the percentage of structurally and numerically aberrant cells at dose level 50μg/mL in the S9 activated 4 hour exposure group (6.0% and 5.0%, respectively), were within the ranges of the percentage of structurally and numerically aberrant cells observed with the S9 activated historical solvent control (0.0-6.5% and 0.0-13.5%, respectively), were within the ranges of the percentage of structurally and numerically aberrant cells observed with the S9 activated historical solvent control (0.0-6.5% and 0.0-13.5% respectively). The statistically significant increase in the percentage of structurally aberrant cells at dose level 50μg/mL in the non-activated 20 hour exposure group (5.0%), was within the range of the percentage of structurally aberrant cells observed with the non-activated historical solvent control (0.0-6.0%). Therefore, the statistically significant increases in structurally and numerically aberrant cells observed in the S9 activated 4 hour and non-activated 200hour exposure groups were considered not biologically significant. The statistically significant increase in the percentage of structurally aberrant cells observed at dose level 150μg/mL in the non-activated 4 hour exposure group (9.0%) was outside the non-activated historical control range and was therefore considered significant. Based on the findings of this study, Santicizer 2148 was concluded to be positive for the induction of structural chromosome aberrations in the absence of S9 activation, and negative for the induction of structural chromosome aberrations in the presence of S9 activation in Chinese hamster ovary (CHO) cells. Santicizer 2148 was concluded to be negative for the induction of numerical chromosome aberrations in the absence and presence of S9 activation in Chinese hamster ovary (CHO) cells.
Referenceopen allclose all
Table 1. Toxicity Test Results with test strain TA100
Amount of test material per plate (mg) |
S-9* |
Toxic Response** |
Solubility† |
0.1 |
- |
N |
S |
0.1 |
+ |
N |
S |
0.3 |
- |
N |
S |
0.3 |
+ |
N |
S |
1.0 |
- |
N |
S |
1.0 |
+ |
N |
S |
3.0 |
- |
T |
I |
3.0 |
+ |
T |
I |
5.0 |
- |
T |
I |
5.0 |
+ |
T |
I |
* = S-9 Mix was prepared using 10% (v/v) rat liver S-9 preparation (MolTox 02337) in the S-9 Mix
** = N = No toxic response; T = Toxicity observed
† = S = Test material soluble; I = Test material insoluble
Table 2. Statistical summary of plate incorporation test results for TA98 and TA100 with and without S9
Strain |
TA98 |
TA98 |
TA100 |
TA100 |
Activation System |
With S-9 |
None |
With S-9 |
None |
Test Date |
09-Feb-90 |
09-Feb-90 |
09-Feb-90 |
02-Mar-90 |
Amount/plate (mg) |
Revertants/plate Mean and standard deviation in ( ) |
|||
0.03 |
38.0 (± 8.3) |
27.7 (± 2.1) |
93.0 (± 11.5) |
123 (± 6.2) |
0.10 |
34 (± 7.5) |
28.7 (± 9.1) |
103.7 (± 3.1) |
114.3 (± 13.0) |
0.30 |
34.7 (± 4.0) |
24.0 (± 3.5) |
95.7 (± 9.1) |
124.0 (± 13.2) |
1.00 |
36.0 (± 5.3) |
25.7 (± 4.5) |
93.3 (± 7.5) |
110.7 (± 20.0) |
3.00 (T, I) † |
35.0 (± 4.0) |
20.5 (± 3.5) |
0.0 (± 0) |
111.5 (± 4.9) |
Solvent Controls |
33.7 (± 6.8) |
26.0 (± 2.3) |
98.0 (± 10.6) |
123.0 (± 10.4) |
Summary Analysis |
||||
Treatment levels with Rev/Plate > Control |
||||
p<0.06 |
0 |
0 |
0 |
0 |
p<0.01 |
0 |
0 |
0 |
0 |
Bartlett’s test |
N |
N |
N |
N |
No. of outliers (Grubb’s test) |
0 |
0 |
0 |
0 |
Dose Response |
N |
N |
N |
N |
Lack of fit test |
N |
N |
N |
N |
ll analyses performed with Log(10) transformed data.
Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level
† = T = Toxicity observed, I = Test material insoluble
Table 3. Statistical summary of plate incorporation test results for TA1535 and TA1537 with and without S9
Strain |
TA1535 |
TA1535 |
TA1537 |
TA1537 |
Activation System |
With S-9 |
None |
With S-9 |
None |
Test Date |
13-Feb-90 |
13-Feb-90 |
13-Feb-90 |
13-Feb-90 |
Amount/plate (mg) |
Revertants/plate Mean and standard deviation in ( ) |
|||
0.03 |
11.7 (± 3.2) |
10.7(± 2.5) |
12.0 (± 1.7) |
9.3 (± 3.1) |
0.10 |
11.0(±1.0) |
9.0 (± 1.0) |
13.3 (± 5.0) |
9.7 (± 6.4) |
0.30 |
10.3(± 1.2) |
8.7 (± 3.1) |
12.3 (± 2.1) |
6.7 (± 1.5) |
1.00 |
11.7(± 3.2) |
11.3 (± 0.6) |
11.0 (± 3.6) |
6.3 (± 0.6) |
3.00 (T, I) † |
12.0 (± 1.0) |
6.0 (± 1.4) |
11.3 (± 1.6) |
7.7 (± 2.1) |
Solvent Controls |
11.9 (± 3.1) |
14.3 (± 1.8) |
9.7 (± 2.6) |
7.2 (± 2.3) |
Summary Analysis |
||||
Treatment levels with Rev/Plate > Control |
||||
p<0.06 |
0 |
0 |
0 |
0 |
p<0.01 |
0 |
0 |
0 |
0 |
Bartlett’s test |
N |
N |
N |
N |
No. of outliers (Grubb’s test) |
0 |
0 |
0 |
0 |
Dose Response |
N |
N |
N |
N |
Lack of fit test |
N |
A |
N |
N |
ll analyses performed with Log(10) transformed data.
Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level; A = Data do not allow lack-of-fit test to be performed
† = T = Toxicity observed, I = Test material insoluble
Table 4. Statistical summary of plate incorporation test results for TA98 and TA100 with and without S-9
Strain |
TA98 |
TA98 |
TA100 |
TA100 |
Activation System |
With S-9 |
None |
With S-9 |
None |
Test Date |
23-Feb-90 |
21-Jun-90 |
23-Feb-90 |
23-Feb-90 |
Amount/plate (mg) |
Revertants/plate Mean and standard deviation in ( ) |
|||
0.03 |
40.0 (± 6.8) |
19.0(± 6.6) |
88.3 (± 17.8) |
96.0 (± 22.9) |
0.10 |
35.0(± 6.6) |
17.6 (± 0.7) |
94.0 (± 16.6) |
99.3 (± 22.1) |
0.30 |
36.6(± 7.0) |
18.3 (± 6.8) |
93.7 (± 0.6) |
109.7 (± 12.5) |
1.00 |
37.3(± 7.0) |
14.7 (± 4.2) |
89.7 (± 11.6) |
(T)115.0 (± 18.7) † |
3.00 (T, I) † |
0.0 (± 0.0) |
17.7 (± 0.6) |
0.0 (± 0.0) |
107.3 (± 8.4) |
Solvent Controls |
37.9 (± 0.0) |
17.3 (± 6.3) |
119.4 (± 14.3) |
112.2 (± 20.4) |
Summary Analysis |
||||
Treatment levels with Rev/Plate > Control |
||||
p<0.06 |
0 |
0 |
0 |
0 |
p<0.01 |
0 |
0 |
0 |
0 |
Bartlett’s test |
N |
N |
* |
N |
No. of outliers (Grubb’s test) |
0 |
0 |
A |
0 |
Dose Response |
N |
N |
A |
N |
Lack of fit test |
N |
N |
A |
N |
ll analyses performed with Log(10) transformed data.
Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level; A = Data do not allow lack-of-fit test to be performed
† = T = Toxicity observed, I = Test material insoluble
Table 5. Statistical summary of plate incorporation test results for TA1535 and TA1537
Strain |
TA1535 |
TA1535 |
TA1537 |
TA1537 |
Activation System |
With S-9 |
None |
With S-9 |
None |
Test Date |
02-Mar-90 |
02-Mar-90 |
02-Mar-90 |
02-Mar-90 |
Amount/plate (mg) |
Revertants/plate Mean and standard deviation in ( ) |
|||
0.03 |
14.47 (± 1.6) |
16.3(± 1.5) |
11.3 (± 2.1) |
12.0 (± 3.0) |
0.10 |
14.3(± 4.6) |
13.3 (± 4.0) |
10.3 (± 3.2) |
12.3 (± 2.1) |
0.30 |
10.7 (± 1.6) |
14.3 (± 4.2) |
10.3 (± 1.6) |
9.3 (± 3.1) |
1.00 |
16.7(± 3.1)* |
12.7 (± 2.1) |
11.7 (± 1.0) |
8.7 (± 0.0) |
3.00 (T, I) † |
14.0 (± 0.0) |
0.0 (± 0.0) |
0.0 (± 0.0) |
6.0 (± 0.0) |
Solvent Controls |
13.0 (± 2.9) |
16.0 (± 2.7) |
14.7 (± 4.2) |
9.9 (± 2.3) |
Summary Analysis |
||||
Treatment levels with Rev/Plate > Control |
||||
p<0.06 |
1 |
0 |
0 |
0 |
p<0.01 |
0 |
0 |
0 |
0 |
Bartlett’s test |
N |
N |
N |
N |
No. of outliers (Grubb’s test) |
0 |
0 |
0 |
0 |
Dose Response |
N |
N |
N |
N |
Lack of fit test |
N |
N |
N |
N |
ll analyses performed with Log(10) transformed data.
Codes used are: * = significant at p<=0.06 level; ** = significant at p<=0.01 level; N = Not significant at p<=0.06 level
† = T = Toxicity observed, I = Test material insoluble
Table 1. Preliminary toxicity test using Santicizer 2148 in absence of exogenous metabolic activation period 4 hour treatment, 16 hour recovery period
Treatment |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
1.78 |
99 |
1.76 |
100 |
|
Santicizer 2148 (μg/mL) |
|
||||
0.5 |
1.72 |
100 |
1.72 |
98 |
2 |
1.5 |
2.05 |
99 |
2.03 |
115 |
-15 |
5 |
1.86 |
100 |
1.86 |
106 |
-6 |
15 |
2.00 |
100 |
2.00 |
114 |
-14 |
50 |
1.87 |
100 |
1.87 |
106 |
-6 |
150 |
0.91 |
99 |
0.90 |
51 |
49 |
500 |
0.07 |
0 |
0.00 |
0 |
100 |
1500 |
0.01 |
0 |
0.00 |
0 |
100 |
5000 |
0.37 |
97 |
0.36 |
20 |
80 |
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative controls.
Table 2. Preliminary toxicity test using santicizer 2148 in the presence of exogenous metabolic activation 4 hour treatment, 16 hour recovery period
Treatment |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
2.60 |
100 |
2.60 |
100 |
|
Santicizer 2148 (μg/mL) |
|
||||
0.5 |
1.95 |
99 |
1.93 |
74 |
26 |
1.5 |
2.10 |
99 |
2.08 |
80 |
20 |
5 |
2.16 |
97 |
2.09 |
81 |
19 |
15 |
1.87 |
99 |
1.85 |
71 |
29 |
50 |
1.80 |
98 |
1.76 |
68 |
32 |
150 |
0.72 |
100 |
0.72 |
28 |
72 |
500 |
0.09 |
0 |
0.00 |
0 |
100 |
1500 |
0.09 |
0 |
0.00 |
0 |
100 |
5000 |
0.61 |
100 |
0.61 |
23 |
77 |
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative controls.
Table 3. Preliminary toxicity test using Santicizer 2148 in the absence of exogenous metabolic activation 20 hour continuous treatment
Treatment |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
2.20 |
99 |
2.18 |
100 |
|
Santicizer 2148 (μg/mL) |
|
||||
0.5 |
2.19 |
97 |
2.12 |
97 |
3 |
1.5 |
2.13 |
97 |
2.07 |
95 |
5 |
5 |
2.25 |
96 |
2.16 |
99 |
1 |
15 |
1.99 |
95 |
1.86 |
87 |
13 |
50 |
1.53 |
97 |
1.48 |
68 |
32 |
150 |
0.26 |
95 |
0.25 |
11 |
89 |
500 |
<0.01 |
0 |
0.00 |
0 |
100 |
1500 |
0.00 |
0 |
0.00 |
0 |
100 |
5000 |
0.09 |
0 |
0.00 |
0 |
100 |
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative controls.
Table 4 . Concurrent toxicity test using Santicizer 2148 in the absence of exogenous metabolic activation 4 hour treatment, 16 hour recovery period
Treatment |
Replicate Flask |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
A |
2.15 |
99 |
2.16 |
100 |
|
B |
2.23 |
98 |
||||
Santicizer 2148,μg/mL |
|
|||||
19 |
A |
2.03 |
96 |
2.17 |
100 |
-1 |
B |
2.44 |
98 |
||||
37.5 |
A |
2.19 |
97 |
2.23 |
103 |
-3 |
B |
2.45 |
95 |
||||
75 |
A |
1.89 |
97 |
1.98 |
92 |
8 |
B |
2.22 |
96 |
||||
150 |
A |
0.85 |
90 |
0.73 |
34 |
66 |
B |
0.83 |
85 |
||||
200 |
A |
0.66 |
65 |
0.44 |
21 |
79 |
B |
0.71 |
64 |
||||
250 |
A |
0.35 |
59 |
0.27 |
13 |
87 |
B |
0.31 |
66 |
||||
300 |
A |
0.57 |
72 |
0.32 |
15 |
85 |
B |
0.34 |
66 |
||||
350 |
A |
0.35 |
21 |
0.13 |
6 |
94 |
B |
0.42 |
45 |
||||
400 |
A |
0.20 |
0 |
0.00 |
0 |
100 |
B |
0.29 |
0 |
||||
MMC,μg/mL |
|
|||||
0.1 |
A |
2.22 |
98 |
2.16 |
100 |
0 |
B |
2.23 |
96 |
||||
0.2 |
A |
2.18 |
96 |
2.08 |
96 |
4 |
B |
2.13 |
97 |
||||
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative controls.
Table 5. Cytogenetic Analysis of CHO cells treated with Santicizer 2148 in the absence of exogenous metabolic activation 4 hour treatment, 16 hour recovery period
Treatment |
Flask |
Mitotic Index* |
Cells Scored |
Cells with Aberrations (%) ** |
Number of Structural Aberrations |
Severely damaged Cells‡ |
Average structural aberration: Per cell‡‡ |
||||||
Numerical |
Structural |
Chromatid-type† |
Chromosome-type†† |
||||||||||
Gaps |
Breaks |
Exch |
Breaks |
Dic |
Ring |
||||||||
DMSO |
A |
4.4 |
100 |
1 |
3 |
0 |
2 |
0 |
1 |
0 |
0 |
0 |
0.030 |
B |
5.8 |
100 |
0 |
2 |
0 |
1 |
0 |
0 |
1 |
0 |
0 |
0.020 |
|
Sanctizier 2148 (μg/mL) |
|
||||||||||||
37.5 |
A |
5.6 |
100 |
0 |
2 |
3 |
0 |
0 |
1 |
0 |
1 |
0 |
0.020 |
B |
6.0 |
100 |
0 |
2 |
0 |
1 |
0 |
1 |
0 |
0 |
0 |
0.020 |
|
75 |
A |
3.8 |
100 |
0 |
4 |
2 |
1 |
0 |
0 |
2 |
1 |
0 |
0.040 |
B |
5.4 |
100 |
2 |
5 |
1 |
4 |
0 |
0 |
1 |
0 |
0 |
0.050 |
|
150 |
A |
2.8 |
100 |
2 |
8 |
7 |
2 |
2 |
6 |
2 |
0 |
0 |
0.120 |
B |
4.8 |
100 |
3 |
10 |
2 |
5 |
1 |
0 |
4 |
0 |
0 |
0.100 |
|
MMC (μg/mL) |
|
||||||||||||
0.2 |
A |
7.8 |
100 |
1 |
11 |
0 |
11 |
2 |
0 |
0 |
0 |
0 |
0.130 |
B |
9.0 |
100 |
1 |
12 |
1 |
12 |
3 |
1 |
0 |
0 |
0 |
0.160 |
|
An additional dose level of 19μg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination. Dose levels 200, 250, 300, 350, and 400μg/mL were not analysed due to excessive toxicity.
* = Mitotic index = number mitotic figures x 100/500 cells counted
** = Numerical: includes polyploid and endoreduplicated cells.; Structural: excludes cells with only gaps
† = Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Exch) include quadriradials, triradials, and complex rearrangements
†† = Chromosome breaks include breaks and acentric fragments; dic, dicentric chromosome
‡ = Severely damaged cells include cells with 1 or more pulverized chromosomes and cells with 10+ aberrations
‡‡ = Severely damaged cells were counted as 10 aberrations
Table 6. Concurrent toxicity test using Santicizer 2148 in the presence of exogenous metabolic activation. 4 hour treatment, 16 hour recovery period
Treatment |
Replicate Flask |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
A |
2.64 |
100 |
2.55 |
100 |
|
B |
2.45 |
100 |
||||
Santicizer 2148,μg/mL |
|
|||||
12.5 |
A |
2.02 |
98 |
2.13 |
84 |
16 |
B |
2.33 |
98 |
||||
25 |
A |
2.09 |
99 |
2.20 |
86 |
14 |
B |
2.37 |
98 |
||||
50 |
A |
0.74 |
97 |
0.92 |
36 |
64 |
B |
1.18 |
96 |
||||
75 |
A |
1.34 |
94 |
1.10 |
43 |
57 |
B |
0.99 |
95 |
||||
100 |
A |
0.83 |
98 |
0.70 |
27 |
73 |
B |
0.60 |
97 |
||||
150 |
A |
0.37 |
92 |
0.29 |
12 |
88 |
B |
0.27 |
91 |
||||
200 |
A |
0.16 |
0 |
0.00 |
0 |
100 |
B |
0.13 |
0 |
||||
250 |
A |
0.07 |
0 |
0.00 |
0 |
100 |
B |
0.07 |
0 |
||||
CP,μg/mL |
|
|||||
10 |
A |
1.72 |
99 |
1.48 |
58 |
42 |
B |
1.27 |
98 |
||||
20 |
A |
1.38 |
96 |
1.20 |
47 |
53 |
B |
1.11 |
97 |
||||
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative controls
Table 7. Cytogenetic analysis of CHO cells treated with Santicizer 2148 in the presence of Exogenous metabolic activation 4 hour treatment, 16 hour recovery period
Treatment |
Flask |
Mitotic Index* |
Cells Scored |
Cells with Aberrations (%) ** |
Number of Structural Aberrations |
Severely damaged Cells‡ |
Average structural aberration: Per cell‡‡ |
||||||
Numerical |
Structural |
Chromatid-type† |
Chromosome-type†† |
||||||||||
Gaps |
Breaks |
Exch |
Breaks |
Dic |
Ring |
||||||||
DMSO |
A |
6.4 |
100 |
2 |
2 |
0 |
0 |
0 |
1 |
1 |
0 |
0 |
0.020 |
B |
6.6 |
100 |
1 |
1 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0.010 |
|
Sanctizier 2148 (μg/mL) |
|
||||||||||||
12.5 |
A |
6.6 |
100 |
3 |
2 |
3 |
1 |
0 |
0 |
0 |
1 |
0 |
0.020 |
B |
7.4 |
100 |
4 |
5 |
1 |
4 |
1 |
0 |
0 |
0 |
0 |
0.050 |
|
25 |
A |
9.6 |
100 |
6 |
2 |
0 |
0 |
0 |
0 |
2 |
0 |
0 |
0.020 |
B |
8.2 |
100 |
1 |
1 |
1 |
0 |
0 |
2 |
0 |
0 |
0 |
0.020 |
|
150 |
A |
6.8 |
100 |
3 |
7 |
2 |
0 |
1 |
2 |
4 |
1 |
0 |
0.080 |
B |
8.2 |
100 |
7 |
5 |
1 |
2 |
0 |
1 |
1 |
1 |
0 |
0.050 |
|
CP (μg/mL) |
|
||||||||||||
10 |
A |
4.8 |
30 |
3 |
67 |
4 |
20 |
6 |
12 |
0 |
0 |
0 |
1.267 |
B |
5.6 |
26 |
3 |
77 |
5 |
33 |
6 |
11 |
0 |
0 |
2 |
2.692 |
|
Dose levels 75, 100, 150, 200, and 250μg/mL were not analysed due to excessive toxicity.
* = Mitotic index = number mitotic figures x 100/500 cells counted
** = Numerical: includes polyploid and endoreduplicated cells.; Structural: excludes cells with only gap
† = Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Exch) include quadriradials, triradials, and complex rearrangements
†† = Chromosome breaks include breaks and acentric fragments; dic, dicentric chromosome
‡ = Severely damaged cells include cells with 1 or more pulverized chromosomes and cells with 10+ aberrations
‡‡ = Severely damaged cells were counted as 10 aberrations
Table 8 . Concurrent toxicity test Santicizer 2148 in the absence of exogenous metabolic activation 20 hour continuous treatment
Treatment |
Replicate Flask |
Cell count (x10^6) |
Cell viability (%)* |
Viable cells per flask (x10^6)** |
Cell growth index (%)† |
Cell growth inhibition (%)‡ |
DMSO |
A |
2.70 |
99 |
2.68 |
100 |
|
B |
2.69 |
100 |
||||
Santicizer 2148,μg/mL |
|
|||||
12.5 |
A |
2.57 |
98 |
2.31 |
86 |
10 |
B |
2.12 |
99 |
||||
25 |
A |
2.30 |
98 |
2.00 |
75 |
25 |
B |
1.79 |
98 |
||||
50 |
A |
1.63 |
97 |
1.44 |
54 |
46 |
B |
1.35 |
96 |
||||
75 |
A |
0.69 |
94 |
0.64 |
24 |
76 |
B |
0.69 |
91 |
||||
100 |
A |
0.81 |
94 |
0.87 |
32 |
68 |
B |
1.02 |
95 |
||||
125 |
A |
0.56 |
91 |
0.44 |
17 |
83 |
B |
0.42 |
90 |
||||
150 |
A |
0.50 |
0 |
0.00 |
0 |
100 |
B |
0.48 |
0 |
||||
200 |
A |
0.40 |
0 |
0.00 |
0 |
100 |
B |
0.44 |
0 |
||||
MMC,μg/mL |
9 |
|||||
0.1 |
A |
2.39 |
96 |
2.31 |
86 |
14 |
B |
0.44 |
95 |
||||
0.2 |
A |
2.32 |
97 |
2.24 |
84 |
16 |
B |
2.25 |
99 |
||||
* = Viability determined by trypan blue dye exclusion
** = Viable cells/flask = cell count x % viable cells
† = Growth index = (cells per flask treated group/cells per flask control group), expressed as %
‡ = Cell growth inhibition = 100% - % cell growth index; not calculated for negative control
Table 9. Cytogenetic analysis of CHO cells treated with Santicizer 2148 in the absence of Exogenous metabolic activation 20 hour continuous treatment
Treatment |
Flask |
Mitotic Index* |
Cells Scored |
Cells with Aberrations (%) ** |
Number of Structural Aberrations |
Severely damaged Cells‡ |
Average structural aberration: Per cell‡‡ |
||||||
Numerical |
Structural |
Chromatid-type† |
Chromosome-type†† |
||||||||||
Gaps |
Breaks |
Exch |
Breaks |
Dic |
Ring |
||||||||
DMSO |
A |
5.2 |
100 |
2 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0.010 |
B |
6.2 |
100 |
2 |
2 |
0 |
1 |
0 |
0 |
1 |
0 |
0 |
0.020 |
|
Sanctizier 2148 (μg/mL) |
|
||||||||||||
25 |
A |
8.8 |
100 |
1 |
4 |
2 |
1 |
0 |
0 |
1 |
1 |
1 |
0.130 |
B |
7.2 |
100 |
2 |
2 |
0 |
0 |
0 |
0 |
2 |
1 |
0 |
0.030 |
|
50 |
A |
6.8 |
100 |
2 |
5 |
0 |
1 |
2 |
0 |
3 |
1 |
0 |
0.070 |
B |
5.8 |
100 |
1 |
5 |
0 |
2 |
1 |
0 |
1 |
0 |
1 |
0.140 |
|
75 |
A |
3.0 |
100 |
2 |
3 |
0 |
1 |
0 |
1 |
1 |
0 |
0 |
0.030 |
B |
2.4 |
100 |
2 |
5 |
2 |
3 |
0 |
0 |
1 |
0 |
1 |
0.140 |
|
MMC (μg/mL) |
|
||||||||||||
0.1 |
A |
8.4 |
100 |
2 |
10 |
0 |
10 |
2 |
7 |
1 |
0 |
0 |
0.200 |
B |
8.0 |
100 |
1 |
14 |
0 |
5 |
7 |
0 |
1 |
0 |
2 |
0.330 |
|
An additional dose level of 12.5μg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination. Dose levels 100, 125, 150, and 200μg/mL were not analysed due to excessive toxicity.
* = Mitotic index = number mitotic figures x 100/500 cells counted
** = Numerical: includes polyploid and endoreduplicated cells.; Structural: excludes cells with only gap
† = Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Exch) include quadriradials, triradials, and complex rearrangements
†† = Chromosome breaks include breaks and acentric fragments; dic, dicentric chromosome
‡ = Severely damaged cells include cells with 1 or more pulverized chromosomes and cells with 10+ aberrations
‡‡ = Severely damaged cells were counted as 10 aberrations
Table 10. Summary
Treatment (μg/mL) |
S9 Activation |
Treatment time (hours) |
Mitotic index |
Cells Scored |
Aberratios per cell (Mean ± SD) † |
Cells with Aberrations (%) |
|
Numerical |
Structural |
||||||
DMSO |
- |
4 |
5.1 |
200 |
0,025 ± 0.157 |
0.5 |
2.5 |
Sancticizer 2148 |
|||||||
37.5 |
- |
4 |
5.8 |
200 |
0.020 ± 0.140 |
0.0 |
2.0 |
75 |
- |
4 |
4.6 |
200 |
0.045 ± 0.208 |
1.0 |
4.5 |
150 |
- |
4 |
3.8 |
200 |
0.110 ± 0.411 |
2.5 |
9.0** |
MMC |
|||||||
0.2 |
- |
4 |
8.4 |
200 |
0.145 ± 0.442 |
1.0 |
11.5 |
DMSO |
+ |
4 |
6.5 |
200 |
0.015 ± 0.122 |
1.5 |
1.5 |
Santicizer 2148 |
|||||||
12.5 |
+ |
4 |
7.0 |
200 |
0.035 ± 0.184 |
3.5 |
3.5 |
25 |
+ |
4 |
8.9 |
200 |
0.020 ± 0.172 |
3.5 |
1.5 |
50 |
+ |
4 |
7.5 |
200 |
0.065 ± 0.267 |
5.0* |
6.0* |
CP |
|||||||
10 |
+ |
4 |
5.2 |
56 |
1.929 ± 2.114 |
3.0 |
71.4** |
DMSO |
- |
20 |
5.7 |
200 |
0.015 ±0.122 |
2.0 |
1.5 |
Santicizer 2148 |
|||||||
25 |
- |
20 |
8.0 |
200 |
0.080 ± 0.732 |
1.5 |
3.0 |
50 |
- |
20 |
6.3 |
200 |
0.105 ± 0.753 |
1.5 |
5.0 |
75 |
- |
20 |
2.7 |
200 |
0.085 ± 0.728 |
2.0 |
4.0 |
MMC |
|||||||
0.1 |
- |
20 |
8.2 |
200 |
0.65 ± 1.127 |
1.5 |
12.0** |
Cells from all treatment conditions were harvested at 20 hours after initiation of treatments
† = Severely damaged cells counted as 10 aberrations
* = p<0.05; ** = p<0.01; Fisher’s exact test
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In Vivo Genetic Toxicity
In vivo mammalian micronucleus assay: Negative
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:
- 1990-01-30 to 1990-02-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- Lot# NBP 4321285
EHL Code: T900009
Purity: 90-95%
Sample stored at room temperature as advised by sample submitter
Clear oily liquid - Species:
- mouse
- Strain:
- CD-1
- Details on species / strain selection:
- Not specified
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Age at study initiation: Not specified
- Weight at study initiation: Not specified
- Assigned to test groups randomly: yes, under following basis: computer generated randomization scheme based on animal weights.
- Fasting period before study: Not specified
- Housing: two per cage prior to dosing and subsequently one per cage after dosing in stainless steel cages with stainless steel mesh bottoms.
- Diet (e.g. ad libitum): Purina Certified Laboratory Rodent Chow No. 5002 (Purina Mills Inc., St. Louis, MO) ad libitum
- Water (e.g. ad libitum): Water (Public water system) ad libitum via an automatic watering system
- Acclimation period: 9 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79°F
- Humidity (%): 40-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
IN-LIFE DATES: Not specified - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Not specified
- Concentration of test material in vehicle: 10 mL/Kg - Details on exposure:
- Preliminary Experiments
Two preliminary experiments were performed for the selection of appropriate doses in the main study. In the range-finding two or more mice of each sex were treated (by intraperitoneal injection) with the test material at doses of 1000, 4000, or 5000 mg/Kg body weight. Control animals were dosed with an appropriate volume of corn oil. Treated animals were observed daily for up to 3 days after dosing and a final observation was made six days after dosing. An additional animal per sex was added to the second range-finding experiment to evaluate the effects of the test material on slide preparations for erythrocyte evaluation.
Micronucleus Assay
Animals were treated by intraperitoneal injections of corn il (solvent control, 10 mL/Kg) or the test material in corn oil. The final treatment doses were 500, 2500, and 5000 mg/Kg body weight. Cyclophosphamide (60 mg/Kg) was used as the positive control. - Duration of treatment / exposure:
- Single dose via intraperitoneal injection
- Frequency of treatment:
- Single
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Control (Corn Oil)
- Dose / conc.:
- 500 mg/kg bw/day
- Dose / conc.:
- 2 500 mg/kg bw/day
- Dose / conc.:
- 5 000 mg/kg bw/day
- No. of animals per sex per dose:
- 15/sex/dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s): Not specified
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 60 mg/Kg - Details of tissue and slide preparation:
- Extraction of Bone Marrow Cells and Slide Preparation
All animals were sacrificed by cervical dislocation and their femora removed. Each bone was opened at the end and flushed with approximately 2 mL of fetal bovine serum in a centrifuge tube. Bone marrow from femora of each animal were pooled for slide preparation. The suspension was centrifuged to remove the serum. A portion of the remaining cells were placed on a clean glass microscope slide and a smear prepared. Following preparation of the smears the slides were allowed to air dry over night and stained with a Hema-Tek II slide staining machine and a Wright-Giesma Stain Pak which included stain, buffer, and rinse solutions.
Distirbution of Slides for Scoring
Slides of bone marrow cells were coded prior to distribution. Scoring for each animal was performed by two indivudals using separate slides.
Scoring of Slides
Micronuclei were identified as uniform, darkly stained, round or oval shaped bodies found in the cytoplasm of polychromatic erythrocytes (PCEs). Bodies in PCEs were refractile, improperly shaped or stained, or which were not in the focal plane of the cell were not scored as micronuclei. Cells containing more than one micronucleus were scored as a single micronucleated cell. The number of micronucleated PCEs per 1000 PCEs and the number of PCEs and normochromatic erythrocytes (NCEs) per 1000 erythrocytes wa determined for each animal. - Statistics:
- The individual test animal was used as the individual unit for analysis of micronucleated PCE/erythrocyte fraction. Micronucleated PC frequencies observed for each animal were transformed as the square root prior to analysis. PCE/Total erythrocyte fractions were not transformed. A Dunnett's t-test (one sided) was used for comparison of treatment group and positive control values with solvent control values. A significance level of p<0.05 was used.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
In the range-finding experiments, one mortality was observed at the 72-hour time point in the second experiment. No other deaths were observed up to 72 hours after dosing. Deaths were observed 6 days after dosing. The highest dose selected for the micronucleus experiment was 5000 mg/Kg body weight. Two additional lower doses, 500 and 2500 mg/Kg body weight were also selected.
RESULTS OF DEFINITIVE STUDY
In the micronucleus experiment, statistically significant (p<0.01) weight losses were observed in male mice dosed with 5000 mg/Kg and sacrificed at 48 and 72 hours and in the females dosed with 5000 mg/Kg and sacrificed at 48 hours.
In animals treated at 2500 mg/Kg, statistically significant (p<0.01) decreases in mean body weight were observed for males and females sacrificed at the 24 hour time point and males sacrificed at the 72 hour time point. Additionally, a statistically significant decrease (p<0.05) in mean body weight was observed for treated males sacrificed at 48 hours and treated females sacrificed at 72 hours.
At the low dose (500 mg/Kg), statistically significant (p<0.01) weight losses were observed in treated males sacrificed at 48 and 72 hours. No statistically significant (p<0.05) decrease in mean body weight was observed for any of the other test material treated animals.
A statistically significant (p<0.01) decrease in PCE/Total erythrocyte ratio was observed for female mice treated with 5000 mg/Kg and sacrificed at 48 and 72 hour time points. Additionally, statistically significant (p<0.05) decreases in the PCE/total erythrocyte ratio was observed for female mice treated with 2500 mg/Kg and 5000 mg/Kg and sacrificed at 72 and 24 hours, respectively. A statistically significant (p<0.05) decrease was also observed in males treated with 500 and 5000 mg/Kg of the test material and sacrificed at 72 hours. No statistically significant (p<0.05) decrease in PCE/total erythrocyte ratio was observed for any of the other test material treated animals.
No statistically significant (p<0.05) increase in the number of micronucleated PCEs was observed in any of the test material treated animals when compared with the control animals. The positive control yielded expected positive responses in micronucleus induction. - Conclusions:
- Based on the results observed, it was concluded that the test material is not a mammalian genotoxicant in vivo in mouse bone marrow cells.
- Executive summary:
A key in vivo mouse bone marrow micronucleus assay was conducted to the evaluate the the potential of the test material (XP-2563) to induce chromosomal effects. The test material (in corn oil) was administered via intraperitoneal injection to groups of male and female CD-1 mice (15/sex/dose) at doses of 0, 500, 2500, or 5000 mg/Kg body weight. Following exposure, the mouse bone marrow was sampled at 24, 48, and 72 hours.
One death was observed in the micronucleus experiment in female mice treated at 5000 mg/Kg body weight. Toxcity, as indicated by a statistically significant decreased mean body weight and statistically significant decreases in the PCE/total erythrocyte ratio in test material treated animals, was observed. The test material did not induce statistically significant decreases in micronucleated PCEs in any of the treated animals. Increases in micronucleated PCE frequency were observed in the positive control group.
Based on the results observed, it was concluded that the test material is not a mammalian genotoxicant in vivo in mouse bone marrow cells.
Reference
Table 1. Range-finding Study Results |
||||||||||||||
Dose mg/Kg |
Number Treated |
Number of Deaths |
Observations |
|||||||||||
3-5 hours |
24 hours |
48 hours |
72 hours |
6 Day Total |
||||||||||
Male |
Female |
Male |
Female |
Male |
Female |
Male |
Female |
Male |
Female |
Male |
Female |
Combineda |
||
1000b |
2 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0/4 |
Normal |
4000c |
3 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
2 |
3/6 |
Normal |
5000b |
2 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
¼ |
Normal |
5000c |
3 |
3 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
2 |
1 |
3/6 |
Normald |
a Number of deaths / Total number of animals treated
b Data from initial range-finding experiment
c Data from second range-finding experiment
d One female appeared unresponsive six days after dosing
Table 2. Micronucleus Assay: MeanBody Weight Change and Animal Observations |
|||||||
Dose mg/Kg |
Sex |
Number Treated |
Mean Body Weight Change (g) ± Standard Deviation |
Deaths |
Observations |
||
24 hours |
48 hours |
72 hours |
|||||
Corn Oil Control |
Male |
15 |
-0.9 ± 0.5 |
-1.0 ± 0.4 |
-0.5 ± 0.2 |
0 |
Normal |
Female |
15 |
-0.8 ± 1.1 |
-1.4 ± 0.6 |
-0.6 ± 0.9 |
0 |
Normal |
|
|
|||||||
500 mg/Kg |
Male |
15 |
-3.3 ± 4.2 |
-1.9 ± 0.5 |
-1.7 ± 0.7 |
0 |
Normal |
Female |
15 |
-0.7 ± 1.0 |
-1.6 ± 0.8 |
-1.4 ± 0.6 |
0 |
Normal |
|
|
|||||||
2500 mg/Kg |
Male |
15 |
-2.2 ± 0.3** |
-2.1 ± 0.7* |
-2.0 ± 0.6** |
0 |
Normal |
Female |
15 |
-2.0 ± 0.6** |
-2.0 ± 1.0 |
2.3 ± 0.9* |
0 |
Normal |
|
|
|||||||
5000 mg/Kg |
Male |
15 |
-2.1 ± 1.2 |
-2.9 ± 1.1** |
3.3 ± 1.8** |
0 |
Normal |
Female |
15 |
-1.3 ± 0.8 |
-3.6 ± 0.7** |
2.2 ± 1.3 |
1 |
Normal |
|
|
|||||||
Cyclophosphamide 60 mg/Kg |
Male |
5 |
-0.9 ± 0.4 |
- ± - |
- ± - |
0 |
Normal |
Female |
5 |
1.2 ± 0.3 |
- ± - |
- ± - |
0 |
Normal |
|
* p≤0.05
** p≤0.01
Table 3. Micronucleus Assay: Summary of PCE Ratios and Micronucleus Data for Treated Animals |
||||||||
Harvest Time |
Sex |
Number |
Mean PCE/Total Erythrocyte Fraction ± Standard Deviation |
Mean Micronucleated PCE / 1000 PCE ± Standard Deviation |
||||
Vehicle Control |
Test material |
Positive Control |
Vehicle Control |
Test material |
Positive Control |
|||
500 mg/Kg |
||||||||
24 |
Male |
5 |
0.54 ± 0.07 |
0.48 ± 0.05 |
0.50 ± 0.05 |
0.2 ± 0.45 |
0.2 ± 0.45 |
21.2 ± a |
Female |
5 |
0.55 ± 0.04 |
0.53 ± 0.04 |
0.52 ± 0.05 |
0.4 ± 0.55 |
0.6 ± 0.89 |
35.6 ± a |
|
|
||||||||
48 |
Male |
5 |
0.51 ± 0.08 |
0.48 ± 0.07 |
|
1.2 ± 1.79 |
1.6 ± 1.34 |
|
Female |
5 |
0.56 ± 0.05 |
0.56 ± 0.05 |
|
1.0 ± 0.71 |
0.8 ± 0.84 |
|
|
|
||||||||
72 |
Male |
5 |
0.57 ± 0.08 |
0.44 ± 0.07* |
|
1.6 ± 2.61 |
1.0 ± 1.41 |
|
Female |
5 |
0.60 ± 0.07 |
0.57 ± 0.05 |
|
1.4 ± 1.14 |
0.6 ± 0.89 |
|
|
2500 mg/Kg |
||||||||
24 |
Male |
5 |
0.54 ± 0.07 |
0.47 ± 0.06 |
0.50 ± 0.05 |
0.2 ± 0.45 |
3.0 ± 3.54 |
21.2 ± 18.a |
Female |
5 |
0.55 ± 0.04 |
0.55 ± 0.06 |
0.52 ± 0.05 |
0.4 ± 0.55 |
0.6 ± 0.89 |
35.6 ± 12.a |
|
|
||||||||
48 |
Male |
5 |
0.51 ± 0.08 |
0.53 ± 0.09 |
|
1.2 ± 1.79 |
1.2 ± 0.84 |
|
Female |
5 |
0.56 ± 0.05 |
0.50 ± 0.07 |
|
1.0 ± 0.71 |
1.0 ± 0.71 |
|
|
|
||||||||
72 |
Male |
5 |
0.57 ± 0.08 |
0.57 ± 0.08 |
|
1.6 ± 2.61 |
0.2 ± 0.45 |
|
Female |
5 |
0.60 ± 0.07 |
0.51 ± 0.06* |
|
1.4 ± 1.14 |
0.6 ± 0.89 |
|
|
5000 mg/Kg |
||||||||
24 |
Male |
5 |
0.54 ± 0.07 |
0.45 ± 0.09 |
0.50 ± 0.05 |
0.2 ± 0.45 |
0.4 ± 0.55 |
21.2 ± 18.a |
Female |
5 |
0.55 ± 0.04 |
0.49 ± 0.04* |
0.52 ± 0.05 |
0.4 ± 0.55 |
0.6 ± 0.89 |
35.6 ± 12.a |
|
|
||||||||
48 |
Male |
5 |
0.51 ± 0.08 |
0.49 ± 0.04 |
|
1.2 ± 1.79 |
1.0 ± 1.41 |
|
Female |
5 |
0.56 ± 0.05 |
0.44 ± 0.03** |
|
1.0 ± 0.71 |
0.6 ± 0.89 |
|
|
|
||||||||
72 |
Male |
5 |
0.57 ± 0.08 |
0.48 ± 0.07* |
|
1.6 ± 2.61 |
0.4 ± 0.55 |
|
Female |
5 |
0.60 ± 0.07 |
0.41 ± 0.06** |
|
1.4 ± 1.14 |
1.2 ± 1.79 |
|
|
* p≤0.05; ** p≤0.01 by one-sided t-test. Square root transformed data used for statistical analysis of micronucleated PCE
a data could illegible in the study report.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In Vitro Bacterial Reverse Mutation Assay
In a key bacterial reverse mutation study (Monsanto Chemical Company, 1990b), the test material (XP 2563) was tested in the plate incorporation assay using test strains TA98, TAlOO, TA1535 and TA1537 of Salmonella typhimurium in the presence and absence of an Aroclor 1254-induced rat liver homogenate (S-9) . In the toxicity screen, toxicity and insolubility was observed at levels of 3 and 5 mg/plate with and without activation. The maximum dose level for mutagenicity testing was selected to be 3 mg/plate.
No significant mutagenicity was observed in both the initial assays and the subsequent confirmation assays. Results therefore suggest that XP 2563 is not a mutagen in Salmonella typhimurium under the experimental conditions tested.
In Vitro Chromosome Aberration Assay
In a key study (BioReliance, 2000a), the test material (Santicizer 2148), was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9-activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. Dimethyl sulfoxide (DMSO) was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at a concentration of 500 mg/mL, the maximum concentration used.
In the preliminary toxicity assay, the maximum dose tested was 5000μg/mL. Visible precipitate was observed in treatment medium at dose levels of > 150μg/L. Dose levels of < 50μg/mL were soluble in treatment medium. Selection of doe levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity, i.e., at least 50% cell growth inhibition, was observed at dose levels of >500 and > 150μg/mL in the non-activated and S9-activateed 4 hour exposure groups respectively, and at dose levels > 150μg/mL in the non-activated 20 hour continuous exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 19 to 400μg/mL for the non-activated 4 hour exposure group, from 12.5 go 250μg/mL for the S9-activated 4 hour exposure group, and from 12.5 to 200μg/mL for the non-activated 20 hour continuous exposure group.
In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system, and all cells were harvested at 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at concentrations of > 100μg/mL. Concentrations of < 75μg/mL were soluble in the treatment medium. Toxicity (cell growth inhibition) was 66% and 76% at the highest dose level s evaluated for chromosome aberrations, 150 and 75μg/mL, in the non-activated 4 hour and 20 hour exposure groups respectively. Toxicity (cell growth inhibition) was approximately 64% at the highest dose level evaluated for chromosome aberrations, 50μg/mL, in the S9-activated study. Initially, the non-activated and S9-activated 4 hour exposure groups were scored for structural and numerical chromosome aberrations. Statistically significant increases in structural chromosome aberrations were observed in the non-activated and S(-activated 4 hour exposure groups relative to the solvent control group, at dose levels 150μg/mL in the non-activated 4 hour exposure group (p<0.01, Fisher’s exact test), and at dose level 50μg/mL in the S9 activated 4 hour exposure group (p<0.05, Fisher’s exact test). The Cochran-Armitage test was also positive for a dose response in the non-activated and the S9-activated test systems (p<0.05). No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 4 hour exposure group relative to the solvent control group, at dose level 50μg/mL (p<0.05, Fisher’s exact test). The Cochran-Armitage test was negative for a dose response in the S9 activated test system (p>0.05). The non-activated 20 hour exposure group was then evaluated for chromosome aberrations. A statistically significant increase in structural chromosome aberrations was observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, at dose level 50μg/mL (p<0.05, Fisher’s exact test). The Cochran-Armitage was negative for a dose response (p>0.05). No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, regardless of dose level (p>0.05, Fisher’s exact test).
The statistically significant increases in the percentage of structurally and numerically aberrant cells at dose level 50μg/mL in the S9 activated 4 hour exposure group (6.0% and 5.0%, respectively), were within the ranges of the percentage of structurally and numerically aberrant cells observed with the S9 activated historical solvent control (0.0-6.5% and 0.0-13.5%, respectively), were within the ranges of the percentage of structurally and numerically aberrant cells observed with the S9 activated historical solvent control (0.0-6.5% and 0.0-13.5% respectively). The statistically significant increase in the percentage of structurally aberrant cells at dose level 50μg/mL in the non-activated 20 hour exposure group (5.0%), was within the range of the percentage of structurally aberrant cells observed with the non-activated historical solvent control (0.0-6.0%). Therefore, the statistically significant increases in structurally and numerically aberrant cells observed in the S9 activated 4 hour and non-activated 200hour exposure groups were considered not biologically significant. The statistically significant increase in the percentage of structurally aberrant cells observed at dose level 150μg/mL in the non-activated 4 hour exposure group (9.0%) was outside the non-activated historical control range and was therefore considered significant. Based on the findings of this study, Santicizer 2148 was concluded to be positive for the induction of structural chromosome aberrations in the absence of S9 activation, and negative for the induction of structural chromosome aberrations in the presence of S9 activation in Chinese hamster ovary (CHO) cells. Santicizer 2148 was concluded to be negative for the induction of numerical chromosome aberrations in the absence and presence of S9 activation in Chinese hamster ovary (CHO) cells.
In Vivo Mammalian Micronucleus Assay
A key in vivo mouse bone marrow micronucleus assay was conducted to the evaluate the the potential of the test material (XP-2563) to induce chromosomal effects (Monsanto Company, 1990c). The test material (in corn oil) was administered via intraperitoneal injection to groups of male and female CD-1 mice (15/sex/dose) at doses of 0, 500, 2500, or 5000 mg/Kg body weight. Following exposure, the mouse bone marrow was sampled at 24, 48, and 72 hours.
One death was observed in the micronucleus experiment in female mice treated at 5000 mg/Kg body weight. Toxcity, as indicated by a statistically significant decreased mean body weight and statistically significant decreases in the PCE/total erythrocyte ratio in test material treated animals, was observed. The test material did not induce statistically significant decreases in micronucleated PCEs in any of the treated animals. Increases in micronucleated PCE frequency were observed in the positive control group.
Based on the results observed, it was concluded that the test material is not a mammalian genotoxicant in vivo in mouse bone marrow cells.
Justification for classification or non-classification
Santicizer 2148 Plasticizer does not meet the criteria for classification for genetic toxicity under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 ortheUN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
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