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EC number: 247-161-5 | CAS number: 25646-71-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vivo
Administrative data
- Endpoint:
- genetic toxicity in vivo, other
- Remarks:
- a combined micronucleus and alkaline comet test
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 Sept to 03 Nov 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 023
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 474. Mammalian Erythrocyte Micronucleus Test and OECD Guideline 489. In Vivo Mammalian Alkaline Comet Assay.
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Mammalian comet assay and mammalian erythrocyte micronucleus assay
Test material
- Reference substance name:
- N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate
- EC Number:
- 247-161-5
- EC Name:
- N-(2-(4-amino-N-ethyl-m-toluidino)ethyl)methanesulphonamide sesquisulphate
- Cas Number:
- 25646-71-3
- Molecular formula:
- C12H21N3O2S.3/2H2O4S
- IUPAC Name:
- bis(N-{2-[(4-amino-3-methylphenyl)(ethyl)amino]ethyl}methanesulfonamide); tris(sulfuric acid)
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Crl: WI(Han)
- Details on species / strain selection:
- The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 152.0-160.8g
- Assigned to test groups randomly: Yes. Animals in poor health or at extremes of body weight range were not assigned to groups. Females (used in the dose ranging finding experiment only) were nulliparous and non-pregnant.
- Housing: Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
Up to 5 animals of the same sex and same dosing group were housed together.
- Diet: Ad libitum, except during designated procedures. SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
- Water: Freely available to each animal via water bottles. Municipal tap water.
- Acclimation period: 5 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22
- Humidity (%): 45 to 67
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 09 Sept 2022 To: 13 Oct 2022
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle used: Water
- Justification for choice of solvent/vehicle: Stability analyses performed previously in conjunction with Study No. 20134629 demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the study.
- Concentration of test material in vehicle: 5, 10 and 20 mg/mL
- Amount of vehicle: 10 mL/kg
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in Milli-Q water. This resulted in light pink solutions for all formulations. Test material concentrations were dosed within 5.5 hours after preparation.
Dose formulation analysis for concentration (all groups) and homogeneity (50 and 200 mg/kg bw/day groups) was performed on a single occasion. - Duration of treatment / exposure:
- 3 consecutive days
- Frequency of treatment:
- Daily
- Post exposure period:
- Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 50 mg/kg bw/day
- Remarks:
- Group 2
- Dose / conc.:
- 100 mg/kg bw/day
- Remarks:
- Group 3
- Dose / conc.:
- 200 mg/kg bw/day
- Remarks:
- Group 4
- No. of animals per sex per dose:
- Five male rats were used in each treatment group, with the exception of the highest dose group (8 animals). Three male rats in the vehicle control and highest dose group were used for bioanalysis.
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- ethylmethanesulphonate (EMS)
Justification for choice of positive control(s): EMS is a known mutagen and is recommended in the OECD 489 test guideline for use with any target tissue.
- Route of administration: Oral
- Doses / concentrations: 200 mg/kg bw dissolved in physiological saline.
Positive control slides taken from male animals previously dosed with positive control cyclophosphamide (CP) (19 mg/kg bw/day at 10 mL/kg bw in physiological saline dosed orally 48 hours prior to sampling), as part of Test Facility Study No. 20337827 were also used.
Examinations
- Tissues and cell types examined:
- Liver, glandular stomach, duodenum and bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION
Selection of an adequate dose-range for the main test was based on a dose-range finding (DRF) study. The test procedure and conditions were similar to those applied in the main test. In the dose-range finding study, 3 dose-groups were used to define the maximum tolerated dose (MTD) based on the toxic signs observed after dosing with different doses of the test material. One dose group, comprising of 3 males and 3 females, was dosed for three consecutive days (once daily) with the highest concentration of test material that was used for the main study. The other 2 groups consisted of 1 male and 1 female animal. The observation period after dosing was one to three days. During this period mortality and physical condition were recorded at least once a day.
The clinical signs of the DRF for the current OECD 489/474, did not indicate that the MTD had been reached. However, 200 mg/kg bw/day was considered to be a suitable top dose level for the main study. Based on the information provided by the Sponsor, in a previous study (OECD 422), all females were killed in extremis at 150 mg/kg bw/day and at 75 mg/kg bw/day showed clinical signs and adverse effects at necropsy. In an acute oral study, the LD50 was > 75 mg/kg bw, but < 300 mg/kg bw. Additionally, the OECD 422 DRF and acute oral study showed that the systemic toxicity is similar between males and females and therefore it was acceptable to run the main study with males only.
TREATMENT AND SAMPLING TIMES
The animals were dosed with vehicle or the test material for three consecutive days and twice with EMS. Bioanalysis blood samples were taken from 3 additional control and top dose males on Day 2 at 0, 1, 2, 4, 6, and 24 hrs post dose.
Approximately 3-4 hours after the third treatment with the test material bone marrow was isolated for the micronucleus test. In addition duodenum, glandular stomach and liver were collected/isolated and examined for DNA damage with the alkaline comet assay.
IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
Mortality: All animals were assessed twice daily from arrival to termination
Clinical observations: All animals were assessed daily from start of treatment to termination
Bodyweights: All animals were weighed prior to dosing
BIOANALYTICAL SAMPLE COLLECTION
Blood samples were taken from satelite animals control and top dose males on Day 2 at 0 hr (pre-dose) and 1, 2, 4, 6, 24 hrs post-dose via jugular vein puncture or if not feasible the retro-orbital sinus (under isoflurane anaesthesia). Samples were collected on ice. Resultant plasma was directly transferred into the stabilizer solution. Plasma/stabilizer solution must be in 1/1 (v/v) ratio.
Bioanalytical Sample Processing:
Immediately following the blood collection, samples were centrifuged within 15 minutes at approximately 2000 g for 10 minutes at 4-8 °C and the resultant plasma was separated, immediately transferred to uniquely labelled clear Micronic V-bottomed 1.4 mL tubes containing enough stabilizer solution to achieve 1:1 dilution (e.g. 50 µL of plasma was transferred to a tube containing 50 µL of stabilizer solution). Samples were mixed by gently inverting the tube few times and frozen immediately over dry ice or in a freezer set to maintain -80 °C.
Bioanalytical Sample Analysis:
Plasma samples were analyzed for concentration of Test Material using a LC-MS/MS method (liquid chromatography with tandem mass spectrometry) (Charles River project number 20365406).
Statistical analyses including regression analysis and descriptive statistics including arithmetic means and standard deviations, accuracy and precision were performed.
DETAILS OF SLIDE PREPARATION
Liver cells
A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+ and Mg2+ free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+ and Mg2+ free) and kept on ice.
Glandular stomach cells
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca2+, Mg2+ free, Life Technologies, Breda, the Netherlands). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis. The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consisted of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use). The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.
Duodenum cells
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodeunum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consisted of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca2+, Mg2+ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.
Sampling, fixation and storage of tissue for histotechnology and histopathology:
Part of the liver, stomach, duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin, together with the part of the tail containing the identification number.
Preparation of Comet Slides:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 10 to 60 minutes in the refrigerator in the dark until a clear ring appears at the edge of the comet slide area.
Lysis, Electrophoresis and Staining of the Slides:
The cells on the slides were overnight (approximately 16 to 18 hrs) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes (glandular stomach and duodenum) or 30 minutes (liver) at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 – 1 Volt/cm. The electrophoresis was performed for 20 to 30 minutes under constant cooling (actual temperature 4.0 °C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.
Isolation of Bone Marrow:
Bone marrow were sampled 48 hrs after the first dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum. The cell suspensions were collected and centrifuged at 216 g for 5 min.
Preparation of Bone Marrow Smears:
The supernatant was removed with a Pasteur pipette. Approximately 500 µL serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
Staining of the Bone Marrow Smears:
The slides were automatically stained using the “Wright-stain-procedure” in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining was based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
Positive control slides taken from male animals previously dosed with positive control (cyclophosphamide, 19 mg/kg bw/day at 10 mL/kg bw dosed orally 48 hours prior to sampling), as part of Test Facility Study No. 20337827, were added to the study slides for evaluation as scoring controls. Data from these animals was entered in the study as a positive control group for use in statistical evaluation. Arbitrary animal numbers were given based on the standard animal identification scheme. Slides from the positive control will be archived together with the study.
METHOD OF ANALYSIS
Comet Scoring:
To prevent bias, slides were randomly coded (per tissue) before examination of the comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a comet assay IV image analysis system (Instem, Staffordshire, United Kingdom). One hundred fifty comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of comets were used:
• Only horizontal orientated comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control.
Analysis of the bone marrow smears for micronuclei:
To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The proportion of immature erythrocytes was determined by counting and differentiating at least the first 500 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.
- Evaluation criteria:
- Comet Assay:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg bw/day
Micronucleus Test:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. - Statistics:
- ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.
A test material is considered positive in the comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative in the comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
A test material is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- DOSE FORMULATION ANALYSIS
Accuracy:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e., mean accuracies between 90% and 110%).
No test item was detected in the vehicle control group formulations.
Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%).
RESULTS OF RANGE-FINDING STUDY
The results of this dose-range finding study are presented in Table 1 (Any other information on results incl. tables).
No abnormalities were observed, except one female dosed at 200 mg/kg bw/day, which showed hunched posture, rough coat and eyes partly closed on Day 2 post-dose and on Day 3 pre- and post-dose.
RESULTS OF DEFINITIVE STUDY
Based on information provided by the sponsor and the results of the dose-range finding study dose levels of 50, 100 and 200 mg/kg bw/day were selected as appropriate doses for the main test. The clinical signs of the DRF for the current OECD 489/474, did not indicate that the MTD had been reached. However, 200 mg/kg bw/day was considered to be a suitable top dose level for the main study. Based on the information provided by the Sponsor, in a previous study (OECD 422), all females were killed in extremis at 150 mg/kg bw/day and at 75 mg/kg bw/day showed clinical signs and adverse effects at necropsy. In an acute oral study, the LD50 was >75 mg/kg bw, but <300 mg/kg bw. Additionally, the OECD 422 DRF and acute oral study showed that the systemic toxicity is similar between males and females and therefore it was acceptable to run the main study with males only.
Five male animals were used in each treatment group, with the exception of the positive control and TK animals and the highest dose group (3 and 8 animals per group, respectively). The mean body weights per group were recorded immediately prior to dosing and are presented in Table 2 (Any other information on results incl. tables).
Mortality and Toxic Signs:
The animals of the groups treated with 50, 100 and 200 mg/kg bw/day and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Micronucleated Polychromatic Erythrocytes:
The mean number of micronucleated polychromatic erythrocytes per group and the mean proportion of immature erythrocytes are presented in Table 3 (Any other information on results incl. tables). The individual data are described in Table 7 (Any other information on results incl. tables). The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database (Table 28, Any other information on results incl. tables).
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database (Table 29, Any other information on results incl. tables). Hence, all criteria for an acceptable assay were met.
Proportion of Immature Erythrocytes:
The animals of the groups which were treated with test material showed no decrease in the proportion of immature erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the proportion of immature erythrocytes, demonstrating toxic effects on erythropoiesis.
Comet Slide Analysis:
Comet slides were prepared and analyzed. An overview of the mean Tail Intensity is presented in Table 4 - 6 (Any other information on results incl. tables). The detailed data of the treatment groups is presented in Table 8-10 (Any other information on results incl. tables). The detailed data of the individual rats is presented in Table 11 - 25 (Any other information on results incl. tables). No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum, glandular stomach and liver cells of test material treated male treated animals compared to the vehicle treated animals. In addition, there were no Hedgehogs observed in vehicle and test material treated groups. The mean Tail Intensity in duodenum, glandular stomach and liver cells of vehicle-treated rats was 4.01 ± 1.19% (mean ± SD), 7.44 ± 1.76% (mean ± SD) and 3.35 ± 0.89% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Table 26, Any other information on results incl. tables). The positive control EMS induced a significant increase and showed a mean Tail Intensity of 44.08 ± 2.50% (mean ± SD, p<0.001 Students t test), 58.37 ± 4.46% (mean ± SD, p<0.001 Students t test) and 79.49 ± 0.82% (mean ± SD, p<0.001 Students t test) in male animals in duodenum, glandular stomach and liver cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database (Table 27, Any other information on results incl. tables). Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.
Bioanalysis:
Blood was sampled 1, 2, 4, 6, and 24 h after the second dose of toxicokinetic animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed no measurable amount of test material in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure. The bioanalytical phase report (Test Facility Study No. 20365399) tables are presented in the field "Overall remarks, attachments section".
Any other information on results incl. tables
Table 1: Mortality and Toxic Signs in the Dose-range Finding Study
Group | Sex |
Animal Number
|
Dose mg/kg bw/day | Toxic signs* | ||||
Day 1 | Day 2 | Day 3 | ||||||
|
|
|
| Post-dose | Pre-dose | Post-dose | Pre-dose | Post-dose |
|
|
|
|
|
|
|
|
|
1 | Male | 101 | 75 | B | B | B | B | B |
1 | Female | 102 | 75 | B | B | B | B | B |
|
|
|
|
|
|
|
|
|
2 | Male | 103 | 100 | B | B | B | B | B |
2 | Female | 104 | 100 | B | B | B | B | B |
|
|
|
|
|
|
|
|
|
3 | Male | 105 | 200 | B | B | B | B | B |
3 | Female | 106 | 200 | B | B | J, N, X | J, N, X | J, N, X |
3 | Male | 107 | 200 | B | B | B | B | B |
3 | Male | 108 | 200 | B | B | B | B | B |
3 | Female | 109 | 200 | B | B | B | B | N |
3 | Female | 110 | 200 | B | B | B | B | N |
|
|
|
|
|
|
|
|
|
* Legend 'Mortality and toxic signs':
B = showed no abnormalities; J = hunched posture; N = rough coat; X= eyes partly closed.
Table 2: Mean Body Weight Immediately Prior to Dosing
Group code * | Dose (mg/kg bw/day) | Day 1 | Day 2 | Day 3 | ||||||
1 | 0 | 160.8 | ± | 8.6 | 167.2 | ± | 9.2 | 162.2 | ± | 9.4 |
2 | 50 | 157.4 | ± | 5.1 | 163.0 | ± | 4.5 | 159.2 | ± | 5.9 |
3 | 100 | 152.0 | ± | 11.9 | 157.6 | ± | 12.9 | 153.4 | ± | 12.5 |
4 | 200 | 159.5 | ± | 9.9 | 161.8 | ± | 12.9 | 159.3 | ± | 14.2 |
5 | 200 (EMS) | # |
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| 160.0 | ± | 9.2 | 155.0 | ± | 10.0 |
TK 1 | 0 | 156.3 | ± | 12.2 | 162.7 | ± | 12.7 | $ |
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TK 4 | 200 | 156.7 | ± | 8.0 | 160.0 | ± | 12.5 | $ |
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# Not dosed. was started on Day 2.
$ Not weighed.
* Positive control slides taken from male animals previously dosed with positive control CP (cyclophosphamide, 19 mg/kg bw/day at 10 mL/kg bw as part of Test Facility Study No. 20337827).
Table 3: Mean Number of Micronucleated Polychromatic Erythrocytes and Proportion of Immature Erythrocytes
Group | Treatment | Number of Animals | Dose (mg/kg bw/day) | Number of micronucleated polychromatic erythrocytes | Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.) (#) | ||||
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| MALES |
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1 | Vehicle Control | 5 | 0 | 4.0 | ± | 1.6 | 0.56 | ± | 0.05 |
2 | Test Material | 5 | 50 | 3.8 | ± | 2.0 | 0.54 | ± | 0.04 |
3 | Test Material | 5 | 100 | 5.4 | ± | 2.1 | 0.55 | ± | 0.05 |
4 | Test Material | 5 | 200 | 5.4 | ± | 1.5 | 0.59 | ± | 0.01 |
5 | CP | 3 | 19 | 66.7 | ± | 28.7($) | 0.49 | ± | 0.04($) |
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Vehicle control = Milli-Q water.
CP = Cyclophosphamide.
(*) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(#) The proportion was determined from at least the first 500 erythrocytes counted.
($) Significantly different from corresponding control group (Students t test, P < 0.001).
Table 4: Overview Tail Intensity in Duodenum Cells of Male Rats
Tail Intensity (%) | S.D. | |
Vehicle Control | 4.01 | 1.19 |
Test Material 50 mg/kg bw/day | 4.34 | 1.54 |
Test Material 100 mg/kg bw/day | 3.34 | 1.30 |
Test Material 200 mg/kg bw/day | 4.12 | 1.00 |
EMS 200 mg/kg bw/day | 44.08 | 2.50 |
Table 5: Overview Tail Intensity in Glandular Stomach Cells of Male Rats
Tail Intensity (%) | S.D. | |
Vehicle Control | 7.44 | 1.76 |
Test Material 50 mg/kg bw/day | 5.19 | 2.13 |
Test Material 100 mg/kg bw/day | 4.43 | 1.36 |
Test Material 200 mg/kg bw/day | 6.30 | 1.89 |
EMS 200 mg/kg bw/day | 58.37 | 4.46 |
Table 6: Overview Tail Intensity in Liver Cells of Male Rats
Tail Intensity (%) | S.D. | |
Vehicle Control | 3.35 | 0.89 |
Test Material 50 mg/kg bw/day | 3.22 | 1.06 |
Test Material 100 mg/kg bw/day | 3.16 | 0.81 |
Test Material 200 mg/kg bw/day | 2.60 | 1.42 |
EMS 200 mg/kg bw/day | 79.49 | 0.82 |
Table 7: Individual Data Micronucleus Assay
Individual data (males) (Group 1: oral intubation of the vehicle) (Group 2: oral intubation at 50 mg/kg bw/day) (Group 3: oral intubation at CD3 at 100 mg/kg bw/day) (Group 4: oral intubation at 200 mg/kg bw/day) (Group 6: oral intubation of cyclophosphamide at 19 mg/kg bw/day)
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Group |
Animal number | Number of | Number of normochromatic erythrocytes(*) | Proportion of immature erythrocytes (*) | Number of micronucleated polychromatic erythrocytes | Number of polychromatic erythrocytes scored for micronuclei |
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| 1 | 1 | 307 | 213 | 0.59 | 4 | 4001 |
| 1 | 2 | 326 | 223 | 0.59 | 6 | 4005 |
| 1 | 3 | 319 | 208 | 0.61 | 3 | 4002 |
| 1 | 4 | 283 | 282 | 0.50 | 5 | 4000 |
| 1 | 5 | 269 | 246 | 0.52 | 2 | 4000 |
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| 2 | 6 | 281 | 253 | 0.53 | 4 | 4001 |
| 2 | 7 | 275 | 287 | 0.49 | 2 | 4000 |
| 2 | 8 | 353 | 299 | 0.54 | 7 | 4000 |
| 2 | 9 | 292 | 263 | 0.53 | 2 | 4000 |
| 2 | 10 | 302 | 200 | 0.60 | 4 | 4000 |
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| 3 | 11 | 308 | 198 | 0.61 | 5 | 4000 |
| 3 | 12 | 259 | 253 | 0.51 | 7 | 4000 |
| 3 | 13 | 325 | 218 | 0.60 | 4 | 4004 |
| 3 | 14 | 269 | 256 | 0.51 | 8 | 4006 |
| 3 | 15 | 278 | 262 | 0.51 | 3 | 4001 |
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| 4 | 16 | 312 | 239 | 0.57 | 6 | 4000 |
| 4 | 17 | 315 | 218 | 0.59 | 6 | 4000 |
| 4 | 18 | 299 | 214 | 0.58 | 7 | 4000 |
| 4 | 19 | 338 | 234 | 0.59 | 3 | 4003 |
| 4 | 20 | 324 | 214 | 0.60 | 5 | 4002 |
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| 6 | 117 | 251 | 254 | 0.50 | 93 | 4000 |
| 6 | 118 | 271 | 250 | 0.52 | 71 | 4000 |
| 6 | 119 | 246 | 311 | 0.44 | 36 | 4000 |
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(*) The proportion was determined from the first 500 erythrocytes counted.
Table 8: Mean Tail Intensity in Duodenum Cells
Vehicle Controls |
| Tail Intensity (%) |
Group 1 | Rat 1 | 4.16 |
Rat 2 | 5.76 | |
Rat 3 | 4.07 | |
Rat 4 | 2.43 | |
Rat 5 | 3.64 | |
Mean | 4.01 | |
S.D. | 1.19 | |
Test Material 50 mg/kg bw/day |
| Tail Intensity (%) |
Group 2 | Rat 6 | 4.69 |
Rat 7 | 2.80 | |
Rat 8 | 4.76 | |
Rat 9 | 6.54 | |
Rat 10 | 2.92 | |
Mean | 4.34 | |
S.D. | 1.54 | |
Test Material 100 mg/kg bw/day |
| Tail Intensity (%) |
Group 3 | Rat 11 | 3.20 |
Rat 12 | 2.37 | |
Rat 13 | 5.53 | |
Rat 14 | 3.27 | |
Rat 15 | 2.34 | |
Mean | 3.34 | |
S.D. | 1.30 | |
Test Material 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 4 | Rat 16 | 4.06 |
Rat 17 | 5.82 | |
Rat 18 | 3.62 | |
Rat 19 | 3.25 | |
Rat 20 | 3.86 | |
Mean | 4.12 | |
S.D. | 1.00 | |
EMS 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 5 | Rat 24 | 43.81 |
Rat 25 | 46.70 | |
Rat 26 | 41.73 | |
Mean | 44.08 | |
S.D. | 2.50 |
Table 9: Mean Tail Intensity in Glandular Stomach Cells
Vehicle Controls |
| Tail Intensity (%) |
Group 1 | Rat 1 | 8.83 |
Rat 2 | 9.79 | |
Rat 3 | 6.20 | |
Rat 4 | 5.79 | |
Rat 5 | 6.60 | |
Mean | 7.44 | |
S.D. | 1.76 | |
Test Material 50 mg/kg bw/day |
| Tail Intensity (%) |
Group 2 | Rat 6 | 4.98 |
Rat 7 | 7.04 | |
Rat 8 | 5.69 | |
Rat 9 | 6.61 | |
Rat 10 | 1.66 | |
Mean | 5.19 | |
S.D. | 2.13 | |
Test Material 100 mg/kg bw/day |
| Tail Intensity (%) |
Group 3 | Rat 11 | 5.69 |
Rat 12 | 5.64 | |
Rat 13 | 2.39 | |
Rat 14 | 4.42 | |
Rat 15 | 3.99 | |
Mean | 4.43 | |
S.D. | 1.36 | |
Test Material 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 4 | Rat 16 | 8.01 |
Rat 17 | 8.52 | |
Rat 18 | 4.08 | |
Rat 19 | 5.19 | |
Rat 20 | 5.73 | |
Mean | 6.30 | |
S.D. | 1.89 | |
EMS 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 5 | Rat 24 | 53.80 |
Rat 25 | 62.71 | |
Rat 26 | 58.62 | |
Mean | 58.37 | |
S.D. | 4.46 |
Table 10: Mean Tail Intensity in Liver Cells
Vehicle Controls |
| Tail Intensity (%) |
Group 1 | Rat 1 | 3.23 |
Rat 2 | 2.57 | |
Rat 3 | 4.16 | |
Rat 4 | 2.43 | |
Rat 5 | 4.36 | |
Mean | 3.35 | |
S.D. | 0.89 | |
Test Material 50 mg/kg bw/day |
| Tail Intensity (%) |
Group 2 | Rat 6 | 2.98 |
Rat 7 | 2.95 | |
Rat 8 | 4.97 | |
Rat 9 | 3.11 | |
Rat 10 | 2.08 | |
Mean | 3.22 | |
S.D. | 1.06 | |
Test Material 100 mg/kg bw/day |
| Tail Intensity (%) |
Group 3 | Rat 11 | 3.68 |
Rat 12 | 3.54 | |
Rat 13 | 3.99 | |
Rat 14 | 2.28 | |
Rat 15 | 2.30 | |
Mean | 3.16 | |
S.D. | 0.81 | |
Test Material 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 4 | Rat 16 | 3.80 |
Rat 17 | 4.06 | |
Rat 18 | 2.79 | |
Rat 19 | 1.58 | |
Rat 20 | 0.74 | |
Mean | 2.60 | |
S.D. | 1.42 | |
EMS 200 mg/kg bw/day |
| Tail Intensity (%) |
Group 5 | Rat 24 | 80.11 |
Rat 25 | 79.79 | |
Rat 26 | 78.56 | |
Mean | 79.49 | |
S.D. | 0.82 |
Table 11: Individual Rat Data in Duodenum cells (Group 1: 0 mg/kg bw/day)
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|
|
Rat 1 | Slide | Tail Intensity (%) |
52a2 | 3.25 | |
52b1 | 5.37 | |
52a1 | 3.87 | |
Mean | 4.16 | |
S.D. | 1.09 | |
Rat 2 | Slide | Tail Intensity (%) |
64a2 | 6.78 | |
64b1 | 5.38 | |
64a1 | 5.10 | |
mean | 5.76 | |
S.D. | 0.90 | |
Rat 3 | Slide | Tail Intensity (%) |
67a2 | 4.76 | |
67b1 | 4.55 | |
67a1 | 2.89 | |
Mean | 4.07 | |
S.D. | 1.03 | |
Rat 4 | Slide | Tail Intensity (%) |
60a2 | 3.24 | |
60b1 | 1.95 | |
60a1 | 2.12 | |
Mean | 2.43 | |
S.D. | 0.70 | |
Rat 5 | Slide | Tail Intensity (%) |
47a2 | 2.33 | |
47b1 | 3.35 | |
47a1 | 5.23 | |
Mean | 3.64 | |
S.D. | 1.47 |
Table 12: Individual Rat Data in Duodenum Cells (Group 2: Test Material 50 mg/kg bw/day)
Rat 6 | Slide | Tail Intensity (%) |
62a2 | 4.14 | |
62b1 | 4.18 | |
62a1 | 5.74 | |
Mean | 4.69 | |
S.D. | 0.91 | |
Rat 7 | Slide | Tail Intensity (%) |
50a2 | 3.64 | |
50b1 | 2.91 | |
50a1 | 1.85 | |
mean | 2.80 | |
S.D. | 0.90 | |
Rat 8 | Slide | Tail Intensity (%) |
65a2 | 5.09 | |
65b1 | 3.83 | |
65a1 | 5.35 | |
Mean | 4.76 | |
S.D. | 0.81 | |
Rat 9 | Slide | Tail Intensity (%) |
55a2 | 6.71 | |
55b1 | 5.51 | |
55a1 | 7.39 | |
Mean | 6.54 | |
S.D. | 0.95 | |
Rat 10 | Slide | Tail Intensity (%) |
59a1 | 2.39 | |
59b1 | 2.54 | |
59a2 | 3.82 | |
Mean | 2.92 | |
S.D. | 0.78 |
Table 13: Individual Rat Data in Duodenum Cells (Group 3: Test Material 100 mg/kg bw/day)
Rat 11 | Slide | Tail Intensity (%) |
61b1 | 2.46 | |
61a1 | 2.89 | |
61a2 | 4.27 | |
Mean | 3.20 | |
S.D. | 0.95 | |
Rat 12 | Slide | Tail Intensity (%) |
69a2 | 1.49 | |
69b1 | 2.51 | |
69a1 | 3.11 | |
mean | 2.37 | |
S.D. | 0.82 | |
Rat 13 | Slide | Tail Intensity (%) |
66b1 | 5.90 | |
66a1 | 5.24 | |
66a2 | 5.46 | |
Mean | 5.53 | |
S.D. | 0.33 | |
Rat 14 | Slide | Tail Intensity (%) |
53a2 | 2.47 | |
53b1 | 3.13 | |
53a1 | 4.22 | |
Mean | 3.27 | |
S.D. | 0.88 | |
Rat 15 | Slide | Tail Intensity (%) |
49a2 | 3.13 | |
49b1 | 1.46 | |
49a1 | 2.43 | |
Mean | 2.34 | |
S.D. | 0.84 |
Table 14: Individual Rat Data in Duodenum Cells (Group 4: Test Material 200 mg/kg bw/day)
Rat 16 | Slide | Tail Intensity (%) |
56a2 | 4.25 | |
56b1 | 3.33 | |
56a1 | 4.61 | |
Mean | 4.06 | |
S.D. | 0.66 | |
Rat 17 | Slide | Tail Intensity (%) |
63a2 | 6.19 | |
63b1 | 5.12 | |
63a1 | 6.14 | |
mean | 5.82 | |
S.D. | 0.60 | |
Rat 18 | Slide | Tail Intensity (%) |
68a2 | 3.42 | |
68b1 | 2.40 | |
68a1 | 5.04 | |
Mean | 3.62 | |
S.D. | 1.33 | |
Rat 19 | Slide | Tail Intensity (%) |
58a2 | 2.75 | |
58b1 | 1.30 | |
58a1 | 5.69 | |
Mean | 3.25 | |
S.D. | 2.24 | |
Rat 20 | Slide | Tail Intensity (%) |
51a2 | 4.26 | |
51b1 | 4.05 | |
51a1 | 3.27 | |
Mean | 3.86 | |
S.D. | 0.52 |
Table 15: Individual Rat Data in Duodenum Cells (Group 5: EMS 200 mg/kg bw/day)
Rat 24 | Slide | Tail Intensity (%) |
48a2 | 41.03 | |
48b1 | 45.93 | |
48a1 | 44.47 | |
Mean | 43.81 | |
S.D. | 2.52 | |
Rat 25 | Slide | Tail Intensity (%) |
57a2 | 48.00 | |
57b1 | 41.69 | |
57a1 | 50.41 | |
Mean | 46.70 | |
S.D. | 4.50 | |
Rat 26 | Slide | Tail Intensity (%) |
54a2 | 44.57 | |
54b1 | 46.78 | |
54a1 | 33.83 | |
Mean | 41.73 | |
S.D. | 6.93 | |
Table 16: Individual Rat Data in Glandular Stomach Cells (Group 1: 0 mg/kg bw/day)
Rat 1 | Slide | Tail Intensity (%) |
31a2 | 12.57 | |
31b1 | 6.45 | |
31a1 | 7.46 | |
Mean | 8.83 | |
S.D. | 3.29 | |
Rat 2 | Slide | Tail Intensity (%) |
44a2 | 12.35 | |
44b1 | 7.45 | |
44a1 | 9.56 | |
mean | 9.79 | |
S.D. | 2.46 | |
Rat 3 | Slide | Tail Intensity (%) |
46a2 | 3.36 | |
46b1 | 7.50 | |
46a1 | 7.74 | |
Mean | 6.20 | |
S.D. | 2.46 | |
Rat 4 | Slide | Tail Intensity (%) |
39a2 | 7.57 | |
39b1 | 5.72 | |
39a1 | 4.09 | |
Mean | 5.79 | |
S.D. | 1.74 | |
Rat 5 | Slide | Tail Intensity (%) |
27a2 | 3.97 | |
27b1 | 8.86 | |
27a1 | 6.97 | |
Mean | 6.60 | |
S.D. | 2.46 |
Table 17: Individual Rat Data in Glandular Stomach Cells (Group 2: Test Material 50 mg/kg bw/day)
Rat 6 | Slide | Tail Intensity (%) |
32a2 | 5.40 | |
32b1 | 6.45 | |
32a1 | 3.07 | |
Mean | 4.98 | |
S.D. | 1.73 | |
Rat 7 | Slide | Tail Intensity (%) |
37a2 | 8.26 | |
37b1 | 5.43 | |
37a1 | 7.44 | |
mean | 7.04 | |
S.D. | 1.46 | |
Rat 8 | Slide | Tail Intensity (%) |
43a2 | 7.92 | |
43b1 | 5.00 | |
43a1 | 4.15 | |
Mean | 5.69 | |
S.D. | 1.98 | |
Rat 9 | Slide | Tail Intensity (%) |
25a2 | 8.86 | |
25b1 | 7.77 | |
25a1 | 3.19 | |
Mean | 6.61 | |
S.D. | 3.01 | |
Rat 10 | Slide | Tail Intensity (%) |
33a2 | 3.57 | |
33b1 | 1.17 | |
33a1 | 0.24 | |
Mean | 1.66 | |
S.D. | 1.72 |
Table 18: Individual Rat Data in Glandular Stomach Cells (Group 3: Test Material 100 mg/kg bw/day)
Rat 11 | Slide | Tail Intensity (%) |
28a2 | 6.86 | |
28b1 | 3.27 | |
28a1 | 6.94 | |
Mean | 5.69 | |
S.D. | 2.10 | |
Rat 12 | Slide | Tail Intensity (%) |
38a2 | 3.99 | |
38b1 | 8.62 | |
38a1 | 4.30 | |
mean | 5.64 | |
S.D. | 2.59 | |
Rat 13 | Slide | Tail Intensity (%) |
41a2 | 2.30 | |
41b1 | 1.21 | |
41a1 | 3.66 | |
Mean | 2.39 | |
S.D. | 1.22 | |
Rat 14 | Slide | Tail Intensity (%) |
45a2 | 3.85 | |
45b1 | 5.30 | |
45a1 | 4.11 | |
Mean | 4.42 | |
S.D. | 0.77 | |
Rat 15 | Slide | Tail Intensity (%) |
34a2 | 4.62 | |
34b1 | 4.14 | |
34a1 | 3.21 | |
Mean | 3.99 | |
S.D. | 0.72 |
Table 19: Individual Rat Data in Glandular Stomach Cells (Group 4: Test Material 200 mg/kg bw/day)
Rat 16 | Slide | Tail Intensity (%) |
26a2 | 11.26 | |
26b1 | 7.55 | |
26a1 | 5.22 | |
Mean | 8.01 | |
S.D. | 3.05 | |
Rat 17 | Slide | Tail Intensity (%) |
36a2 | 8.56 | |
36b1 | 7.83 | |
36a1 | 9.15 | |
mean | 8.52 | |
S.D. | 0.66 | |
Rat 18 | Slide | Tail Intensity (%) |
42a2 | 3.52 | |
42b1 | 3.39 | |
42a1 | 5.33 | |
Mean | 4.08 | |
S.D. | 1.09 | |
Rat 19 | Slide | Tail Intensity (%) |
29a2 | 8.77 | |
29b1 | 3.00 | |
29a1 | 3.80 | |
Mean | 5.19 | |
S.D. | 3.12 | |
Rat 20 | Slide | Tail Intensity (%) |
40a2 | 4.79 | |
40b1 | 7.04 | |
40a1 | 5.35 | |
Mean | 5.73 | |
S.D. | 1.17 |
Table 20: Individual Rat Data in Glandular Stomach Cells (Group 5: EMS 200 mg/kg bw/day)
Rat 24 | Slide | Tail Intensity (%) |
35a2 | 54.07 | |
35b1 | 55.68 | |
35a1 | 51.64 | |
Mean | 53.80 | |
S.D. | 2.04 | |
Rat 25 | Slide | Tail Intensity (%) |
24a1 | 60.34 | |
24b1 | 66.72 | |
24a2 | 61.07 | |
Mean | 62.71 | |
S.D. | 3.49 | |
Rat 26 | Slide | Tail Intensity (%) |
30a2 | 59.16 | |
30b1 | 53.23 | |
30a1 | 63.47 | |
Mean | 58.62 | |
S.D. | 5.14 | |
Table 21: Individual Rat Data in Liver Cells (Group 1: 0 mg/kg bw/day)
Rat 1 | Slide | Tail Intensity (%) |
16a2 | 3.14 | |
16b1 | 2.36 | |
16a1 | 4.20 | |
Mean | 3.23 | |
S.D. | 0.93 | |
Rat 2 | Slide | Tail Intensity (%) |
11a2 | 2.01 | |
11b1 | 3.31 | |
11a1 | 2.40 | |
mean | 2.57 | |
S.D. | 0.67 | |
Rat 3 | Slide | Tail Intensity (%) |
7a2 | 4.55 | |
7b1 | 4.91 | |
7a1 | 3.01 | |
Mean | 4.16 | |
S.D. | 1.01 | |
Rat 4 | Slide | Tail Intensity (%) |
20a2 | 2.27 | |
20a1 | 3.18 | |
20b | 1.85 | |
Mean | 2.43 | |
S.D. | 0.68 | |
Rat 5 | Slide | Tail Intensity (%) |
1a2 | 3.18 | |
1b1 | 4.41 | |
1a1 | 5.48 | |
Mean | 4.36 | |
S.D. | 1.15 |
Table 22: Individual Rat Data in Liver Cells (Group 2: Test Material 50 mg/kg bw/day)
Rat 6 | Slide | Tail Intensity (%) |
8b1 | 2.76 | |
8a1 | 1.92 | |
8a2 | 4.27 | |
Mean | 2.98 | |
S.D. | 1.19 | |
Rat 7 | Slide | Tail Intensity (%) |
17a2 | 2.99 | |
17b1 | 3.51 | |
17a1 | 2.34 | |
mean | 2.95 | |
S.D. | 0.59 | |
Rat 8 | Slide | Tail Intensity (%) |
22a2 | 6.23 | |
22b1 | 5.85 | |
22a1 | 2.84 | |
Mean | 4.97 | |
S.D. | 1.86 | |
Rat 9 | Slide | Tail Intensity (%) |
2a2 | 3.06 | |
2b1 | 2.15 | |
2a1 | 4.14 | |
Mean | 3.11 | |
S.D. | 1.00 | |
Rat 10 | Slide | Tail Intensity (%) |
13a2 | 1.36 | |
13b1 | 2.72 | |
13a1 | 2.17 | |
Mean | 2.08 | |
S.D. | 0.69 |
Table 23: Individual Rat Data in Liver Cells (Group 3: Test Material 100 mg/kg bw/day)
Rat 11 | Slide | Tail Intensity (%) |
10a2 | 3.89 | |
10b1 | 3.05 | |
10a1 | 4.10 | |
Mean | 3.68 | |
S.D. | 0.56 | |
Rat 12 | Slide | Tail Intensity (%) |
18a2 | 3.73 | |
18b1 | 3.38 | |
18a1 | 3.51 | |
mean | 3.54 | |
S.D. | 0.17 | |
Rat 13 | Slide | Tail Intensity (%) |
21a2 | 4.14 | |
21b1 | 2.62 | |
21a1 | 5.21 | |
Mean | 3.99 | |
S.D. | 1.30 | |
Rat 14 | Slide | Tail Intensity (%) |
5a2 | 2.51 | |
5b1 | 3.04 | |
5a1 | 1.28 | |
Mean | 2.28 | |
S.D. | 0.91 | |
Rat 15 | Slide | Tail Intensity (%) |
14a2 | 2.25 | |
14b1 | 2.13 | |
14a1 | 2.53 | |
Mean | 2.30 | |
S.D. | 0.21 |
Table 24: Individual Rat Data in Liver Cells (Group 4: Test Material 200 mg/kg bw/day)
Rat 16 | Slide | Tail Intensity (%) |
19a2 | 4.14 | |
19b1 | 3.44 | |
19a1 | 3.83 | |
Mean | 3.80 | |
S.D. | 0.35 | |
Rat 17 | Slide | Tail Intensity (%) |
15a1 | 5.48 | |
15b1 | 3.45 | |
15a2 | 3.24 | |
mean | 4.06 | |
S.D. | 1.24 | |
Rat 18 | Slide | Tail Intensity (%) |
23a2 | 2.10 | |
23b1 | 2.38 | |
23a1 | 3.89 | |
Mean | 2.79 | |
S.D. | 0.96 | |
Rat 19 | Slide | Tail Intensity (%) |
9a2 | 1.15 | |
9b1 | 2.55 | |
9a1 | 1.05 | |
Mean | 1.58 | |
S.D. | 0.84 | |
Rat 20 | Slide | Tail Intensity (%) |
3a1 | 1.14 | |
3b1 | 0.52 | |
3a2 | 0.57 | |
Mean | 0.74 | |
S.D. | 0.34 |
Table 25: Individual Rat Data in Liver Cells (Group 5: EMS 200 mg/kg bw/day)
Rat 24 | Slide | Tail Intensity (%) |
4a2 | 80.18 | |
4b1 | 81.13 | |
4a1 | 79.01 | |
Mean | 80.11 | |
S.D. | 1.06 | |
Rat 25 | Slide | Tail Intensity (%) |
12b1 | 84.05 | |
12a1 | 76.19 | |
12a2 | 79.14 | |
Mean | 79.79 | |
S.D. | 3.97 | |
Rat 26 | Slide | Tail Intensity (%) |
6a2 | 77.18 | |
6b1 | 77.68 | |
6a1 | 80.82 | |
Mean | 78.56 | |
S.D. | 1.98 | |
Table 26: Historical Negative Control Data for Comet Assay
| Duodenum Males and Females | Liver Males and Females | Glandular Stomach Males and Females |
Mean | 7.0 | 2.9 | 6.7 |
SD | 2.8 | 1.3 | 2.9 |
n | 39 | 50 | 33 |
Lower control limit (95% control limits) | 1.4 | 0.3 | 1.0 |
Upper control limit (95% control limits) | 12.5 | 5.5 | 12.4 |
SD = Standard deviation
n = Number of observations
Liver, Stomach, Duodenum: Historical control data from experiments performed in November 2019 – November 2022
Table 27: Historical Positive Control Data for Comet Assay
| Duodenum Males and Females | Liver Males and Females | Glandular Stomach Males and Females |
Mean | 50.5 | 80.6 | 57.7 |
SD | 9.5 | 7.8 | 7.8 |
n | 39 | 50 | 33 |
Lower control limit (95% control limits) | 32.0 | 65.3 | 42.3 |
Upper control limit (95% control limits) | 69.1 | 95.8 | 73.0 |
SD = Standard deviation
n = Number of observations
Liver, Stomach, Duodenum: Historical control data from experiments performed in November 2019 – November 2022
Table 28: Historical Negative Control Data for Micronucleus Studies
| Male |
Mean Number of Micronucleated cells per 4000 cells | 3.7 |
SD | 1.6 |
N | 70 |
Lower Control Limit (95% Control Limits) | 1 |
Upper Control Limit (95% Control Limits) | 7 |
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between November 2019 and November 2022.
Table 29: Historical Positive Control Data for Micronucleus Studies
| Male |
Mean Number of Micronucleated cells per 4000 cells | 33.7 |
SD | 22.7 |
N | 65 |
Lower Control Limit (95% Control Limits) | -11 |
Upper Control Limit (95% Control Limits) | 78 |
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between November 2019 and November 2022.
Applicant's summary and conclusion
- Conclusions:
- It was concluded that the test substance is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 200 mg/kg bw/day (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in the report. Additionally, the comet assay was found to be valid and the test substance is not genotoxic in the comet assay in duodenum, glandular stomach and liver cells when sampled approximately 3-4 hours post dosing, for male rats that were dosed via oral gavage for three consecutive days up to a dose of 200 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions described in the report.
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