TY - JOUR
AU - Harbo, Sam, J
AB - Abstract The ICH revised the S3A guidance allowing blood to be microsampled for toxicokinetic analysis from the main study cohorts of rats in general toxicology studies. The resulting changes in the hemogram have been examined in healthy animals but the ability to read through the data when there are toxicological changes has not been thoroughly examined in the literature. To address this, a toxicology study in Sprague Dawley rats was conducted where animals received repeated doses of saline or valproic acid by IP injection daily for 7 days. Animals in both treatment groups were unbled, serially bled (6 bleeds/animal at 0.1 ml/bleed) or compositely bled (2 bleeds/animal at 0.6 ml/bleed) on days 1 and 7 for TK analysis. No statistically significant changes in the clinical pathology were observed for either the serial bleed or composite bleed animals when compared with their respective unbled control; however, a 4%–7% decrease in erythrocyte counts following serial bleeding and a 5%–19% decrease following composite bleeding was observed. When all the clinical pathology and organ weight data were equivalence tested, both the serial bleed and composite bleed results were equivalent to their unbled controls except for the erythroid parameters in the composite bleed group. Toxicokinetic analysis of the blood samples resulted in comparable concentration-time curves, regardless of the method of blood collection. Under these study conditions, the results show blood microsamples can be collected from the core or recovery cohort of animals in a toxicology study without impacting the toxicological interpretation in rats. biotransformation and toxicokinetics, exposure assessment, risk assessment, safety evaluation A general toxicology study designed to examine the safety of a drug candidate or other chemical agent should include a concurrent toxicokinetic (TK) analysis, which is used to examine the relationship between the dose and the resulting exposure in support of the toxicological observations. Because TK analysis requires multiple blood specimens to be collected from the animal over a short period of time, there may be toxicological effects resulting from both the administered drug and the acute loss of blood. For smaller animals such as rodents, the amount of blood loss can be consequential, potentially leading to increased moribundity or mortality as well as increased susceptibility to toxic effects. A common practice to ensure the robustness of the toxicity data is to isolate the TK bleeds from the main study cohorts through the use of a concomitantly dosed satellite group from which only TK data will be derived. For rats, a dose group of 9 animals/sex is commonly used where animals are bled in groups of 3 at a given timepoint until all animals have been bled once, then the process is repeated. When all blood specimens have been collected, each rat may be bled 1–3 times and the data can be processed into a single concentration-time (C-t) curve that is a composite of all 9 rats. Research studies conducted in humans or animals should always be designed with consideration of the 3 R’s: replacement, refinement, and reduction. At this time, there is no acceptable replacement for animal studies in pharmaceutical safety assessment; however, studies may be designed to reduce animal usage. In the case of toxicology studies in the rat, the satellite cohort constitutes a significant proportion of the total study animals. If the TK blood specimens could be collected from the main study cohort, the satellite cohort could be eliminated, which would constitute a 60% reduction in animal usage for exploratory studies and 40% for regulated studies (Caron et al., 2015). This would also refine the toxicological data since blood concentrations and toxicological observations would be collected in the same animals thus removing interanimal variability from the interpretation. In addition to addressing the 3 R’s, eliminating the satellite cohort would reduce the time, effort, cost, and test material to conduct the study. The success of this design is predicated on the blood collections being toxicologically inconsequential; otherwise the interpretation may be confounded. As the sensitivity of bioanalytical assays continues to improve, the amount of blood required for analysis continues to decrease. The use of low volume samples (microsamples) allows an entire TK profile to be generated from a single rat obviating the need for the traditional composite method. Since the toxicological burden due to blood loss would also greatly decrease, the possibility of shrinking or eliminating the satellite TK group should be considered. The methods used to collect blood microsamples, such as tail vein and saphenous bleeds, are less stressful to animals compared with techniques for larger volumes of blood collection such as jugular puncture and retro-orbital bleeds; the latter requiring anesthesia which could act as another confounding study variable. Several publications have investigated the feasibility of using microsampled bleeds in toxicology studies. These studies, conducted in naïve animals, demonstrated the microsamples had only very minor effects on standard hematological measurements and suggest they would not impact the overall analysis and conclusion of toxicological studies (Caron et al., 2015; Powles-Glover et al., 2014). This study was intended to build on this foundation by incorporating TK microsamples in a toxicology study examining saline-treated animals and animals administered a toxic dose of an approved drug, sodium valproate. Within each treatment group, a cohort of animals was unbled, serially or compositely bled. All 3 valproate-treated cohorts were then compared with their corresponding saline control cohorts (eg, valproate no bleed [NB] vs saline NB) to examine whether the interpretation of drug toxicity was affected. Animals were then compared within a given treatment to the unbled animals (eg, valproate serial bleed [SB] vs valproate NB) to determine if the blood sampling had a statistically significant effect on any measurements. Animals were dosed for 7 days with a serial or composite TK profile generated on dosing days 1 and 7. The blood microsamples were expected to have an impact on the hemogram but not enough to impact the toxicological interpretation of the study. A 14-day recovery period was included to determine if any changes related to the blood loss were reversible. MATERIALS AND METHODS Animals Sprague Dawley rats Crl: CD(SD) (58 male and 58 female) were purchased from Charles River Labs Kingston (Stone Ridge, New York) and acclimated for 7 days prior to study initiation. Animals were approximately 9 weeks old at study initiation with male body weights between 275 and 330 g and female body weights between 195 and 250 g. Animals were stratified into groups based on body weight. Study groups were organized based on treatment and further subdivided based on bleeding regimen as shown in Table 1. Animals were socially housed with 2 or 3 animals from the same group and sex per cage. Animals were housed in polycarbonate cages with wood chip bedding (Sani-Chips, P.J. Murphy Forest Products Corporation, Montville, New Jersey) and provided with polycarbonate huts or tubes as enrichment. Certified Rodent Diet 5002 (LabDiet, St Louis, Missouri) and water from the West Jefferson municipal water source were provided ad libitum. Temperature and humidity ranges of the study room were maintained at 72°F ± 3°F and 50% ± 15%, respectively. The light cycle was maintained at 12 h on/12 h off. General procedures for animal care and housing met AAALAC International recommendations and the current requirements stated in the “Guide for Care and Use of Laboratory Animals” (National Research Council, 2011). All study activities were approved by Battelle’s Institutional Animal Care and Use Committee prior to conduct. Table 1. Study Groups and Animal Numbers Used to Conduct the Study Group Dosea (mg/kg/day) Blood Volume Collected for TK (Days 1 and 7) Animal Numbers Coreb Recoveryc Male Female Male Female Saline CB 0 2 × 0.6 ml 9 9 — — Saline SB 0 6 × 0.1 ml 5 5 5 5 Saline NB 0 0 ml 5 5 5 5 Valproate CB 500 2 × 0.6 ml 9 9 — — Valproate SB 500 6 × 0.1 ml 5 5 5 5 Valproate NB 500 0 ml 5 5 5 5 Group Dosea (mg/kg/day) Blood Volume Collected for TK (Days 1 and 7) Animal Numbers Coreb Recoveryc Male Female Male Female Saline CB 0 2 × 0.6 ml 9 9 — — Saline SB 0 6 × 0.1 ml 5 5 5 5 Saline NB 0 0 ml 5 5 5 5 Valproate CB 500 2 × 0.6 ml 9 9 — — Valproate SB 500 6 × 0.1 ml 5 5 5 5 Valproate NB 500 0 ml 5 5 5 5 Abbreviations: CB, composite bleeds; SB, serial bleeds; NB, no bleeds. a Dose volume was 5 ml/kg/day. b Necropsy day 8. c Necropsy day 22. View Large Table 1. Study Groups and Animal Numbers Used to Conduct the Study Group Dosea (mg/kg/day) Blood Volume Collected for TK (Days 1 and 7) Animal Numbers Coreb Recoveryc Male Female Male Female Saline CB 0 2 × 0.6 ml 9 9 — — Saline SB 0 6 × 0.1 ml 5 5 5 5 Saline NB 0 0 ml 5 5 5 5 Valproate CB 500 2 × 0.6 ml 9 9 — — Valproate SB 500 6 × 0.1 ml 5 5 5 5 Valproate NB 500 0 ml 5 5 5 5 Group Dosea (mg/kg/day) Blood Volume Collected for TK (Days 1 and 7) Animal Numbers Coreb Recoveryc Male Female Male Female Saline CB 0 2 × 0.6 ml 9 9 — — Saline SB 0 6 × 0.1 ml 5 5 5 5 Saline NB 0 0 ml 5 5 5 5 Valproate CB 500 2 × 0.6 ml 9 9 — — Valproate SB 500 6 × 0.1 ml 5 5 5 5 Valproate NB 500 0 ml 5 5 5 5 Abbreviations: CB, composite bleeds; SB, serial bleeds; NB, no bleeds. a Dose volume was 5 ml/kg/day. b Necropsy day 8. c Necropsy day 22. View Large Test Article Normal saline for injection was acquired from Hospira (lot No. of 78-065-DK, Lake Forest, Illinois) and used as received. Pharmaceutical-grade sodium valproate (100 mg/ml) for injection was manufactured by Fresenius Kabi (lot Nos. 6013148 and 6013179; product No. 439405, Bad Homburg, Germany) and used as received. Dosing Animals were administered 5 ml/kg of normal saline or valproic acid (100 mg/ml, 500 mg/kg) for 6 days based on their body weight on day 1. Animals were weighed again on day 7 and administered their final dose based on the new body weight. Dosing was done at approximately the same time each morning via IP injection. Clinical Measurements and Observations Animals were weighed 3–4 days before study initiation for randomization. Animals were weighed again on study days 1 and 7 to calculate doses. Recovery body weights were measured on study days 14 and 21. Fasted, terminal body weights (TBWs) were measured prior to necropsy on study day 8 or 22. Food consumption was measured quantitatively at the cage level at the end of the dosing period and weekly thereafter. Individual consumption was assumed to be equal between housemates and was calculated by dividing the total food consumed per period by the number of rats in the cage. Animals were observed for signs of moribundity and mortality twice daily in the morning and evening throughout the acclimation and study periods. Cageside clinical observations were performed on all animals at least once prior to randomization, prior to the initial dose and once daily for the duration of the study. The observations were performed approximately 2 h following dose administration or at approximately the same time of day during the recovery period. A terminal observation was also recorded on the day of scheduled necropsy. Blood Collection Animals in the composite bleed (CB) groups were anesthetized with carbon dioxide/oxygen (CO2/O2 70/30%) and whole blood specimens of approximately 0.6 ml were collected from the retro-orbital venous plexus on days 1 and 7 into lithium heparin tubes (lot No. S072411, Sarstedt, Nümbrecht, Germany) according the schedule in Table 2. Table 2. CB Schedule in Saline- and Valproate-Treated Rats Target Number of Rats Target Time of Blood Collection (Hours [h] Postdose Administration) 0 ha 0.5 h 1 h 2 h 4 h 8 h First rat/sex/group X X Second rat/sex/group X X Third rat/sex/group X X Fourth rat/sex/group X X Fifth rat/sex/group X X Sixth rat/sex/group X X Seventh rat/sex/group X X Eighth rat/sex/group X X Ninth rat/sex/group X X Target Number of Rats Target Time of Blood Collection (Hours [h] Postdose Administration) 0 ha 0.5 h 1 h 2 h 4 h 8 h First rat/sex/group X X Second rat/sex/group X X Third rat/sex/group X X Fourth rat/sex/group X X Fifth rat/sex/group X X Sixth rat/sex/group X X Seventh rat/sex/group X X Eighth rat/sex/group X X Ninth rat/sex/group X X X—specimen collected. a The 0 h specimen was collected prior to dose administration. View Large Table 2. CB Schedule in Saline- and Valproate-Treated Rats Target Number of Rats Target Time of Blood Collection (Hours [h] Postdose Administration) 0 ha 0.5 h 1 h 2 h 4 h 8 h First rat/sex/group X X Second rat/sex/group X X Third rat/sex/group X X Fourth rat/sex/group X X Fifth rat/sex/group X X Sixth rat/sex/group X X Seventh rat/sex/group X X Eighth rat/sex/group X X Ninth rat/sex/group X X Target Number of Rats Target Time of Blood Collection (Hours [h] Postdose Administration) 0 ha 0.5 h 1 h 2 h 4 h 8 h First rat/sex/group X X Second rat/sex/group X X Third rat/sex/group X X Fourth rat/sex/group X X Fifth rat/sex/group X X Sixth rat/sex/group X X Seventh rat/sex/group X X Eighth rat/sex/group X X Ninth rat/sex/group X X X—specimen collected. a The 0 h specimen was collected prior to dose administration. View Large Animals in the SB groups were placed under a heating lamp for 12 min prior to specimen collection. After warming, the animals were restrained in a polycarbonate restraint tube and the tail was cleaned with 70% isopropyl alcohol. A whole blood specimen of approximately 0.1 ml was then dripped from the tail vein into tubes containing lithium heparin using a 23-gauge butterfly needle with the tubing cut off at the end of the cannula (BD, Franklin Lakes, New Jersey). Blood specimens were collected from each animal prior to dose administration and at approximately 0.5, 1, 2, 4, and 8 h postdosing on days 1 and 7. Blood specimens were mixed gently and placed on wet ice until centrifugation. Specimens were centrifuged for 10 min at 4°C and an rcf of 1800 × g. Plasma was separated, frozen and stored at approximately −80°C until analysis. Liquid Chromatographic Analysis With Tandem Mass Spectrometry Sodium valproate standard fortification solutions were prepared by serial dilution of 2 independent weighings of sodium valproate reference standard (European Pharmacopoeia, Strasbourg, France). The fortification solutions were diluted 20-fold with Sprague Dawley rat plasma (lithium heparin anticoagulant, BioIVT, Westbury, New York) to produce matrix calibration standards with concentrations of 0.187 (designated as the method lower limit of quantitation), 0.325, 0.933, 1.63, 4.66, 8.13, 14.9, and 16.3 µg/ml valproic acid. Plasma standards were extracted in duplicate with one replicate analyzed at the beginning and one at the end of each injection sequence of sample extracts. A linear regression with 1/x weighting was used to model calibration data normalized to internal standard (IS) multiple reaction monitoring (MRM) peak area response. Plasma sample and standard extracts were prepared by protein precipitation of 20 µl plasma with 100 µl methanol (HPLC grade, Sigma-Aldrich, St Louis, Missouri) containing 2000 µg/ml valproic acid-d6 IS (Cerilliant, Round Rock, Texas). The extraction procedure involved vortexing the plasma and solvent for 5 min followed by centrifugation for 10 min at 21 000 × g. The resulting supernatants were then transferred to auto-sampler vials. Analysis of extracts was carried out using a liquid chromatograph (Nexera X2, Shimadzu, Kyoto, Japan) coupled to a mass spectrometer (MS, API 5500, Sciex, Framington, Massachusetts). An injected sample extract volume of 2 µl was separated via mobile phase gradient using a Kinetex, C8, 50 × 2.1 mm, 2.6 µm chromatography column (Phenomenex, Torrance, California). The mobile phases were 10 mM ammonium acetate (HPLC grade, Fisher Scientific, Waltham, Massachusetts) in ASTM Type 1 water (designated mobile phase A and methanol (designated mobile phase B) with a total flow rate of 0.3 ml/min. A mobile phase gradient with a 2.0-min ramp from 25% B to 95% B followed by a 2.0-min hold at 95% B and a 0.1-min return to 25% B was used for all analyses. Mass spectrometry interface and analyte MRM parameters were an adaptation of those reported in the literature (Pucci et al., 2005). Postcolumn mobile phase was introduced to the MS via TurboIon Spray interface. MS detection was carried out in the negative ionization mode with MRM. The precursor/product ion pairs for MRM were m/z = 143/143 for valproate and m/z = 149/149 for IS. TK Analysis TK analysis was performed using a dose of 500 mg/kg, the nominal collection time points, and the measured plasma concentrations of sodium valproate. Since all of the actual sample collection time points were within 5% of target from 0 to 4 h or within 15 min of the target 8-h timepoint, the nominal timepoints were considered sufficient estimates. The resulting C-t profiles were evaluated using the noncompartmental analysis (NCA) module in the WinNonlin software program (Version 7.0, Certara, Princeton, New Jersey). For a given C-t profile, the values that were interpreted to define the terminal linear phase were identified by the WinNonlin algorithm to estimate the first order rate constant associated with the terminal linear phase. Ideally, the terminal phase should be defined by at least 3 data points following the Cmax. For 5 of the serial curves the terminal phase was set manually since the Cmax needed to be included as the first point in the terminal phase. This happened as a result of a late Cmax (eg, 2 h) or due to the loss of a data point in the terminal phase due to collection or analytical issues. Of the 5 affected curves from the valproate SB group, 2 were day 1 males, 2 were day 1 females, and 1 was a day 7 male. All of the day 1 issues were due to a missing timepoint on the terminal phase whereas the day 7 issue was due to a late Cmax. Since the drug effects were known prior to the day 7 bleeds, the animals were warmed longer to improve circulation which facilitated the bleeds and resolved the issue. To generate the composite C-t curves, the measured plasma concentrations of sodium valproate at a given timepoint were averaged. Plasma concentrations that were reported to be below the limit of quantitation or above the limit of quantitation (ALOQ) were not included in the TK analysis. For the 2 values that were ALOQ, there was insufficient volume to conduct a subsequent dilution. C-t profiles were considered to be adequately characterized when the following criteria were met: (1) the coefficient of determination (r2) for the terminal linear phase was ≥0.85, (2) the time of the last observed concentration was >3 times the half-life, and (3) the total area under the concentration-time curve (AUC∞) had <20% of the area extrapolated. Clinical Pathology Specimens for clinical pathology evaluation were collected on days 8 and 22 from the core and recovery cohorts, respectively. Animals were fasted overnight prior to blood collection for clinical pathology evaluation. Animals were anesthetized with 70% carbon dioxide in oxygen and blood for hematology and clinical chemistry analysis was collected via the retro-orbital venous plexus whereas blood for coagulation analysis was collected from the posterior vena cava or abdominal aorta. All acceptable specimens were analyzed for the parameters listed in Table 3. Hematology parameters were measured on an Advia 120 (Siemens, Munich, Germany; serum chemistry parameters were measured on a Cobas 6000 c501 (Roche, Basel, Switzerland); coagulation parameters were measured on a Stago Compact Max (Stago, Cedex, France). For hematology specimens, slides to examine cell morphology were prepared from unclotted samples when any of the following criteria were met: WBC > 25 k/ml, lymphocytes <50%, neutrophils <5%, basophils >2%, eosinophils >5%, large unstained cells >3%, hemoglobin (HGB) <9.0 g/dl, platelets <500 k/μl, and reticulocytes >500 k/μl. Table 3. Clinical and Anatomic Pathology Assessments Conducted at Each Necropsy Hematology Basophil count (BASO, 103 cells/μl) Platelet count (PLT, 103 cells/μl) Cell morphology Reticulocyte count, absolute (RETIC, 103 cells/μl) Eosinophil count (EOS, 103 cells/μl) Mean corpuscular hemoglobin (MCH, pg) Erythrocyte count (RBC, 106 cells/μl) Mean corpuscular hemoglobin concentration (MCHC, g/dl) Hematocrit (HCT, %) Mean corpuscular volume (MCV, fl) Hemoglobin (HGB, g/dl) Mean platelet volume (MPV, fl) Leukocyte count, total (WBC, 103 cells/μl) Monocyte count (MONO, 103 cells/μl) Large unstained cell count (LUC, 103 cells/μl) Lymphocyte count (LYMPH, 103 cells/μl) Neutrophil count (NEUT, 103 cells/μl) Serum Chemistry Alanine aminotransferase (ALT, U/l) Creatinine (mg/dl) Albumin (g/dl) Globulin, calculated (g/dl) Albumin/globulin (A/G) ratio, calculated Glucose (mg/dl) Alkaline phosphatase (ALKP, U/l) Lactate dehydrogenase (LDH, U/l) Aspartate aminotransferase (AST, U/l) Phosphorus (P, mg/dl) Bilirubin, direct (mg/dl) Potassium (K, mmol/l) Bilirubin, total (mg/dl) Sodium (Na, mmol/l) Calcium (mg/dl) Total protein (g/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Chloride (Cl, mmol/l) Urea nitrogen (BUN, mg/dl) Creatine kinase (CK, U/l) Coagulation Activated partial thromboplastin time (APTT, seconds) Prothrombin time (PT, seconds) Fibrinogen (mg/dl) Anatomic Pathology Tissues collected Tissues weighed Tissues processed to slides and evaluated Epididymides Epididymides Liver Kidneys Kidneys Testes Liver Liver Ovaries Ovaries Prostate gland Prostate gland Testes Testes Uterus (with cervix) Uterus (with cervix) Hematology Basophil count (BASO, 103 cells/μl) Platelet count (PLT, 103 cells/μl) Cell morphology Reticulocyte count, absolute (RETIC, 103 cells/μl) Eosinophil count (EOS, 103 cells/μl) Mean corpuscular hemoglobin (MCH, pg) Erythrocyte count (RBC, 106 cells/μl) Mean corpuscular hemoglobin concentration (MCHC, g/dl) Hematocrit (HCT, %) Mean corpuscular volume (MCV, fl) Hemoglobin (HGB, g/dl) Mean platelet volume (MPV, fl) Leukocyte count, total (WBC, 103 cells/μl) Monocyte count (MONO, 103 cells/μl) Large unstained cell count (LUC, 103 cells/μl) Lymphocyte count (LYMPH, 103 cells/μl) Neutrophil count (NEUT, 103 cells/μl) Serum Chemistry Alanine aminotransferase (ALT, U/l) Creatinine (mg/dl) Albumin (g/dl) Globulin, calculated (g/dl) Albumin/globulin (A/G) ratio, calculated Glucose (mg/dl) Alkaline phosphatase (ALKP, U/l) Lactate dehydrogenase (LDH, U/l) Aspartate aminotransferase (AST, U/l) Phosphorus (P, mg/dl) Bilirubin, direct (mg/dl) Potassium (K, mmol/l) Bilirubin, total (mg/dl) Sodium (Na, mmol/l) Calcium (mg/dl) Total protein (g/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Chloride (Cl, mmol/l) Urea nitrogen (BUN, mg/dl) Creatine kinase (CK, U/l) Coagulation Activated partial thromboplastin time (APTT, seconds) Prothrombin time (PT, seconds) Fibrinogen (mg/dl) Anatomic Pathology Tissues collected Tissues weighed Tissues processed to slides and evaluated Epididymides Epididymides Liver Kidneys Kidneys Testes Liver Liver Ovaries Ovaries Prostate gland Prostate gland Testes Testes Uterus (with cervix) Uterus (with cervix) View Large Table 3. Clinical and Anatomic Pathology Assessments Conducted at Each Necropsy Hematology Basophil count (BASO, 103 cells/μl) Platelet count (PLT, 103 cells/μl) Cell morphology Reticulocyte count, absolute (RETIC, 103 cells/μl) Eosinophil count (EOS, 103 cells/μl) Mean corpuscular hemoglobin (MCH, pg) Erythrocyte count (RBC, 106 cells/μl) Mean corpuscular hemoglobin concentration (MCHC, g/dl) Hematocrit (HCT, %) Mean corpuscular volume (MCV, fl) Hemoglobin (HGB, g/dl) Mean platelet volume (MPV, fl) Leukocyte count, total (WBC, 103 cells/μl) Monocyte count (MONO, 103 cells/μl) Large unstained cell count (LUC, 103 cells/μl) Lymphocyte count (LYMPH, 103 cells/μl) Neutrophil count (NEUT, 103 cells/μl) Serum Chemistry Alanine aminotransferase (ALT, U/l) Creatinine (mg/dl) Albumin (g/dl) Globulin, calculated (g/dl) Albumin/globulin (A/G) ratio, calculated Glucose (mg/dl) Alkaline phosphatase (ALKP, U/l) Lactate dehydrogenase (LDH, U/l) Aspartate aminotransferase (AST, U/l) Phosphorus (P, mg/dl) Bilirubin, direct (mg/dl) Potassium (K, mmol/l) Bilirubin, total (mg/dl) Sodium (Na, mmol/l) Calcium (mg/dl) Total protein (g/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Chloride (Cl, mmol/l) Urea nitrogen (BUN, mg/dl) Creatine kinase (CK, U/l) Coagulation Activated partial thromboplastin time (APTT, seconds) Prothrombin time (PT, seconds) Fibrinogen (mg/dl) Anatomic Pathology Tissues collected Tissues weighed Tissues processed to slides and evaluated Epididymides Epididymides Liver Kidneys Kidneys Testes Liver Liver Ovaries Ovaries Prostate gland Prostate gland Testes Testes Uterus (with cervix) Uterus (with cervix) Hematology Basophil count (BASO, 103 cells/μl) Platelet count (PLT, 103 cells/μl) Cell morphology Reticulocyte count, absolute (RETIC, 103 cells/μl) Eosinophil count (EOS, 103 cells/μl) Mean corpuscular hemoglobin (MCH, pg) Erythrocyte count (RBC, 106 cells/μl) Mean corpuscular hemoglobin concentration (MCHC, g/dl) Hematocrit (HCT, %) Mean corpuscular volume (MCV, fl) Hemoglobin (HGB, g/dl) Mean platelet volume (MPV, fl) Leukocyte count, total (WBC, 103 cells/μl) Monocyte count (MONO, 103 cells/μl) Large unstained cell count (LUC, 103 cells/μl) Lymphocyte count (LYMPH, 103 cells/μl) Neutrophil count (NEUT, 103 cells/μl) Serum Chemistry Alanine aminotransferase (ALT, U/l) Creatinine (mg/dl) Albumin (g/dl) Globulin, calculated (g/dl) Albumin/globulin (A/G) ratio, calculated Glucose (mg/dl) Alkaline phosphatase (ALKP, U/l) Lactate dehydrogenase (LDH, U/l) Aspartate aminotransferase (AST, U/l) Phosphorus (P, mg/dl) Bilirubin, direct (mg/dl) Potassium (K, mmol/l) Bilirubin, total (mg/dl) Sodium (Na, mmol/l) Calcium (mg/dl) Total protein (g/dl) Cholesterol (mg/dl) Triglycerides (mg/dl) Chloride (Cl, mmol/l) Urea nitrogen (BUN, mg/dl) Creatine kinase (CK, U/l) Coagulation Activated partial thromboplastin time (APTT, seconds) Prothrombin time (PT, seconds) Fibrinogen (mg/dl) Anatomic Pathology Tissues collected Tissues weighed Tissues processed to slides and evaluated Epididymides Epididymides Liver Kidneys Kidneys Testes Liver Liver Ovaries Ovaries Prostate gland Prostate gland Testes Testes Uterus (with cervix) Uterus (with cervix) View Large Anatomic Pathology According to Table 3, a subset of tissues that was expected to exhibit drug-related toxicity was collected. Tissues were examined grossly and weighed prior to fixation. All collected tissues were fixed in 10% neutral-buffered formalin (NBF), with the exception of testes/epididymides which were preserved in Modified Davidson’s fixative and subsequently transferred to 10% NBF. The liver (both sexes) and testes were processed for routine hematoxylin and eosin staining and evaluated microscopically by a board-certified veterinary pathologist. Statistical Analysis Statistical analysis using Provantis Body weights, clinical pathology, and organ weights were stratified by sex and compared for test article effects and bleed effects by analysis of variance (ANOVA) using Provantis (Instem, Stone, UK). For all data, normality was determined by the Shapiro-Wilks test and homogeneity of variances by Levene’s test. Data may have been log-transformed to meet parametric assumptions. For parametric data determined to be normally distributed and homogeneous among groups, an ANOVA F-test was used to determine whether there were differences among the group means. If the ANOVA F-test was significant, then tests for differences between the control and each of the comparison groups were conducted using Dunnett’s test, which adjusts for multiple comparisons. For nonparametric data that were not normally distributed and/or nonhomogeneous, a Kruskal-Wallis test was used to determine whether there were differences among the group means. If the Kruskal-Wallis test was significant, then tests for differences between the control and each of the comparison groups were conducted using Wilcoxon tests and the Bonferroni-Holm method to correct for multiple comparisons. All statistical tests were performed at the 0.05 level of significance (p < .05), after accounting for multiple comparisons where indicated. Equivalence testing Body weights, clinical pathology, and organ weights were compared within a treatment modality to assess bleed effects based on ANOVA models with main effects for treatment and bleed group, sex where appropriate, and all interactions. The ANOVA models were used to estimate the difference between the 2 treatments with 90% CIs for equivalence testing. Unlike the Provantis statistics, statistical comparisons were not stratified by sex. To assess equivalence between CB and SB groups and between SB and NB groups, the estimated mean difference between the comparators of interest and 90% CI were compared with an equivalence region of ±2 times the root mean square error. The equivalence region allows for a difference in means of approximately ± σ and the expected width of the 90% CI based on sample sizes of 5–10. The 2 comparators were determined to be equivalent at a 0.05 significance level if the 90% CI fell entirely within the equivalence region. Log-transformed data were back-transformed after analysis for more easily interpretable results, so mean shifts between groups represent ratios of the 2 means. SAS version 9.4 (SAS, Cary, North Carolina) was used for all statistical analysis. RESULTS Body Weights The group mean body weights with respect to time are provided in Supplementary Table 1. At the initiation of the study, animals of both sexes showed no statistical differences in body weight with a male body weight range of 277.5–328.9 g for the saline groups and 276.1–323.1 g for the valproate treatment groups. Female saline groups had a body weight range of 198.3–242.3 g while the valproate treatment groups had a range of 206.6–245.5 g. Over the 7-day dosing period, group mean body weights in the saline-treated males and females increased while group mean body weights in the valproate-treated males and females decreased leading to statistically significant differences for all bleed groups. After the dosing period, the valproate-treated animals exhibited a faster growth rate compared with their saline-treated controls resulting in similar group mean weights within the sexes. On day 14, there were no statistical differences in group mean body weights for males or females in the SB comparison. There were statistical differences for both males and females in the NB comparison. By day 21, only the NB comparison in the males was still significant though the difference in the means continued to narrow. Clinical Observations No abnormal clinical observations were recorded for the saline-treated groups of either sex. Valproate-treated animals were observed as ataxic and lethargic within 5–10 min of dosing which transitioned to a coma in agreement with published observations (Zieve and Lyftogt, 1989). While in this state, animals were unresponsive to being handled and did not react to the light or heat of the heating lamp. Effects began to resolve by 4 h with full recovery by 8 h. No differential effects were observed between the SB or CB groups to the unbled control; however, 1 male rat was found dead following the sixth dose of valproic acid in the CB group, and 1 female rat was euthanized due to moribundity following the second dose of valproic acid in the CB group. There were no unscheduled deaths in the SB or NB treatment groups. Blood Collection Specimens were successfully collected from all surviving rats in the CB groups. Blood collection via the tail vein was less successful as 17/240 total samples were missed or shorted. Allowing the valproate-treated animals to sit for a longer time under the heat lamp, or bringing them closer to the heat lamp, facilitated blood collection. TK Analysis All composite TK curves (4/4) were successfully modeled using the NCA module of WinNonlin and yielded respective half-lives of 1.89 and 1.53 h on days 1 and 7 for male rats, and 2.72 and 1.44 h on days 1 and 7 for the females. The males had an estimated AUC∞ of 2560 h·μg/ml on day 1 and 3040 h·μg/ml on day 7; females had corresponding AUC∞ estimates of 4250 and 3080 h·μg/ml. Samples from the SB groups were analyzed individually. Most of the curves (38/40) were successfully modeled, although 5 of these curves required the Cmax to be used in the estimation of the terminal linear phase. The results from all 5 of these animals were comparable with the group averages based on Cmax, AUC and t1/2, thus this did not impact the data. The analyzed curves provide a range of half-lives from 1.16–2.17 h for the males and 0.94–2.94 h for females across study days. The observed Cmax values ranged from 587 to 1240 μg/ml for the males and 732–1580 μg/ml for the females across study days. The AUC∞ ranged from 2500 to 4350 for the males and 3190–6650 μg·h/ml for the females. The serial data were also averaged at each timepoint within each sex, consistent with what is done for composite profile generation. This pooled serial bleed (PSB) data provide an average curve to describe the population that is slightly different than averaging the individually analyzed data. The curves generated from pooling the data resulted in respective half-lives of 1.83 and 1.54 h on days 1 and 7 for male rats, and 2.09 and 1.64 h on days 1 and 7 for the females. A summary of the analyzed TK data is found in Table 4. The individual C-t curves generated from the males on days 1 and 7 are presented in the top of Figure 1 and the females are presented in the bottom of Figure 1. The composite curves and PSB curves are presented together in Figure 2. Table 4. Group Mean Estimates and Ranges of Noncompartmental Pharmacokinetic Parameters for SB, PSB, and CB animals Bleed Cmax Range Tmax t1/2 Range AUC∞ Range μg/ml μg/ml h h h h·μg/ml h·μg/ml Males Day 1 SB 962 587–1190 0.85 1.85 1.31–2.17 3250 2500–4350 PSB 931 — 1 1.83 — 3190 — CB 891 — 1 1.89 — 2560 — Day 7 SB 1000 687–1240 0.85 1.56 1.16–1.96 3580 2690–4000 PSB 940 — 1 1.54 — 3570 — CB 792 — 0.5 1.53 — 3040 — Females Day 1 SB 1080 829–1280 0.85 2.14 1.11–2.94 4820 3240–6650 PSB 1020 — 1 2.09 — 4720 — CB 871 — 1 2.72 — 4250 — Day 7 SB 1090 732–1580 0.85 1.66 0.94–2.41 3810 3190–5430 PSB 952 1 1.64 — 3920 — CB 839 1 1.44 — 3080 — Bleed Cmax Range Tmax t1/2 Range AUC∞ Range μg/ml μg/ml h h h h·μg/ml h·μg/ml Males Day 1 SB 962 587–1190 0.85 1.85 1.31–2.17 3250 2500–4350 PSB 931 — 1 1.83 — 3190 — CB 891 — 1 1.89 — 2560 — Day 7 SB 1000 687–1240 0.85 1.56 1.16–1.96 3580 2690–4000 PSB 940 — 1 1.54 — 3570 — CB 792 — 0.5 1.53 — 3040 — Females Day 1 SB 1080 829–1280 0.85 2.14 1.11–2.94 4820 3240–6650 PSB 1020 — 1 2.09 — 4720 — CB 871 — 1 2.72 — 4250 — Day 7 SB 1090 732–1580 0.85 1.66 0.94–2.41 3810 3190–5430 PSB 952 1 1.64 — 3920 — CB 839 1 1.44 — 3080 — View Large Table 4. Group Mean Estimates and Ranges of Noncompartmental Pharmacokinetic Parameters for SB, PSB, and CB animals Bleed Cmax Range Tmax t1/2 Range AUC∞ Range μg/ml μg/ml h h h h·μg/ml h·μg/ml Males Day 1 SB 962 587–1190 0.85 1.85 1.31–2.17 3250 2500–4350 PSB 931 — 1 1.83 — 3190 — CB 891 — 1 1.89 — 2560 — Day 7 SB 1000 687–1240 0.85 1.56 1.16–1.96 3580 2690–4000 PSB 940 — 1 1.54 — 3570 — CB 792 — 0.5 1.53 — 3040 — Females Day 1 SB 1080 829–1280 0.85 2.14 1.11–2.94 4820 3240–6650 PSB 1020 — 1 2.09 — 4720 — CB 871 — 1 2.72 — 4250 — Day 7 SB 1090 732–1580 0.85 1.66 0.94–2.41 3810 3190–5430 PSB 952 1 1.64 — 3920 — CB 839 1 1.44 — 3080 — Bleed Cmax Range Tmax t1/2 Range AUC∞ Range μg/ml μg/ml h h h h·μg/ml h·μg/ml Males Day 1 SB 962 587–1190 0.85 1.85 1.31–2.17 3250 2500–4350 PSB 931 — 1 1.83 — 3190 — CB 891 — 1 1.89 — 2560 — Day 7 SB 1000 687–1240 0.85 1.56 1.16–1.96 3580 2690–4000 PSB 940 — 1 1.54 — 3570 — CB 792 — 0.5 1.53 — 3040 — Females Day 1 SB 1080 829–1280 0.85 2.14 1.11–2.94 4820 3240–6650 PSB 1020 — 1 2.09 — 4720 — CB 871 — 1 2.72 — 4250 — Day 7 SB 1090 732–1580 0.85 1.66 0.94–2.41 3810 3190–5430 PSB 952 1 1.64 — 3920 — CB 839 1 1.44 — 3080 — View Large Figure 1. View largeDownload slide Individual concentration-time curves for male (top) and female (bottom) rats treated with valproic acid and bled serially via the tail vein on dosing days 1 and 7. Figure 1. View largeDownload slide Individual concentration-time curves for male (top) and female (bottom) rats treated with valproic acid and bled serially via the tail vein on dosing days 1 and 7. Figure 2. View largeDownload slide Concentration-time curves for male (top) and female (bottom) rats treated with valproic acid on dosing days 1 and 7. For pooled serial bleeds, all concentrations for a given sex were averaged at each timepoint to generate a single average curve; for composite bleeds the concentrations from 3 animals/sex were averaged at each timepoint. Figure 2. View largeDownload slide Concentration-time curves for male (top) and female (bottom) rats treated with valproic acid on dosing days 1 and 7. For pooled serial bleeds, all concentrations for a given sex were averaged at each timepoint to generate a single average curve; for composite bleeds the concentrations from 3 animals/sex were averaged at each timepoint. Clinical Pathology Drug effects Drug effects were examined by comparing the valproate-treated groups to their respective saline control groups, eg, valproate SB versus saline SB. All statistically significant differences determined by Provantis are presented in Table 5; however, only toxicologically relevant differences will be discussed. Tabular summaries of the clinical pathology data at the end of the dosing and recovery periods are provided in Table 5 and SupplementaryTable 2, respectively. Table 5. Statistically Significant Differences in Clinical Pathology Parameters Potentially Due to Drug Effects at the End of the 7-Day Dosing Period NB SB CB Parameter Saline NB Valproate NB Saline SB Valproate SB Saline CB Valproate CB Core Males  ALKP 232 ± 57 101 ± 19* 217 ± 39 107 ± 9* 214 ± 36 120 ± 31*  Total protein 6.0 ± 0.2 5.3 ± 0.2* 6.2 ± 0.3 5.3 ± 0.2* 6.2 ± 0.3 5.6 ± 0.3*  Albumin 4.2 ± 0.1 3.7 ± 0.3* 4.4 ± 0.2 3.7 ± 0.3* 4.2 ± 0.2 3.9 ± 0.4  Chloride 101 ± 1 104 ± 1* 102 ± 0 105 ± 1* 102 ± 1 103 ± 3  MCHC 31.5 ± 0.6 32.8 ± 0.6* 31.2 ± 0.8 32.9 ± 0.7* 31.6 ± 0.5 32 ± 0.6  WBC 7.24 ± 2.4 4.47 ± 0.75* 9.52 ± 2.34 3.93 ± 1.02* 9.90 ± 2.65 6.83 ± 3.35  LYMPH 6.08 ± 2.18 3.27 ± 0.51* 7.67 ± 1.33 2.97 ± 0.88* 7.98 ± 2.11 4.40 ± 1.57*  PLT 1005 ± 47 726 ± 239* 1100 ± 136 710 ± 89* 1103 ± 165 907 ± 184*  HCT 45.4 ± 2.4 43.9 ± 3.6 46.4 ± 1.7 41.5 ± 1.5* 43.5 ± 1.9 40.2 ± 4.1  RETIC 252.1 ± 32.9 135 ± 121.8 327.1 ± 45.9 127.2 ± 84.8* 423.7 ± 73.5 297.2 ± 163*  LUC 0.07 ± 0.03 0.07 ± 0.02 0.09 ± 0.02 0.05 ± 0.02* 0.10 ± 0.04 0.12 ± 0.07  MCV 63.9 ± 1.7 61.6 ± 2.3 65.7 ± 1.4 60.8 ± 1* 64.6 ± 2.8 62.2 ± 1.6  Globulin 1.8 ± 0.3 1.6 ± 0.3 1.8 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 1.7 ± 0.2*  Cholesterol 55 ± 12 52 ± 7 58 ± 13 44 ± 8 59 ± 8 49 ± 3*  CK 466 ± 132 369 ± 324 336 ± 136 620 ± 446 420 ± 162 265 ± 130*  Glucose 92 ± 9 88 ± 11 114 ± 34 88 ± 16 122 ± 22 91 ± 12*  Potassium 6.8 ± 0.2 6.4 ± 0.6 6.5 ± 0.5 6.1 ± 0.3 6.7 ± 0.5 6.0 ± 0.6*  EOS 0.02 ± 0.02 0.03 ± 0.04 0.05 ± 0.03 0.06 ± 0.04 0.11 ± 0.1 0.03 ± 0.03*  BASO 0.02 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.02 ± 0.01* Core Females  ALKP 171 ± 33 97 ± 28* 155 ± 38 89 ± 28* 156 ± 35 114 ± 21*  Total Protein 6.4 ± 0.2 5.3 ± 0.2* 6.4 ± 0.3 5.2 ± 0.3* 6.3 ± 0.4 5.4 ± 0.2*  Albumin 4.6 ± 0.2 3.8 ± 0.3* 4.5 ± 0.2 3.8 ± 0.4* 4.5 ± 0.3 4.0 ± 0.2*  Globulin 1.8 ± 0.1 1.4 ± 0.2* 1.9 ± 0.2 1.4 ± 0.2* 1.9 ± 0.1 1.4 ± 0.2*  Potassium 5.9 ± 0.3 5.1 ± 0.5* 6.4 ± 0.3 5.3 ± 0.4* 6.3 ± 0.5 5.3 ± 0.7*  HCT 46.0 ± 1.2 38.8 ± 5.1* 42.6 ± 1 36.0 ± 2.2* 38.1 ± 3.2 34.1 ± 4.4*  MPV 8.4 ± 0.2 9.2 ± 0.6* 8.3 ± 0.2 9.1 ± 0.3* 8.2 ± 0.3 9.0 ± 0.3*  MCHC 33.3 ± 0.2 34.7 ± 0.3* 32.7 ± 0.6 34.3 ± 0.7* 33.1 ± 0.7 34.0 ± 0.9*  WBC 7.26 ± 1.69 4.03 ± 1.07* 10.00 ± 3.43 4.15 ± 1.01* 7.81 ± 2.86 2.99 ± 0.71*  LYMPH 6.07 ± 1.22 2.93 ± 0.91* 8.60 ± 3.18 3.37 ± 1.03* 6.70 ± 2.58 2.21 ± 0.41*  RETIC 218.7 ± 40.7 17.2 ± 21.4* 245.0 ± 53.0 31.7 ± 25.3* 370.5 ± 51.3 140.2 ± 165.6*  EOS 0.08 ± 0.05 0 ± 0.01* 0.11 ± 0.04 0.01 ± 0.01* 0.10 ± 0.07 0.01 ± 0.01*  PLT 1174 ± 88 721 ± 93* 1350 ± 206 646 ± 124* 1244 ± 134 776 ± 208*  HGB 15.3 ± 0.4 13.5 ± 1.8* 14.0 ± 0.4 12.4 ± 0.6* 12.6 ± 0.9 11.6 ± 1.3  LDH 381 ± 127 148 ± 67* 208 ± 61 131 ± 18* 315 ± 188 190 ± 64  ALT 46 ± 8 34 ± 6* 39 ± 7 33 ± 4 36 ± 7 36 ± 11  AST 106 ± 9 82 ± 11* 92 ± 10 97 ± 13 91 ± 16 114 ± 39  Cholesterol 58 ± 9 40 ± 9* 68 ± 19 48 ± 7 67 ± 13 43 ± 8*  Monocytes 0.19 ± 0.1 0.14 ± 0.09 0.20 ± 0.07 0.09 ± 0.04* 0.18 ± 0.1 0.08 ± 0.04*  BASO 0.02 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01* 0.03 ± 0.02 0.01 ± 0.00*  Creatinine 0.5 ± 0.0 0.4 ± 0.1 0.5 ± 0.0 0.4 ± 0.0* 0.5 ± 0.1 0.4 ± 0.0*  RBC 7.70 ± 0.15 6.67 ± 0.97 7.03 ± 0.33 6.23 ± 0.18* 6.26 ± 0.59 6.03 ± 0.44  MCV 59.7 ± 1.9 58.2 ± 1.3 60.7 ± 1.9 57.8 ± 1.9* 60.9 ± 1.7 58.9 ± 2.6  Neutrophils 1.13 ± 0.08 1.2 ± 0.1 1.04 ± 0.03 NM 1.49 ± 0.39 1.09a  PT 9.8 ± 0.4 10.6 ± 1.3 9.9 ± 0.4 10.5 ± 0.3* 9.9 ± 0.3 10.1 ± 0.4  A/G Ratio 2.5 ± 0.2 2.8 ± 0.5 2.5 ± 0.3 2.8 ± 0.6 2.4 ± 0.2 3.0 ± 0.6*  LUC 0.09 ± 0.04 0.07 ± 0.04 0.16 ± 0.1 0.1 ± 0.06 0.09 ± 0.05 0.04 ± 0.02* NB SB CB Parameter Saline NB Valproate NB Saline SB Valproate SB Saline CB Valproate CB Core Males  ALKP 232 ± 57 101 ± 19* 217 ± 39 107 ± 9* 214 ± 36 120 ± 31*  Total protein 6.0 ± 0.2 5.3 ± 0.2* 6.2 ± 0.3 5.3 ± 0.2* 6.2 ± 0.3 5.6 ± 0.3*  Albumin 4.2 ± 0.1 3.7 ± 0.3* 4.4 ± 0.2 3.7 ± 0.3* 4.2 ± 0.2 3.9 ± 0.4  Chloride 101 ± 1 104 ± 1* 102 ± 0 105 ± 1* 102 ± 1 103 ± 3  MCHC 31.5 ± 0.6 32.8 ± 0.6* 31.2 ± 0.8 32.9 ± 0.7* 31.6 ± 0.5 32 ± 0.6  WBC 7.24 ± 2.4 4.47 ± 0.75* 9.52 ± 2.34 3.93 ± 1.02* 9.90 ± 2.65 6.83 ± 3.35  LYMPH 6.08 ± 2.18 3.27 ± 0.51* 7.67 ± 1.33 2.97 ± 0.88* 7.98 ± 2.11 4.40 ± 1.57*  PLT 1005 ± 47 726 ± 239* 1100 ± 136 710 ± 89* 1103 ± 165 907 ± 184*  HCT 45.4 ± 2.4 43.9 ± 3.6 46.4 ± 1.7 41.5 ± 1.5* 43.5 ± 1.9 40.2 ± 4.1  RETIC 252.1 ± 32.9 135 ± 121.8 327.1 ± 45.9 127.2 ± 84.8* 423.7 ± 73.5 297.2 ± 163*  LUC 0.07 ± 0.03 0.07 ± 0.02 0.09 ± 0.02 0.05 ± 0.02* 0.10 ± 0.04 0.12 ± 0.07  MCV 63.9 ± 1.7 61.6 ± 2.3 65.7 ± 1.4 60.8 ± 1* 64.6 ± 2.8 62.2 ± 1.6  Globulin 1.8 ± 0.3 1.6 ± 0.3 1.8 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 1.7 ± 0.2*  Cholesterol 55 ± 12 52 ± 7 58 ± 13 44 ± 8 59 ± 8 49 ± 3*  CK 466 ± 132 369 ± 324 336 ± 136 620 ± 446 420 ± 162 265 ± 130*  Glucose 92 ± 9 88 ± 11 114 ± 34 88 ± 16 122 ± 22 91 ± 12*  Potassium 6.8 ± 0.2 6.4 ± 0.6 6.5 ± 0.5 6.1 ± 0.3 6.7 ± 0.5 6.0 ± 0.6*  EOS 0.02 ± 0.02 0.03 ± 0.04 0.05 ± 0.03 0.06 ± 0.04 0.11 ± 0.1 0.03 ± 0.03*  BASO 0.02 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.02 ± 0.01* Core Females  ALKP 171 ± 33 97 ± 28* 155 ± 38 89 ± 28* 156 ± 35 114 ± 21*  Total Protein 6.4 ± 0.2 5.3 ± 0.2* 6.4 ± 0.3 5.2 ± 0.3* 6.3 ± 0.4 5.4 ± 0.2*  Albumin 4.6 ± 0.2 3.8 ± 0.3* 4.5 ± 0.2 3.8 ± 0.4* 4.5 ± 0.3 4.0 ± 0.2*  Globulin 1.8 ± 0.1 1.4 ± 0.2* 1.9 ± 0.2 1.4 ± 0.2* 1.9 ± 0.1 1.4 ± 0.2*  Potassium 5.9 ± 0.3 5.1 ± 0.5* 6.4 ± 0.3 5.3 ± 0.4* 6.3 ± 0.5 5.3 ± 0.7*  HCT 46.0 ± 1.2 38.8 ± 5.1* 42.6 ± 1 36.0 ± 2.2* 38.1 ± 3.2 34.1 ± 4.4*  MPV 8.4 ± 0.2 9.2 ± 0.6* 8.3 ± 0.2 9.1 ± 0.3* 8.2 ± 0.3 9.0 ± 0.3*  MCHC 33.3 ± 0.2 34.7 ± 0.3* 32.7 ± 0.6 34.3 ± 0.7* 33.1 ± 0.7 34.0 ± 0.9*  WBC 7.26 ± 1.69 4.03 ± 1.07* 10.00 ± 3.43 4.15 ± 1.01* 7.81 ± 2.86 2.99 ± 0.71*  LYMPH 6.07 ± 1.22 2.93 ± 0.91* 8.60 ± 3.18 3.37 ± 1.03* 6.70 ± 2.58 2.21 ± 0.41*  RETIC 218.7 ± 40.7 17.2 ± 21.4* 245.0 ± 53.0 31.7 ± 25.3* 370.5 ± 51.3 140.2 ± 165.6*  EOS 0.08 ± 0.05 0 ± 0.01* 0.11 ± 0.04 0.01 ± 0.01* 0.10 ± 0.07 0.01 ± 0.01*  PLT 1174 ± 88 721 ± 93* 1350 ± 206 646 ± 124* 1244 ± 134 776 ± 208*  HGB 15.3 ± 0.4 13.5 ± 1.8* 14.0 ± 0.4 12.4 ± 0.6* 12.6 ± 0.9 11.6 ± 1.3  LDH 381 ± 127 148 ± 67* 208 ± 61 131 ± 18* 315 ± 188 190 ± 64  ALT 46 ± 8 34 ± 6* 39 ± 7 33 ± 4 36 ± 7 36 ± 11  AST 106 ± 9 82 ± 11* 92 ± 10 97 ± 13 91 ± 16 114 ± 39  Cholesterol 58 ± 9 40 ± 9* 68 ± 19 48 ± 7 67 ± 13 43 ± 8*  Monocytes 0.19 ± 0.1 0.14 ± 0.09 0.20 ± 0.07 0.09 ± 0.04* 0.18 ± 0.1 0.08 ± 0.04*  BASO 0.02 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01* 0.03 ± 0.02 0.01 ± 0.00*  Creatinine 0.5 ± 0.0 0.4 ± 0.1 0.5 ± 0.0 0.4 ± 0.0* 0.5 ± 0.1 0.4 ± 0.0*  RBC 7.70 ± 0.15 6.67 ± 0.97 7.03 ± 0.33 6.23 ± 0.18* 6.26 ± 0.59 6.03 ± 0.44  MCV 59.7 ± 1.9 58.2 ± 1.3 60.7 ± 1.9 57.8 ± 1.9* 60.9 ± 1.7 58.9 ± 2.6  Neutrophils 1.13 ± 0.08 1.2 ± 0.1 1.04 ± 0.03 NM 1.49 ± 0.39 1.09a  PT 9.8 ± 0.4 10.6 ± 1.3 9.9 ± 0.4 10.5 ± 0.3* 9.9 ± 0.3 10.1 ± 0.4  A/G Ratio 2.5 ± 0.2 2.8 ± 0.5 2.5 ± 0.3 2.8 ± 0.6 2.4 ± 0.2 3.0 ± 0.6*  LUC 0.09 ± 0.04 0.07 ± 0.04 0.16 ± 0.1 0.1 ± 0.06 0.09 ± 0.05 0.04 ± 0.02* Statistically significant differences observed in the NB comparison are prioritized followed by significant differences in the serial bleed comparison followed by the composite bleed comparison. Abbreviation: NM, not measurable. a Only a single measurement was recorded so a standard deviation could not be calculated. * p < .05 when comparing treated versus untreated animals of a single sex within a given bleeding regimen (eg, valproate SB vs saline SB). Bold added for emphasis. View Large Table 5. Statistically Significant Differences in Clinical Pathology Parameters Potentially Due to Drug Effects at the End of the 7-Day Dosing Period NB SB CB Parameter Saline NB Valproate NB Saline SB Valproate SB Saline CB Valproate CB Core Males  ALKP 232 ± 57 101 ± 19* 217 ± 39 107 ± 9* 214 ± 36 120 ± 31*  Total protein 6.0 ± 0.2 5.3 ± 0.2* 6.2 ± 0.3 5.3 ± 0.2* 6.2 ± 0.3 5.6 ± 0.3*  Albumin 4.2 ± 0.1 3.7 ± 0.3* 4.4 ± 0.2 3.7 ± 0.3* 4.2 ± 0.2 3.9 ± 0.4  Chloride 101 ± 1 104 ± 1* 102 ± 0 105 ± 1* 102 ± 1 103 ± 3  MCHC 31.5 ± 0.6 32.8 ± 0.6* 31.2 ± 0.8 32.9 ± 0.7* 31.6 ± 0.5 32 ± 0.6  WBC 7.24 ± 2.4 4.47 ± 0.75* 9.52 ± 2.34 3.93 ± 1.02* 9.90 ± 2.65 6.83 ± 3.35  LYMPH 6.08 ± 2.18 3.27 ± 0.51* 7.67 ± 1.33 2.97 ± 0.88* 7.98 ± 2.11 4.40 ± 1.57*  PLT 1005 ± 47 726 ± 239* 1100 ± 136 710 ± 89* 1103 ± 165 907 ± 184*  HCT 45.4 ± 2.4 43.9 ± 3.6 46.4 ± 1.7 41.5 ± 1.5* 43.5 ± 1.9 40.2 ± 4.1  RETIC 252.1 ± 32.9 135 ± 121.8 327.1 ± 45.9 127.2 ± 84.8* 423.7 ± 73.5 297.2 ± 163*  LUC 0.07 ± 0.03 0.07 ± 0.02 0.09 ± 0.02 0.05 ± 0.02* 0.10 ± 0.04 0.12 ± 0.07  MCV 63.9 ± 1.7 61.6 ± 2.3 65.7 ± 1.4 60.8 ± 1* 64.6 ± 2.8 62.2 ± 1.6  Globulin 1.8 ± 0.3 1.6 ± 0.3 1.8 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 1.7 ± 0.2*  Cholesterol 55 ± 12 52 ± 7 58 ± 13 44 ± 8 59 ± 8 49 ± 3*  CK 466 ± 132 369 ± 324 336 ± 136 620 ± 446 420 ± 162 265 ± 130*  Glucose 92 ± 9 88 ± 11 114 ± 34 88 ± 16 122 ± 22 91 ± 12*  Potassium 6.8 ± 0.2 6.4 ± 0.6 6.5 ± 0.5 6.1 ± 0.3 6.7 ± 0.5 6.0 ± 0.6*  EOS 0.02 ± 0.02 0.03 ± 0.04 0.05 ± 0.03 0.06 ± 0.04 0.11 ± 0.1 0.03 ± 0.03*  BASO 0.02 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.02 ± 0.01* Core Females  ALKP 171 ± 33 97 ± 28* 155 ± 38 89 ± 28* 156 ± 35 114 ± 21*  Total Protein 6.4 ± 0.2 5.3 ± 0.2* 6.4 ± 0.3 5.2 ± 0.3* 6.3 ± 0.4 5.4 ± 0.2*  Albumin 4.6 ± 0.2 3.8 ± 0.3* 4.5 ± 0.2 3.8 ± 0.4* 4.5 ± 0.3 4.0 ± 0.2*  Globulin 1.8 ± 0.1 1.4 ± 0.2* 1.9 ± 0.2 1.4 ± 0.2* 1.9 ± 0.1 1.4 ± 0.2*  Potassium 5.9 ± 0.3 5.1 ± 0.5* 6.4 ± 0.3 5.3 ± 0.4* 6.3 ± 0.5 5.3 ± 0.7*  HCT 46.0 ± 1.2 38.8 ± 5.1* 42.6 ± 1 36.0 ± 2.2* 38.1 ± 3.2 34.1 ± 4.4*  MPV 8.4 ± 0.2 9.2 ± 0.6* 8.3 ± 0.2 9.1 ± 0.3* 8.2 ± 0.3 9.0 ± 0.3*  MCHC 33.3 ± 0.2 34.7 ± 0.3* 32.7 ± 0.6 34.3 ± 0.7* 33.1 ± 0.7 34.0 ± 0.9*  WBC 7.26 ± 1.69 4.03 ± 1.07* 10.00 ± 3.43 4.15 ± 1.01* 7.81 ± 2.86 2.99 ± 0.71*  LYMPH 6.07 ± 1.22 2.93 ± 0.91* 8.60 ± 3.18 3.37 ± 1.03* 6.70 ± 2.58 2.21 ± 0.41*  RETIC 218.7 ± 40.7 17.2 ± 21.4* 245.0 ± 53.0 31.7 ± 25.3* 370.5 ± 51.3 140.2 ± 165.6*  EOS 0.08 ± 0.05 0 ± 0.01* 0.11 ± 0.04 0.01 ± 0.01* 0.10 ± 0.07 0.01 ± 0.01*  PLT 1174 ± 88 721 ± 93* 1350 ± 206 646 ± 124* 1244 ± 134 776 ± 208*  HGB 15.3 ± 0.4 13.5 ± 1.8* 14.0 ± 0.4 12.4 ± 0.6* 12.6 ± 0.9 11.6 ± 1.3  LDH 381 ± 127 148 ± 67* 208 ± 61 131 ± 18* 315 ± 188 190 ± 64  ALT 46 ± 8 34 ± 6* 39 ± 7 33 ± 4 36 ± 7 36 ± 11  AST 106 ± 9 82 ± 11* 92 ± 10 97 ± 13 91 ± 16 114 ± 39  Cholesterol 58 ± 9 40 ± 9* 68 ± 19 48 ± 7 67 ± 13 43 ± 8*  Monocytes 0.19 ± 0.1 0.14 ± 0.09 0.20 ± 0.07 0.09 ± 0.04* 0.18 ± 0.1 0.08 ± 0.04*  BASO 0.02 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01* 0.03 ± 0.02 0.01 ± 0.00*  Creatinine 0.5 ± 0.0 0.4 ± 0.1 0.5 ± 0.0 0.4 ± 0.0* 0.5 ± 0.1 0.4 ± 0.0*  RBC 7.70 ± 0.15 6.67 ± 0.97 7.03 ± 0.33 6.23 ± 0.18* 6.26 ± 0.59 6.03 ± 0.44  MCV 59.7 ± 1.9 58.2 ± 1.3 60.7 ± 1.9 57.8 ± 1.9* 60.9 ± 1.7 58.9 ± 2.6  Neutrophils 1.13 ± 0.08 1.2 ± 0.1 1.04 ± 0.03 NM 1.49 ± 0.39 1.09a  PT 9.8 ± 0.4 10.6 ± 1.3 9.9 ± 0.4 10.5 ± 0.3* 9.9 ± 0.3 10.1 ± 0.4  A/G Ratio 2.5 ± 0.2 2.8 ± 0.5 2.5 ± 0.3 2.8 ± 0.6 2.4 ± 0.2 3.0 ± 0.6*  LUC 0.09 ± 0.04 0.07 ± 0.04 0.16 ± 0.1 0.1 ± 0.06 0.09 ± 0.05 0.04 ± 0.02* NB SB CB Parameter Saline NB Valproate NB Saline SB Valproate SB Saline CB Valproate CB Core Males  ALKP 232 ± 57 101 ± 19* 217 ± 39 107 ± 9* 214 ± 36 120 ± 31*  Total protein 6.0 ± 0.2 5.3 ± 0.2* 6.2 ± 0.3 5.3 ± 0.2* 6.2 ± 0.3 5.6 ± 0.3*  Albumin 4.2 ± 0.1 3.7 ± 0.3* 4.4 ± 0.2 3.7 ± 0.3* 4.2 ± 0.2 3.9 ± 0.4  Chloride 101 ± 1 104 ± 1* 102 ± 0 105 ± 1* 102 ± 1 103 ± 3  MCHC 31.5 ± 0.6 32.8 ± 0.6* 31.2 ± 0.8 32.9 ± 0.7* 31.6 ± 0.5 32 ± 0.6  WBC 7.24 ± 2.4 4.47 ± 0.75* 9.52 ± 2.34 3.93 ± 1.02* 9.90 ± 2.65 6.83 ± 3.35  LYMPH 6.08 ± 2.18 3.27 ± 0.51* 7.67 ± 1.33 2.97 ± 0.88* 7.98 ± 2.11 4.40 ± 1.57*  PLT 1005 ± 47 726 ± 239* 1100 ± 136 710 ± 89* 1103 ± 165 907 ± 184*  HCT 45.4 ± 2.4 43.9 ± 3.6 46.4 ± 1.7 41.5 ± 1.5* 43.5 ± 1.9 40.2 ± 4.1  RETIC 252.1 ± 32.9 135 ± 121.8 327.1 ± 45.9 127.2 ± 84.8* 423.7 ± 73.5 297.2 ± 163*  LUC 0.07 ± 0.03 0.07 ± 0.02 0.09 ± 0.02 0.05 ± 0.02* 0.10 ± 0.04 0.12 ± 0.07  MCV 63.9 ± 1.7 61.6 ± 2.3 65.7 ± 1.4 60.8 ± 1* 64.6 ± 2.8 62.2 ± 1.6  Globulin 1.8 ± 0.3 1.6 ± 0.3 1.8 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 1.7 ± 0.2*  Cholesterol 55 ± 12 52 ± 7 58 ± 13 44 ± 8 59 ± 8 49 ± 3*  CK 466 ± 132 369 ± 324 336 ± 136 620 ± 446 420 ± 162 265 ± 130*  Glucose 92 ± 9 88 ± 11 114 ± 34 88 ± 16 122 ± 22 91 ± 12*  Potassium 6.8 ± 0.2 6.4 ± 0.6 6.5 ± 0.5 6.1 ± 0.3 6.7 ± 0.5 6.0 ± 0.6*  EOS 0.02 ± 0.02 0.03 ± 0.04 0.05 ± 0.03 0.06 ± 0.04 0.11 ± 0.1 0.03 ± 0.03*  BASO 0.02 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.02 ± 0.01* Core Females  ALKP 171 ± 33 97 ± 28* 155 ± 38 89 ± 28* 156 ± 35 114 ± 21*  Total Protein 6.4 ± 0.2 5.3 ± 0.2* 6.4 ± 0.3 5.2 ± 0.3* 6.3 ± 0.4 5.4 ± 0.2*  Albumin 4.6 ± 0.2 3.8 ± 0.3* 4.5 ± 0.2 3.8 ± 0.4* 4.5 ± 0.3 4.0 ± 0.2*  Globulin 1.8 ± 0.1 1.4 ± 0.2* 1.9 ± 0.2 1.4 ± 0.2* 1.9 ± 0.1 1.4 ± 0.2*  Potassium 5.9 ± 0.3 5.1 ± 0.5* 6.4 ± 0.3 5.3 ± 0.4* 6.3 ± 0.5 5.3 ± 0.7*  HCT 46.0 ± 1.2 38.8 ± 5.1* 42.6 ± 1 36.0 ± 2.2* 38.1 ± 3.2 34.1 ± 4.4*  MPV 8.4 ± 0.2 9.2 ± 0.6* 8.3 ± 0.2 9.1 ± 0.3* 8.2 ± 0.3 9.0 ± 0.3*  MCHC 33.3 ± 0.2 34.7 ± 0.3* 32.7 ± 0.6 34.3 ± 0.7* 33.1 ± 0.7 34.0 ± 0.9*  WBC 7.26 ± 1.69 4.03 ± 1.07* 10.00 ± 3.43 4.15 ± 1.01* 7.81 ± 2.86 2.99 ± 0.71*  LYMPH 6.07 ± 1.22 2.93 ± 0.91* 8.60 ± 3.18 3.37 ± 1.03* 6.70 ± 2.58 2.21 ± 0.41*  RETIC 218.7 ± 40.7 17.2 ± 21.4* 245.0 ± 53.0 31.7 ± 25.3* 370.5 ± 51.3 140.2 ± 165.6*  EOS 0.08 ± 0.05 0 ± 0.01* 0.11 ± 0.04 0.01 ± 0.01* 0.10 ± 0.07 0.01 ± 0.01*  PLT 1174 ± 88 721 ± 93* 1350 ± 206 646 ± 124* 1244 ± 134 776 ± 208*  HGB 15.3 ± 0.4 13.5 ± 1.8* 14.0 ± 0.4 12.4 ± 0.6* 12.6 ± 0.9 11.6 ± 1.3  LDH 381 ± 127 148 ± 67* 208 ± 61 131 ± 18* 315 ± 188 190 ± 64  ALT 46 ± 8 34 ± 6* 39 ± 7 33 ± 4 36 ± 7 36 ± 11  AST 106 ± 9 82 ± 11* 92 ± 10 97 ± 13 91 ± 16 114 ± 39  Cholesterol 58 ± 9 40 ± 9* 68 ± 19 48 ± 7 67 ± 13 43 ± 8*  Monocytes 0.19 ± 0.1 0.14 ± 0.09 0.20 ± 0.07 0.09 ± 0.04* 0.18 ± 0.1 0.08 ± 0.04*  BASO 0.02 ± 0.02 0.01 ± 0.01 0.03 ± 0.02 0.01 ± 0.01* 0.03 ± 0.02 0.01 ± 0.00*  Creatinine 0.5 ± 0.0 0.4 ± 0.1 0.5 ± 0.0 0.4 ± 0.0* 0.5 ± 0.1 0.4 ± 0.0*  RBC 7.70 ± 0.15 6.67 ± 0.97 7.03 ± 0.33 6.23 ± 0.18* 6.26 ± 0.59 6.03 ± 0.44  MCV 59.7 ± 1.9 58.2 ± 1.3 60.7 ± 1.9 57.8 ± 1.9* 60.9 ± 1.7 58.9 ± 2.6  Neutrophils 1.13 ± 0.08 1.2 ± 0.1 1.04 ± 0.03 NM 1.49 ± 0.39 1.09a  PT 9.8 ± 0.4 10.6 ± 1.3 9.9 ± 0.4 10.5 ± 0.3* 9.9 ± 0.3 10.1 ± 0.4  A/G Ratio 2.5 ± 0.2 2.8 ± 0.5 2.5 ± 0.3 2.8 ± 0.6 2.4 ± 0.2 3.0 ± 0.6*  LUC 0.09 ± 0.04 0.07 ± 0.04 0.16 ± 0.1 0.1 ± 0.06 0.09 ± 0.05 0.04 ± 0.02* Statistically significant differences observed in the NB comparison are prioritized followed by significant differences in the serial bleed comparison followed by the composite bleed comparison. Abbreviation: NM, not measurable. a Only a single measurement was recorded so a standard deviation could not be calculated. * p < .05 when comparing treated versus untreated animals of a single sex within a given bleeding regimen (eg, valproate SB vs saline SB). Bold added for emphasis. View Large For males in the NB group, statistically significant decreases in alkaline phosphatase (−56%), total protein (−12%), and albumin (−12%) were observed on day 8. In the hemogram, statistically significant decreases in leukocytes (−38%), lymphocytes (−46%), and platelets (−28%) were also observed. Comparable effects were observed in both the SB comparisons with further decreases in the hematocrit and reticulocyte count. Statistically significant differences were noted for several other parameters, but these were minor or not considered test article-related. On day 8, female rats in the NB group had a greater number of statistically significant changes compared with the males. Alkaline phosphatase (−43%), total protein (−17%), albumin (−17%), and globulin (−22%) were decreased as well as alanine aminotransferase (−26%) and aspartate aminotransferase (−23%). In the hemogram, leukocyte (−44%), lymphocyte (−52%), reticulocyte (−92%), and platelet counts (−39%) were decreased with a subsequent increase in mean platelet volume (+10%). Decreases in hemoglobin (−12%) and the hematocrit (−16%) were observed as well as decreases in erythrocyte counts (−13%); however, the latter were not statistically significant. Comparable effects were observed in both the SB and CB groups, but the decrease in mean erythrocyte counts in the SB group (−11%) were statistically significant. After 14 days of recovery, the differences in clinical pathology between the treated and untreated animals were minimal. For males in the NB comparison, statistical differences were noted in the globulin (−34%) and albumin/globulin ratio (+100%) measurements which were not observed in the SB comparison. The total leukocyte and lymphocyte counts remained lower in both valproate-treated groups; this was not statistically significant in the NB group but was significant in the SB group. Bleed Effects Bleed effects were examined by comparing the SB and CB groups to their respective unbled control, eg, valproate SB or valproate CB versus valproate NB. Statistically significant differences determined by Provantis are presented in Table 6. Tabular summaries of the data at the end of the dosing and recovery periods are provided in Table 6 and Supplementary Table 3, respectively. For the saline-treated males, no statistically significant effects were observed in the animals that were serially bled or compositely bled, although the erythrocyte counts continually decreased as more blood was taken, with a corresponding increase in reticulocytes. For saline-treated females, no statistically significant changes were observed in the serially bled animals though statistically significant decreases in erythrocyte count (−19%), hemoglobin (−18%), and hematocrit (−17%) were observed in the CB group. Table 6. Statistically Significant Differences in Clinical Pathology Parameters Potentially Due to Bleed Effects at the End of the 7-Day Dosing Period Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Males  ALKP −8% −6% 232 ± 57 101 ± 19* +6% +19%  Total Protein +3% +3% 6.0 ± 0.2 5.3 ± 0.2* 0% +6%  RBC −5% −1% 7.10 ± 0.49 7.12 ± 0.42 −4% −9%  HGB −4% +1% 14.3 ± 0.6 14.4 ± 1.1 −5% −10%  HCT −4% +2% 45.4 ± 2.4 43.9 ± 3.6 −5% −8%  RETIC +68% +30% 252.1 ± 32.9 135.0 ± 121.8 −6% +120%*  MCHC 0% −1% 31.5 ± 0.6 32.8 ± 0.6* 0% −2%  LYMPH +31% +26% 6.08 ± 2.18 3.27 ± 0.51* −9% +33%  PLT +10% +9% 1005 ± 47 726 ± 239* −2% +25%  MPV +2% 0% 9.0 ± 0.5 9.9 ± 0.7* −2% −9%   Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Females  ALKP −8% −6% 171 ± 33 97 ± 28* +6% +19%  LDH −17% −45% 381 ± 127 148 ± 67* −8% +18%  Total Protein −2% 0% 6.4 ± 0.2 5.3 ± 0.2* −2% +2%  Albumin −2% −2% 4.6 ± 0.2 3.8 ± 0.3* 0% +5%  RBC −19%* −9% 7.70 ± 0.15 6.67 ± 0.97* −7% −13%  HGB −18%* −9% 15.3 ± 0.4 13.5 ± 1.8* −8% −14%  HCT −17%* −7% 46.0 ± 1.2 38.8 ± 5.1* −7% −12%  RETIC +69% +12% 218.7 ± 40.7 17.2 ± 21.4* +84% +715%*  LYMPH +10% +42% 6.07 ± 1.22 2.93 ± 0.91* +15% −25%  EOS +25% +38% 0.08 ± 0.05 0.00 ± 0.01* 0.01 0.01  PLT +3% +15% 1174 ± 88 520 ± 294* +7% +49%  MPV −2% −1% 8.4 ± 0.2 9.2 ± 0.6* −1% −2%  APTT −18%* −9% 11.5 ± 0.6 12.1 ± 1.3 −5% −22%* Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Males  ALKP −8% −6% 232 ± 57 101 ± 19* +6% +19%  Total Protein +3% +3% 6.0 ± 0.2 5.3 ± 0.2* 0% +6%  RBC −5% −1% 7.10 ± 0.49 7.12 ± 0.42 −4% −9%  HGB −4% +1% 14.3 ± 0.6 14.4 ± 1.1 −5% −10%  HCT −4% +2% 45.4 ± 2.4 43.9 ± 3.6 −5% −8%  RETIC +68% +30% 252.1 ± 32.9 135.0 ± 121.8 −6% +120%*  MCHC 0% −1% 31.5 ± 0.6 32.8 ± 0.6* 0% −2%  LYMPH +31% +26% 6.08 ± 2.18 3.27 ± 0.51* −9% +33%  PLT +10% +9% 1005 ± 47 726 ± 239* −2% +25%  MPV +2% 0% 9.0 ± 0.5 9.9 ± 0.7* −2% −9%   Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Females  ALKP −8% −6% 171 ± 33 97 ± 28* +6% +19%  LDH −17% −45% 381 ± 127 148 ± 67* −8% +18%  Total Protein −2% 0% 6.4 ± 0.2 5.3 ± 0.2* −2% +2%  Albumin −2% −2% 4.6 ± 0.2 3.8 ± 0.3* 0% +5%  RBC −19%* −9% 7.70 ± 0.15 6.67 ± 0.97* −7% −13%  HGB −18%* −9% 15.3 ± 0.4 13.5 ± 1.8* −8% −14%  HCT −17%* −7% 46.0 ± 1.2 38.8 ± 5.1* −7% −12%  RETIC +69% +12% 218.7 ± 40.7 17.2 ± 21.4* +84% +715%*  LYMPH +10% +42% 6.07 ± 1.22 2.93 ± 0.91* +15% −25%  EOS +25% +38% 0.08 ± 0.05 0.00 ± 0.01* 0.01 0.01  PLT +3% +15% 1174 ± 88 520 ± 294* +7% +49%  MPV −2% −1% 8.4 ± 0.2 9.2 ± 0.6* −1% −2%  APTT −18%* −9% 11.5 ± 0.6 12.1 ± 1.3 −5% −22%* Valproate NB is compared with Saline NB to determine the drug effect. The SB and CB groups are compared with their respective treated or untreated NB group to examine the bleed effect. * p < .05 for a single sex when comparing valproate NB to saline NB (drug effect) or when comparing a given bleeding regimen to the unbled control within a given treatment regimen to examine the bleed effect (eg, valproate SB vs valproate NB). Bold added for emphasis. View Large Table 6. Statistically Significant Differences in Clinical Pathology Parameters Potentially Due to Bleed Effects at the End of the 7-Day Dosing Period Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Males  ALKP −8% −6% 232 ± 57 101 ± 19* +6% +19%  Total Protein +3% +3% 6.0 ± 0.2 5.3 ± 0.2* 0% +6%  RBC −5% −1% 7.10 ± 0.49 7.12 ± 0.42 −4% −9%  HGB −4% +1% 14.3 ± 0.6 14.4 ± 1.1 −5% −10%  HCT −4% +2% 45.4 ± 2.4 43.9 ± 3.6 −5% −8%  RETIC +68% +30% 252.1 ± 32.9 135.0 ± 121.8 −6% +120%*  MCHC 0% −1% 31.5 ± 0.6 32.8 ± 0.6* 0% −2%  LYMPH +31% +26% 6.08 ± 2.18 3.27 ± 0.51* −9% +33%  PLT +10% +9% 1005 ± 47 726 ± 239* −2% +25%  MPV +2% 0% 9.0 ± 0.5 9.9 ± 0.7* −2% −9%   Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Females  ALKP −8% −6% 171 ± 33 97 ± 28* +6% +19%  LDH −17% −45% 381 ± 127 148 ± 67* −8% +18%  Total Protein −2% 0% 6.4 ± 0.2 5.3 ± 0.2* −2% +2%  Albumin −2% −2% 4.6 ± 0.2 3.8 ± 0.3* 0% +5%  RBC −19%* −9% 7.70 ± 0.15 6.67 ± 0.97* −7% −13%  HGB −18%* −9% 15.3 ± 0.4 13.5 ± 1.8* −8% −14%  HCT −17%* −7% 46.0 ± 1.2 38.8 ± 5.1* −7% −12%  RETIC +69% +12% 218.7 ± 40.7 17.2 ± 21.4* +84% +715%*  LYMPH +10% +42% 6.07 ± 1.22 2.93 ± 0.91* +15% −25%  EOS +25% +38% 0.08 ± 0.05 0.00 ± 0.01* 0.01 0.01  PLT +3% +15% 1174 ± 88 520 ± 294* +7% +49%  MPV −2% −1% 8.4 ± 0.2 9.2 ± 0.6* −1% −2%  APTT −18%* −9% 11.5 ± 0.6 12.1 ± 1.3 −5% −22%* Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Males  ALKP −8% −6% 232 ± 57 101 ± 19* +6% +19%  Total Protein +3% +3% 6.0 ± 0.2 5.3 ± 0.2* 0% +6%  RBC −5% −1% 7.10 ± 0.49 7.12 ± 0.42 −4% −9%  HGB −4% +1% 14.3 ± 0.6 14.4 ± 1.1 −5% −10%  HCT −4% +2% 45.4 ± 2.4 43.9 ± 3.6 −5% −8%  RETIC +68% +30% 252.1 ± 32.9 135.0 ± 121.8 −6% +120%*  MCHC 0% −1% 31.5 ± 0.6 32.8 ± 0.6* 0% −2%  LYMPH +31% +26% 6.08 ± 2.18 3.27 ± 0.51* −9% +33%  PLT +10% +9% 1005 ± 47 726 ± 239* −2% +25%  MPV +2% 0% 9.0 ± 0.5 9.9 ± 0.7* −2% −9%   Untreated Bleed Effect Control Drug Effect Treated Bleed Effect Parameter Saline CB Saline SB Saline NB Valproate NB Valproate SB Valproate CB Core Females  ALKP −8% −6% 171 ± 33 97 ± 28* +6% +19%  LDH −17% −45% 381 ± 127 148 ± 67* −8% +18%  Total Protein −2% 0% 6.4 ± 0.2 5.3 ± 0.2* −2% +2%  Albumin −2% −2% 4.6 ± 0.2 3.8 ± 0.3* 0% +5%  RBC −19%* −9% 7.70 ± 0.15 6.67 ± 0.97* −7% −13%  HGB −18%* −9% 15.3 ± 0.4 13.5 ± 1.8* −8% −14%  HCT −17%* −7% 46.0 ± 1.2 38.8 ± 5.1* −7% −12%  RETIC +69% +12% 218.7 ± 40.7 17.2 ± 21.4* +84% +715%*  LYMPH +10% +42% 6.07 ± 1.22 2.93 ± 0.91* +15% −25%  EOS +25% +38% 0.08 ± 0.05 0.00 ± 0.01* 0.01 0.01  PLT +3% +15% 1174 ± 88 520 ± 294* +7% +49%  MPV −2% −1% 8.4 ± 0.2 9.2 ± 0.6* −1% −2%  APTT −18%* −9% 11.5 ± 0.6 12.1 ± 1.3 −5% −22%* Valproate NB is compared with Saline NB to determine the drug effect. The SB and CB groups are compared with their respective treated or untreated NB group to examine the bleed effect. * p < .05 for a single sex when comparing valproate NB to saline NB (drug effect) or when comparing a given bleeding regimen to the unbled control within a given treatment regimen to examine the bleed effect (eg, valproate SB vs valproate NB). Bold added for emphasis. View Large For valproate-treated males, no statistically significant differences were observed in the SB group when compared with the unbled, treated group. For the CB males, only reticulocyte counts increased by a statistically significant amount (+120%). In the treated animals, erythroid parameters (erythrocyte count, hemoglobin and hematocrit) tended to decrease as increasing volumes of blood were removed, but no statistically significant differences were noted. Valproate-treated females also showed similar decreasing trends in the erythroid parameters without statistical significance. Reticulocytes were significantly elevated in the CB females though the results were highly variable (140.2 ± 165.6 x 103 cells/μl). Following a 14-day recovery, there were no notable differences between the saline SB and saline NB groups (Supplementary Table 3). The only notable differences in the treated animals were the protein parameters (albumin/globulin ratio in the serial bled males. In this case, the serial bled males had decreased concentrations of albumin (−23%) but increased concentrations of globulin (+33%) and no differences in total protein. The erythroid parameters were all comparable between groups. All of the core and recovery clinical pathology parameters were equivalence tested within treatment groups to determine if the TK bleeds affected the parameter estimates. The serial bled animals in both treatment groups were statistically equivalent to their unbled controls for all parameters (Figure 3). For compositely bled animals, the 90% CIs were within the equivalence region for most parameters in both the saline- and valproate-treated groups (Figure 3), except erythrocyte count, hemoglobin, hematocrit, and reticulocyte count, which were outside the equivalence region for one or both treatments. Statistical equivalence plots for the remaining clinical pathology parameters are provided in the Supplementary Figures 1–5. For both treatments, both the serial bled and compositely bled groups were shown to be equivalent to the unbled controls for serum chemistry, differential leukocyte counts, coagulation, liver weight and related serum chemistry parameters, and kidney weight and related serum chemistry parameters, with few exceptions; protein composition and AST in the valproate-treated, recovery cohort were the only other clinical pathology parameters that were not determined to be statistically equivalent. Figure 3. View largeDownload slide Statistical equivalence region (±2σ) in the erythroid parameters comparing bled animals to their unbled control within a given treatment. Error bars represent the 90% CI for a given measurement. Figure 3. View largeDownload slide Statistical equivalence region (±2σ) in the erythroid parameters comparing bled animals to their unbled control within a given treatment. Error bars represent the 90% CI for a given measurement. Anatomic Pathology Tabular summaries of the anatomic pathology data, including organ weights and histological evaluation, are provided in Supplementary Table 4 (core necropsy) and Supplementary Table 5 (recovery necropsy). At the end of the dosing period, the valproate-treated males and females exhibited minor hepatic vacuolization and a single incident of hepatic necrosis in the females. Absolute liver weights in the NB (−15%) and CB (−10%) males were statistically lower than the corresponding saline-treated rats. When terminal liver weights were normalized to TBW, the differences were no longer significant. For the valproate-treated females, a statistically significant decrease in absolute liver weight was only observed in the compositely bled group (−14%). Again when normalized to TBW there was no statistical difference in the means. After 14 days of recovery, statistically significant liver weight decreases were observed in the valproate-treated, NB males (−18% absolute weight, −10% liver/TBW). For the valproate-treated, SB males, a 14% decrease in absolute liver weight and 8% decrease in liver/TBW were observed although neither was statistically significant. No statistically significant effects were observed in the absolute or normalized liver weights of females at the end of recovery. Absolute kidney weights were notably smaller at the end of the dosing period in both the male and female valproate-treated, NB groups as well as the females of the valproate-treated, SB group. The decreased kidney weights were not statistically significant and the effects were less apparent when normalized to TBW. Following 14 days of recovery there were no notable effects on the kidney or kidney/TBW measurements in any comparison. For the treated females, statistically significant decreases in mean absolute ovarian weight were noted in the SB group (−25%); notable but not statistically significant decreases in absolute weight were also observed in the unbled (−20%) and compositely bled females (−21%). When normalized to TBW, differences from control were not apparent. At the end of the recovery period, no differences in absolute ovarian weight or ovarian weight normalized to TBW were noted. For treated males, statistically significant decreases in absolute testicular weights were observed in the NB group (−19%), SB group (−33%), and CB group (−29%). When normalized to TBW, statistically significant decreases were still observed in the SB (−26%) and CB groups (−20%). Statistically significant decreases in absolute epididymal weights were observed only in the NB males (−17%) and no statistical differences were observed when normalized to TBW. After 14 days of recovery, statistically significant decreases in mean testicular weight were noted in the NB males (−34%) and SB males (−46%). When normalized to TBW, statistically significant decreases were still noted in both groups; −27% for the NB males and −43% for the SB males. Mean decreases in absolute epididymal weights were noted in the NB (−26%) and SB (−29%) groups. When normalized to TBW, a 19% mean decrease was noted in the NB males and 24% decrease in the SB males. When the testes were examined histologically, atrophy of the seminiferous tubules was noted with an incidence of 22% in the CB males, 60% in the NB males, and 80% in the SB males at the end of the dosing period. There was no evidence of atrophy in the untreated males. At the end of the recovery period, atrophy of the seminiferous tubules was noted in 60% of the NB, valproate-treated males and 100% of the SB, valproate-treated males. Atrophy was only noted in one of the untreated males (10%). Statistically significant decreases in absolute prostate and prostate/TBW were observed in all valproate-treated males. NB males had a 40% decrease in absolute weights and 29% decrease in normalized prostate weights; SB males had a mean decrease of 35% in absolute weight and 27% in prostate/TBW; CB males had a mean decrease of 37% in absolute weight and 30% decrease in prostate/TBW. Statistically significant effects on the absolute or normalized prostate weights were not observed at the end of the recovery period. All organ weights were equivalence tested to determine if the TK bleeds had an impact on organ weight. The equivalence plots are provided in the supplemental information (Supplementary Figs. 4–6). At the end of the dosing period, the 90% CI for most organ weights were within the equivalence regions for both the valproate- and saline-treated animals. Several exceptions where the 90% CI did not fall entirely within the equivalence region for one or both treatments include the kidney/TBW as well as the absolute and body weight-normalized testicular weights. At the end of the recovery period, the 90% CI for all of the organs were within the equivalence region for one or both treatments except the absolute and body weight-normalized weights of the testes and epididymides. This was true for both the saline- and valproate-treated males. DISCUSSION Study Considerations In rats, valproic acid has been shown to affect the liver (Lahneche et al., 2017; Tong et al., 2005), testes and prostate (Nishimura et al., 2000; Sukhorum and Iamsaard, 2017), platelets (Szupera et al., 2000), leukocytes (Almodovar-Cuevas et al., 1985), and fetal development (Vorhees, 1987). These effects are also noted on the package insert (Depakene, 2017). The dose of drug was selected based on evidence from the literature indicating it may cause 20% mortality in addition to microscopically observable liver toxicity (Abdou et al., 2014; Tong et al., 2005). If the animals were able to survive under such conditions while subsequently providing accurate TK data without influencing the interpretation of the clinical or anatomic pathology, then it is expected the effects of microsampling under the conditions of longer-term studies may be negligible. The tail vein bleed has generally been shown as a mild bleeding technique which causes only momentary discomfort while allowing the animal handler to accurately control specimen volume. However in this study, the tail vein bleed itself may have been a significant stressor. Treatment with valproic acid led to systemic effects that may be a result of exaggerated pharmacology. The treatment had a coma-inducing effect that led to poor circulation within the tail vein making it more difficult to access the vein and reducing blood flow from the vein. This frequently led to multiple needle sticks, an increased duration within the restraint tube and an increased exposure to the heat lamp, all potential stressors that could have influence on the clinical pathology data. Overall 17 samples were not able to be collected due to sampling difficulties via the tail vein; however, this only affected the analysis of 2/40 C-t profiles. As an alternative, the saphenous vein may provide a potentially less stressful bleed route that could remove the stress of heating the animals and minimize restraint time while maintaining the low-volume benefits of the tail vein bleed. Drug Effects on Food Consumption and Body Weight After each dose of sodium valproate, animals became comatose and incapable of eating or drinking resulting in a reduction in food consumption (data not shown) during the week of treatment and up to an 11% reduction in group mean body weight during treatment (Supplementary Table 1). These effects are not believed to be direct toxicity from sodium valproate; rather they are also likely effects of the exaggerated pharmacology of the drug. After the drug was removed, food consumption increased and the body weight discrepancies between treated and untreated animals narrowed. The changes in body weight were also not impacted by either the SB or CB as all groups were statistically equivalent to their respective unbled controls (SupplementaryFigure 7). Comparison of Clinical Pathology Serum chemistry analysis showed a treatment-related reduction in alkaline phosphatase, total protein, albumin, and globulin in the males and more significantly in females. Additionally, a significant reduction in lactate dehydrogenase was observed in females. These effects are believed to be secondary to body weight reduction considering there were no other changes to hepatic parameters and hepatotoxicity was not observed macroscopically or microscopically. Consistent with the known effects of valproic acid, statistically significant reductions in leukocyte, lymphocyte, and platelet counts were observed in addition to notable, but not statistically significant reductions in erythrocyte counts. The only observable trends that were attributed to blood loss, whether in the treatment or control groups, were in the erythrocyte counts, hemoglobin, and hematocrit. No statistical differences were observed between the SB and NB animals in either the treatment or control cohorts. Further, statistical equivalence to the NB groups was demonstrated for the SB animals for all of these parameters in both the treated and untreated animals. When larger blood volumes were removed for composite analysis, statistically significant differences in the erythroid parameters were observed in the control but not the treated cohort. When these parameters were equivalence tested, the 90% CI did not fall completely within the equivalence region. Thus the bleeds are likely having an effect in addition to the drug toxicity which may make it difficult to distinguish between the effects on the hemogram. For the SB control animals, statistical equivalence was observed for all of the clinical pathology parameters at core and recovery measurements. For the SB animals treated with valproic acid, equivalence was demonstrated for all clinical pathology parameters at core; however, equivalence could not be demonstrated for several parameters in the recovery measurements of aspartate aminotransferase, albumin and globulin. For these parameters, a faster recovery was observed in the SB group as compared with the NB group. The discrepancies were minor and did not impact the interpretation of the data. Comparison of Organ Weights and Anatomic Pathology Testicular atrophy was noted at the core necropsy at the end of the dosing period which was apparent in the organ weights and histopathology of all of the treated males. The effect became more apparent at the recovery necropsy as atrophy was observed at a higher incidence in both SB and NB males. There was variability in the organ weight data between NB males and SB males so statistical equivalence could not be demonstrated based on the study results. This is likely due to normal variability as the same effect was noted in saline-treated males where testicular toxicity was not observed. Despite this variability in organ weight, testicular toxicity was apparent regardless of blood loss thus there was no impact on the interpretation of the data. Decreased prostate and ovarian weights were observed at the core necropsy which were not observed after a 14-day recovery period. These decreased weights are believed to be sequelae resulting from the loss of body weight. No statistical differences between any of the valproate-treated groups were observed, regardless of blood loss. Last hepatotoxicity was an expected outcome at this dose of valproic acid; however, only minor effects were observed in the organ weights and no effects were observed in the clinical pathology or histology. There were no differences in liver toxicity observed between any of the treated groups, regardless of blood loss. Comparison of TKs The TK data generated from microsampling in this study were consistent with the commonly used composite method. The data were more consistent for males where the group mean estimates of terminal half-life were all within approximately 3% of each other (1.83–1.89 h; day 1) for a given day. Females showed greater variability with up to a 23% difference in the group mean estimate of the terminal half-life (2.09–2.72; day 1). Within the SB group, the estimated t1/2 for males ranged from 1.16 to 2.17 h across the entire study while estimates in females exhibited a wider range of 0.94–2.94 h. Valproic acid is primarily metabolized by glucuronidation and beta oxidation with only minor cytochrome P450-mediated metabolism (Ghodke-Puranik, 2013). Although its pharmacokinetics are subject to drug interactions, it is not known to induce its own clearance (Depakene, 2017). The drug interactions are likely due to induction of uridine 5'-diphospho-glucuronosyltransferases since depletion of carnitine, a cofactor for beta oxidation, did not greatly affect the PK profile in rats (Katayama, 2016). Thus the differences in kinetics from days 1 to 7 observed in the females are likely not due to changes in metabolism. In addition to typical population variability, the observed ranges for half-life may be a secondary effect of the drug toxicity. The hepatic clearance of valproic acid is dependent on the unbound fraction (Loscher, 1978), thus the mean half-life from days 1 to 7 is expected to decrease for both sexes considering the plasma concentration of albumin decreased in valproate-treated animals. These differences in the elimination half-life led to large differences in the overall estimates of exposure for the females (3190–6650 μg·h/ml) which could further explain differences in toxicity. Since composite curves do not provide ranges for TK parameters, this type of analysis cannot be conducted. The day 1 to 7 estimates of AUC can be compared for each sex; however, the AUC in males did not show much of an increase from day 1 to 7 (2560–3040 μg·h/ml) whereas the AUC in females decreased from day 1 to 7 (4250–3080 μg·h/ml). Individual TK information provided by microsampling could be used to further examine toxicological observations since potential outliers in exposure could be used to explain spurious lesions in the gross pathology or significant changes in the clinical pathology. In this study, a female rat in the valproate-treated, SB group had the highest estimated exposure of 6650 μg·h/ml on day 1. The day 7 profile was similar through 2 h, including Cmax, but missed specimens prevented the generation of a full profile for t1/2 or AUC estimation. This animal also exhibited the greatest loss in body weight over the dosing period (−14.5%); had the lowest albumin concentration (3.5 g/dl) at necropsy on day 8; had the lowest observed leukocyte count (3.1 × 103 cells/μl); and the lowest lymphocyte count (2.0 × 103 cells/μl) which may provide a direct link between exposure and toxicity. In this study, the risk of missing microsampled specimens increased due to decreased familiarity with the collection technique in addition to the effects of the drug. Although this did result in an inability to produce 2/40 C-t curves it did not affect the overall TK analysis. If more specimens are missed and serial curves cannot be produced, the results show the individual timepoint data could still be pooled to generate a composite curve. The C-t curves and derived parameters resulting from pooling the individual samples were very similar to what was observed for the composite animals thus the overall risk of microsamples affecting the TK analysis is small. CONCLUSIONS This study was designed to incorporate TK microsampling in a safety assessment study and examine what, if any, effects it may have on the toxicological interpretation. To test the robustness of the hypothesis, the study was designed to maximize the potential for bleed effects by minimizing the turnaround time between the 2 TK profiles, ie, 7 days. Having 2 profiles in a single week pushed the required blood volumes to the maximum recommendations (Diehl et al., 2001; Turner et al., 2011). If microsampling the study animals under these conditions does not impact the interpretation of the toxicological or TK endpoints, then it creates an opportunity to reduce animal use and refine the study design consistent with the scientific and ethical principles of NC3Rs. The results of this study confirm previous investigations that erythroid parameters in the hemogram may change as a result of additional blood sampling. In the context of this study, microsampled specimens of up to 0.1 ml taken 6 times/profile with 2 profiles in a 7-day period (1.2 ml/week) had no statistical effect on the erythroid parameters when compared with an unbled control. This was true under both naïve and treated conditions. Further all of the expected drug toxicities including thrombocytopenia, leukopenia, lymphopenia, and testicular atrophy were observed when treated animals were compared with control animals of the same bleeding regimen eg, valproate SB versus saline SB. This was true when CB were collected as well; however, there were several instances where treatment effects did not show statistical significance and bleed effects were statistically significant. Overall the results show no negative effects on toxicological interpretation when microsamples were collected from the core or recovery animals. As expected, microsampling had a positive impact on the TK analysis since serial C-t curves allow a kineticist to see population variability and provide ranges for TK parameter estimates. This also allowed individual estimates of exposure, AUC∞ and Cmax, to become toxicological data for a specific animal so that it could be directly correlated to other observed effects. This could potentially be useful in understanding any aberrant observations that may only be observed in a single animal. The results of this study alone do not definitively demonstrate microsampling poses no risk to study integrity. It is intended to supplement data collected over many years in the industry where some companies have been early adopters of this technique (Sparrow et al., 2011). The results from this study do support the recent adoption of the ICH S3A revision to TKs allowing microsampling to be used in exploratory and regulated studies. Not all studies will be compatible with microsampling, but it should be considered when applicable as a way to improve study conduct in support of the NC3Rs. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This study was funded using Independent Research and Development funding from the Battelle Memorial Institute. ACKNOWLEDGMENTS The authors would like to acknowledge the work of Sarah Shellenbarger, Ekai Moritz, and Amara Hill for their contributions to this study. 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TI - A Factorial Analysis of Drug and Bleeding Effects in Toxicokinetic Studies
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfz092
DA - 2019-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-factorial-analysis-of-drug-and-bleeding-effects-in-toxicokinetic-RdqQnOR87m
SP - 234
VL - 170
IS - 1
DP - DeepDyve
ER -