Functional Observational Battery Research Articles

Functional observational battery (FOB) tests using caffeine and chlorpromazine hydrochloride in sprague-dawley rats.

Caffeine is believed to exert its therapeutic effects by acting as a nonselective, competitive antagonist of adenosine receptors. Chlorpromazine, a phenothiazine, is a classic psychotropic mediator extensively used in the clinical administration of psychotic disorders. This study aimed to validate the procedures used for performing Functional Observational Battery (FOB) tests, to demonstrate the proficiency and interobserver reliability during the FOB tests and also to assess effect on neurobehavioral parameters using positive controls in rats. The rats were administered with Caffeine in Milli-Q water as oral gavage at the dose of 20 mg/kg and Chlorpromazine HCl in 0.9% Saline as intraperitoneal route at the dose of 20 mg/kg. No inter-personnel variability was observed in home cage, handling, open field and sensory reactivity observations recorded in Proficiency test. In conclusion, the known effects of positive controls; caffeine and chlorpromazine HCl on neurobehavioral/Functional Observational Battery parameters including autonomic, neuromuscular and sensory reactivity tests were detected in the current study. FOB test procedures for neurobehavioral, grip strength and motor activity are adequate for the detection of neurotoxic effects of positive controls. No major inter-personnel variability was observed between study personnel in neurobehavioural observations.

Neurobehavioral assessment of BMEDA by modified Irwin test in Sprague-Dawley rats

The neurobehavioral assessment of N,N-bis(2-mercapatoethly)-N′,N′-diethylenediamine (BMEDA), which can form a chelate with rhenium-188 (188Re) to produce the 188Re-BMEDA-liposome, was evaluated. The purpose of this study was to evaluate the potential neurobehavioral changes by using the functional observational battery observation procedures when intravenous injection of BMEDA to Sprague-Dawley rats. Rats were administered BMEDA at dose levels of 1, 2, and 5 mg/kg. No mortalities were observed. There are some observations related to BMEDA treatment found in the 5 mg/kg dose group at 10 min post-dose. Tremor was observed in one male rat and seven female rats. The increased respiration, vocalization, not easy to handle and/or loss of tone in the limb were observed in both males and females, and increased body temperature was observed in male animals. Based on the results, a single intravenous dose of BMEDA administered to rats resulted in increased respiration, vocalization, not easy to handle and/or loss of tone in the limb increasing at the dose level of 5 mg/kg. No neurobehavioral effects were noted after BMEDA administration up to the dose level of 2 mg/kg. The information of this study will provides a point of reference to design appropriately therapeutic studies for future human use.

Food safety assessment and 28-day toxicity study of the synbiotic medical food consortium SBD121

The human gut microbiome plays a crucial role in immune function. The synbiotic consortium or Defined Microbial Assemblage™ (DMA™) Medical Food product, SBD121, consisting of probiotic microbes and prebiotic fibers was designed for the clinical dietary management of rheumatoid arthritis. A 28-day repeated administration study was performed to evaluate the oral toxicity of SBD121 in male and female rats (age/weight at study start: 60 days/156–264 g) administered levels of 0, 4.96 x 1010, 2.48 x 1011, or 4.96 x 1011 colony forming units (CFU)/kg-bw. No treatment related changes were observed in ophthalmological effects, mortality, morbidity, general health and clinical observations, urinalysis, hematology, serum chemistry, absolute or relative organ weights, gross necropsy, or histopathology. A significant decrease in body weight was reported in females in the low and high-concentration groups, which corresponded in part with a significant decrease in food consumption. Results of the functional observation battery indicated front grip strength was significantly greater in the high-concentration males compared to the controls; however, this effect was not considered adverse. Based on these findings, the administration of the Medical Food SBD121 to male and female rats has a no-observable adverse effect level (NOAEL) at the highest level tested of 4.96 x 1011 CFU/kg-bw.

Toxicological evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids

Very long chain fatty acids (VLCFA) have a chain length ≥24 carbons. Fish contain low levels of these fatty acids. A commercial oil called EPAX® Evolve 05 with an up-concentration of VLCFAs of approximately 10 times, has been developed as a dietary supplement by Epax Norway AS. A series of toxicological studies were performed using mice and rats to determine the safety and toxicity of repeat dosing with a gavage administered VLCFA formulation. The results suggest transient lipid accumulation in kidneys and liver. Lipid accumulation was seen with the test item and with the soya control but was not dose related. Liver and kidney lipid accumulation, whilst present in 14- day repeat dose study, was absent in a 90-day rat study. No treatment-effect was seen in urine analysis in any of the studies. No treatment-related effects were seen with a functional observation battery, ophthalmological examination, haematology, urine analysis, oestrus cycle, thyroid hormones, organ weight, or histopathology. In the 90-day study the liver enzymes ALP, AST and ALT were statistically significantly increased with test item but within control values. There were no associated histological findings in the liver suggesting there was no toxic effect and the normalisation of values for all liver enzymes in the recovery groups suggests an adaptive response rather than a prevailing toxic response. The no-observed-adverse-effect level (NOAEL) was determined as 1200 mg VLCFA/kg b.w./day.

Effect of Vehicles on the Functional Observational Battery by Oral Gavage Administration in Wistar Rats and Need for Historical Control Data

Effect of Vehicles on the Functional Observational Battery by Oral Gavage Administration in Wistar Rats and Need for Historical Control Data

Evaluation of gadolinium-based contrast agents in juvenile CD-1 mice including behavioral evaluations.

Seven gadolinium-based contrast agents (GBCAs), four linear and three macrocyclic, were evaluated for potential effects on development, including behavior of juvenile CD-1 mice. The GBCAs were administered via intravenous injection once daily on postnatal day (PND) 9, 12, 15, 18, and 21 (PND 1 was the day of delivery) at doses up to twice the human equivalent clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). Mice were bled for evaluation of exposure (plasma) to gadolinium (Gd) on PND 9, 12, and 70. At scheduled euthanasia, the liver, spleen, brain, skin (dorsal surface), bone (left femur), and kidneys were excised from up to six mice/sex/group on PND 10, 22, or 70 for the determination of Gd levels and histopathological analysis. All mice were monitored for toxicity, growth and survival, sexual maturation, and behavior. Gd was quantifiable in the brain tissues with levels declining over time. There was no long-term effect on the growth and development for mice exposed to any of the GBCAs. There was no impact on neurodevelopment as assessed by brain histology and validated neurobehavioral tests, including a functional observational battery, motor activity, and learning and memory as evaluated in the Morris water maze. For all GBCAs, the highest dose tested represented the no-observable-adverse-effect level in juvenile mice.

Neurofilament light chain in plasma as a sensitive diagnostic biomarker of peripheral neurotoxicity: In Vivo mouse studies with oxaliplatin and paclitaxel - NeuroDeRisk project

Identifying compounds that are neurotoxic either toward the central or the peripheral nervous systems (CNS or PNS) would greatly benefit early stages of drug development by derisking liabilities and selecting safe compounds. Unfortunately, so far assays mostly rely on histopathology findings often identified after repeated-dose toxicity studies in animals. The European NeuroDeRisk project aimed to provide comprehensive tools to identify compounds likely inducing neurotoxicity. As part of this project, the present work aimed to identify diagnostic non-invasive biomarkers of PNS toxicity in mice. We used two neurotoxic drugs in vivo to correlate functional, histopathological and biological findings. CD1 male mice received repeated injections of oxaliplatin or paclitaxel followed by an assessment of drug exposure in CNS/PNS tissues. Functional signs of PNS toxicity were assessed using electronic von Frey and cold paw immersion tests (oxaliplatin), and functional observational battery, rotarod and cold plate tests (paclitaxel). Plasma concentrations of neurofilament light chain (NF-L) and vascular endothelial growth factor A (VEGF-A) were measured, and histopathological evaluations were performed on a comprehensive list of CNS and PNS tissues. Functional PNS toxicity was observed only in oxaliplatin-treated mice. Histopathological findings were observed dose-dependently only in paclitaxel groups. While no changes of VEGF-A concentrations was recorded, NF-L concentrations were increased only in paclitaxel-treated animals as early as 7 days after the onset of drug administration. These results show that plasma NF-L changes correlated with microscopic changes in the PNS, thus strongly suggesting that NF-L could be a sensitive and specific biomarker of PNS toxicity in mice.

Croton hirtus attenuating streptozotocin-induced neuroinflammation in rats

ObjectiveThe present study was aimed to investigate the neuroprotective effect of Croton hirtus (CH) extract against streptozotocin (STZ) in rats. Methods(i) The sub-chronic toxicity consisted of 24 adult rats of either sex weighing from 160 to 200 g were divided into four groups with six rats in each group. Rats in group 1 received Dimethyl sulfoxide (DMSO) mixed with saline; rats in groups 2, 3, and 4 received 100, 200, and 400 mg/kg of methanolic extract of CH (MECH) orally by gavage administration for 28 d, respectively. The functional observation battery and locomotor activity were graded as part of their neurobehavioral activity and the brain regions, including cortex and hippocampus, were analyzed for neuropathological abnormalities. (ii) The main research consisted of 30 adult male Wistar rats weighing from 160 to 200 g, which were divided into five groups and six rats in each group, including control (I), STZ (II), Donepezil (III), MECH (100 mg/kg, IV), and MECH (200 mg/kg, V) groups. Rats in group I received citrate buffer orally and DMSO mixed with saline for 14 d. Rats in group II received STZ via intracerebroventricular injection (3 mg/kg, bilateral ICV-STZ) on days 1 and 3 followed by DMSO mixed with saline for 14 d. Rats in groups III, IV, and V received STZ administration on days 1 and 3 followed by Donepezil [3 mg/(kg·d), p.o.] and MECH [100 and 200 mg/(kg·d), p.o.] for 14 d. Rats were tested for learning and memory parameters such as Morris water maze (MWM) and passive avoidance test (PAT). They were sacrificed after completing behavioural experiments; brains were harvested to estimate the acetylcholinesterase (AChE), lipid peroxidation (LPO), glutathione (GSH), and superoxide dismutase (SOD) by using UV-Visible Spectrophotometer; caspase-3 was evaluated by total fluorescence emission spectra; amyloid β (Aβ) levels were detected using enzyme-linked immuosorbent assay (ELISA); and histopathological examination was conducted in the CA1 region of the hippocampus. Results(i) The sub-chronic toxicity results revealed that open field test parameters including line crossing, rearing, entering the middle square, defecating, or urinating did not differ significantly in the MECH rats (P > 0.05). The histopathological observation did not show any lesions in the neuronal cells and inflammation in both the regions in MECH rats compared with control rats. (ii) The main study findings demonstrated that STZ-treated rats showed a significant increase in impairment in learning and memory parameters (P < 0.001), the levels of AChE, caspase-3, Aβ (1-40 and 1-42), and LPO were increased significantly (P < 0.001), and significant decrease was found in the levels of SOD (P < 0.001) and GSH (P < 0.01). Moreover, neuronal damage was found in the hippocampus. In contrast, STZ-induced behavioural and biochemical impairments in rats were considerably decreased by treatment with MECH dose-dependently. ConclusionMECH significantly prevented the memory deficit induced by STZ due to antioxidant action. Restoration of cholinergic functioning may be the cause of behavioural improvement. Therefore, MECH may be able to treat cognitive disorders like Alzheimer's disease (AD).

Toxicological safety assessment of HempChoice® hemp oil extract; a proprietary extract consisting of a high concentration of cannabidiol (CBD) in addition to other phytocannabinoids and terpenes derived from CannabissativaL

HempChoice® Hemp Oil Extract (Geocann, LLC) is an extract of the aerial parts of hemp (Cannabis sativa L.) primarily comprised of 55–75% cannabidiol (CBD), 1–15% other phytocannabinoids and 1–15% terpenes. The results of multiple safety studies demonstrated that it was non-mutagenic in an Ames and mammalian cell micronucleus.test and was well tolerated in a 14-day range-finding study at dose levels up to 96.03.mg/kg BW/day. In the 90-day study, no HempChoice® Hemp Oil Extract-related significant changes were noted in weekly BW, daily BW gain, food consumption, functional observational battery or motor activity assessment. In addition, no HempChoice® Hemp Oil Extract related mortalities, abnormal clinical observations and ophthalmological changes were reported. Some HempChoice® Hemp Oil Extract-related changes were reported in the hematology and clinical chemistry parameters evaluated. These changes were not outside the normal range and were considered reversible during the 28-day recovery period. No macroscopic findings were reported, and histopathological changes related to HempChoice® Hemp Oil Extract exposure were limited to adaptive changes in the liver which were not observed in the recovery group animals. The no observed adverse effect level (NOAEL) for HempChoice® Hemp Oil Extract was determined to be 185.90 mg/kg BW/day in male and female Sprague-Dawley rats.

The Irwin Test and Functional Observational Battery (FOB) for Assessing the Effects of Compounds on Behavior, Physiology, and Safety Pharmacology in Rodents.

The modified Irwin procedure or functional observational battery (FOB) can be used to achieve several goals. New chemical entities (NCEs) can be behaviorally screened for nervous system effects at a variety of doses to identify potential therapeutic uses and in the selection of appropriate doses for subsequent assays. NCEs can also be evaluated in the behavioral battery and compared with reference standards to assess liabilities in a new compound class, with an estimated therapeutic index being suggested by the doses used in comparison to therapeutic doses. For the assessment of neurotoxicology, the FOB is often used. The differences between the two assays are subtle. The procedures used are essentially the same, but when considering neurotoxicology, the FOB is often conducted using GLP guidelines, with more animals being used per group, and doses that are low enough to determine a no effect level and high enough to induce marked nervous system behaviors. © 2023 Wiley Periodicals LLC. Basic Protocol: The Irwin test and FOB for assessing the effects of compounds on behavior, physiology, and safety pharmacology in rodents.

Simultaneous assessment of central nervous and respiratory systems using jacketed telemetry in socially-housed rats: Application of the “3Rs” principles in core battery safety pharmacology studies

Central nervous (CNS) and respiratory systems are routinely investigated in safety pharmacology core battery studies. For small molecules, the assessment of both vital organ systems is frequently done in rats in two distinct studies. With the advent of a miniaturized technology of jacketed external telemetry for rats (DECRO system), the simultaneous assessment of modified Irwin's or functional observational battery (FOB) test and respiratory (Resp) studies has become possible within a single study. Therefore, the objectives of this study were to perform the FOB and the Resp studies simultaneously in pair-housed rats fitted with jacketed telemetry, and to assess the feasibility and the outcome of this combination in control, baclofen, caffeine, and clonidine treated groups, i.e., with three agents having both respiratory and CNS effects. Our results provided evidence that performing both Resp and FOB assessment simultaneously in the same rat was feasible and the outcome was successful. The expected CNS and respiratory effects of the 3 reference compounds were accurately captured in each assay confirming the results' relevance. In addition, heart rate and activity level were recorded as additional parameters making this design as an enhanced approach for nonclinical safety assessment in rats. This work provides clear evidence that the “3Rs” principles can be effectively applied in core battery safety pharmacology studies while remaining in compliance with worldwide regulatory guidelines. Both reduction in animal use and refinements in procedures are demonstrated with this model.

Toxicity and human health assessment of an alcohol-to-jet (ATJ) synthetic kerosene developed under an international agreement with Sweden

ABSTRACT Alcohol-to-jet (ATJ) Synthetic Kerosene with Aromatics (SKA) fuels are produced by dehydration and refining of alcohol feed stocks. ATJ SKA fuel known as SB-8 was developed by Swedish Biofuels as a cooperative agreement between Sweden and AFRL/RQTF. SB-8 including standard additives was tested in a 90-day toxicity study with male and female Fischer 344 rats exposed to 0, 200, 700, or 2000 mg/m3 fuel in an aerosol/vapor mixture for 6 hr/day, 5 days/week. Aerosols represented 0.04 and 0.84% average fuel concentration in 700 or 2000 mg/m3 exposure groups. Examination of vaginal cytology and sperm parameters found no marked changes in reproductive health. Neurobehavioral effects were increased rearing activity (motor activity) and significantly decreased grooming (functional observational battery) in 2000 mg/m3 female rats. Hematological changes were limited to elevated platelet counts in 2000 mg/m3 exposed males. Minimal focal alveolar epithelial hyperplasia with increased number of alveolar macrophages was noted in some 2000 mg/m3 males and one female rat. Additional rats tested for genotoxicity by micronucleus (MN) formation did not detect bone marrow cell toxicity or alterations in number of MN; SB-8 was not clastogenic. Inhalation results were similar to effects reported for JP-8. Both JP-8 and SB fuels were moderately irritating under occlusive wrapped conditions but slightly irritating under semi-occlusion. Exposure to SB-8, alone or as 50:50 blend with petroleum-derived JP-8, is not likely to enhance adverse human health risks in the military workplace.

Motor-evoked potentials after focal electrical stimulation predict drug-induced convulsion potentials in rats

Drug-induced convulsions—often caused by the inhibition of GABA receptors and stimulation of glutamate receptors—are difficult to predict in animals. In this study, we attempted to detect the proconvulsant potential using motor-evoked potentials (MEPs) after focal electrical stimulation or upon using a functional observational battery (FOB). Pentylenetetrazole, kainic acid, and pilocarpine were used as convulsion-inducing drugs, and baclofen was used as a negative control. First, each compound was administered to male rats, and the FOB tests were performed. All drugs induced behavioral changes, but no commonality was found. Single electrical stimulation train MEPs were recorded under anesthesia for 60 min (at 5 min intervals) after administration of each drug. A dose-dependent increase in MEPs was observed for each convulsion-inducing drug. Moreover, paired electrical stimulation (conditioned and test) of the cerebral motor cortex was conducted with a 1–15 ms interstimulus interval (ISI), 10 min after administration of the drug. All convulsion-inducing drugs inhibited the short-interval intracortical inhibition (ISI: 3 ms), which may be associated with GABA. Intracortical facilitation (ISI: 11 ms), related to glutamate, was not enhanced by any drug but was inhibited by pilocarpine. Dose correlation was not found in short-interval intracortical inhibition or intracortical facilitation in any drugs. No changes in MEPs were observed after baclofen administration. These results suggest that it is possible to evaluate the convulsion potential and associated mechanisms using MEP, independent of the behavioral changes. The early identification of convulsion potential using this model will lead to more efficient drug development.

Alcoholic neuropathy associated with chronic alcohol intake

Alcoholic neuropathy (AN), a debilitating condition that mainly affects chronic alcohol drinkers, is thought to cause lesions in the peripheral nervous system leading to sensory, autonomic, and motor dysfunctions. Despite many studies, the pathogenesis of these lesions is still not completely understood. We investigated few aspects on the development of alcohol-induced peripheral neuropathy, by assessing sensory, motor and autonomic functions, as well as stereological analysis of axonal fibers and myelin sheath of the sciatic nerve. Twelve male Wistar rats were divided into Control group and Alcohol group that was submitted to Two Bottle-Choice Paradigm of intermittent and voluntary alcohol solution intake (20%; v/v) during eight weeks. At the end of treatment, three different sensorium-motor tests were applied - Tactile Sensitivity, Thermal Sensitivity, and Functional Observational Battery (FOB). Quantitative morphometric analysis of sciatic nerve structures was performed by stereological method. Alcohol concentration in the blood was measured to analyze possible correlation between availability of alcohol in the blood and the magnitude of the peripheral nerve lesion. Our data showed a peripheral effect of chronic alcohol intake associated with hyperalgesia and a process of demyelination with a strong correlation with alcohol consumption. This process was associated with increased tactile sensitivity, with behavioral reflexes such as locomotor hyperactivity, changes in gait and balance, and autonomic reflexes such as piloerection.

Seventy-Day Toxicity Study in Juvenile Sprague-Dawley Rats with Semicarbazide (SEM) from Weaning to Sexual Maturity.

The study was conducted to evaluate the toxicological effects, functional observation battery tests, and sexual maturity of semicarbazide oral gavage administration to juvenile Sprague-Dawley rats for 70 days at 0, 15, 30, and 60 mg/kg/day weaning to sexual maturity. At 60 mg/kg/day, there was a delay in mean age at acquisition of balano-preputial and vaginal patency and a decrease in body weight and food consumption in males. Treatment increased reticulocyte count, aspartate aminotransferase, and alanine aminotransferase levels in both sexes and decreased hematocrit and protein in males. Increased absolute and relative liver and spleen weight in both sexes were observed. Male rats had lower thymus and testes weights, whereas female rats had lower uterine weights. Semicarbazide caused significant changes in sperm motility, sperm count, and sperm abnormality. Histopathologically, semicarbazide caused cortical hypertrophy in adrenals and increased extramedullary hematopoiesis in the spleen; hepatocellular hypertrophy, follicular epithelial hypertrophy in the thyroid, and degeneration of seminiferous tubules in the testis were observed at 60 mg/kg/day when compared to control. Results suggest that 60 mg/kg/day of semicarbazide can exert systemic toxicity in juvenile rats. The no observed adverse effect level (NOAEL) of semicarbazide for juvenile Sprague-Dawley rats was estimated to be 30 mg/kg/day.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Mutagenicity and safety pharmacology of a standardized antidiabetic polyherbal formulation

Synacinn is a standardized polyherbal extract formulated for the treatment of diabetes mellitus and its complications. This study aims to assess the mutagenicity potential of Synacinn by Ames assay and in vivo bone marrow micronucleus (MN) test on Sprague Dawley rat. Human ether-a-go-go-related gene (hERG) assay and Functional Observation Battery (FOB) were done for the safety pharmacology tests. In the Ames assay, Dose Range Finding (DRF) study and mutagenicity assays (+/− S9) were carried out. For the MN test, a preliminary and definitive study were conducted. In-life observations and number of immature and mature erythrocytes in the bone marrow cells were recorded. The hERG assay was conducted to determine the inhibitory effect on hERG potassium channel current expressed in human embryonic kidney cells (HEK293). FOB tests were performed orally (250, 750, and 2000 mg/kg) on Sprague Dawley rats. Synacinn is non-mutagenic against all tested strains of Salmonella typhimurium and did not induce any clastogenicity in the rat bone marrow. Synacinn also did not produce any significant inhibition (p ≤ 0.05) on hERG potassium current. Synacinn did not cause any neurobehavioural changes in rats up to 2000 mg/kg. Thus, no mutagenicity, cardiotoxicity and neurotoxicity effects of Synacinn were observed in this study.

Examination of central nervous system by functional observation battery after massive intravenous infusion of carbon monoxide-bound and oxygen-bound hemoglobin vesicles in rats

Carbon monoxide (CO) is known as a toxic gas inducing “CO poisoning”, which acutely affects the central nervous system (CNS) and which persistently affects brain functions depending on the exposure time and CO concentration. By contrast, in pathological rodent models, intravenous infusion of CO-bound hemoglobin vesicles (CO-HbV) has shown various beneficial effects such as anti-oxidative and anti-inflammatory reactions. This study assessed effects of CO-HbV infusion on CNS using a functional observation battery, sensory reflexes, grip strength, and landing foot splay measurements. The test fluids were CO-HbV and O2-bound HbV (O2-HbV) suspended in saline ([Hb] ​= ​10 ​g/dL), and saline alone for comparison. The rats received either 16 or 32 ​mL/kg of fluid intravenously at 1.5 ​mL/min/kg. Observations were made before infusion, and at 5 ​min, 4, 8, 24, 48 and 72 ​h after infusion. Massive doses of 16 and 32 ​mL/kg respectively corresponded to about 29 and 57% of the whole circulating blood volume (56 ​mL/kg). No toxicological effect was observed in any measurement item for any group in comparison to the control saline infusion group. Histopathological examination of hippocampal tissue at 14 days after infusion showed the number of necrotic cells to be minimal. Results obtained from rats in this experiment suggest that the massive intravenous infusion of CO-HbV yields beneficial anti-oxidative and anti-inflammatory effects without showing CO-poisoning-related symptoms of CNS damage.

Safety assessment of the fungal immunomodulatory protein from Ganoderma microsporum (GMI) derived from engineered Pichia pastoris: Genetic toxicology, a 13-week oral gavage toxicity study, and an embryo-fetal developmental toxicity study in Sprague-Dawley rats

The 12.4 kDa fungal immunomodulatory protein from Ganoderma microsporum (GMI) has bioactivity in vitro and in vivo. This study assessed the safety of GMI derived from engineered Pichia pastoris in Sprague-Dawley rats as a dietary supplement and food ingredient by evaluating subchronic toxicity, teratology, and mutagenicity. The oral gavage administration of 10 mL GMI at 0, 50, 75, or 100 mg GMI/kg body weight/day assayed for 91 consecutive days showed no mortality or moribundity. There were no test article-related findings in animal observations/measurements: cageside observation, detailed clinical observations, body weights, feed consumption, ophthalmic examinations, functional observation battery, clinical chemistry, hematology, coagulations, urinalysis, or terminal necropsy (gross or histopathology findings) suggesting that GMI has no subchronic toxicity. The teratology toxicity study of pregnant female rats orally administered GMI at 0, 50, 75, or 100 mg/kg body weight/day throughout organogenesis (gestation date 6–18) showed no mortality, moribundity, and no test article-related finding to dam or fetus. GMI genotoxicity was not observed by mutagenicity studies of Salmonella typhimurium, in vitro chromosome aberrations, and an in vivo micronucleus test in mice. Overall, no observed-adverse-effect level (NOAEL) was determined for GMI based on the subchronic and teratology studies at 100 mg/kg body weight/day.

Issues in the design, analysis, and application of rodent developmental neurotoxicology studies

Developmental neurotoxicity (DNT) studies could benefit from revisions to study design, data analysis, and some behavioral test methods to enhance reproducibility. The Environmental Protection Agency (EPA) reviewed 69 studies submitted to the Office of Pesticide Programs. Two of the behavioral tests identified the lowest observable adverse effect level (LOAEL) 20 and 13 times, respectively, while the other two tests identified the LOAEL only 3 and 4 times, respectively. The EPA review showed that the functional observational battery (FOB) was least effective at detecting the LOAEL, whereas tests of learning and memory (L&M) had methodological shortcomings. Human neurodevelopmental toxicity studies over the past 30 years show that most of the adverse effects are on higher cognitive functions such as L&M. The results of human studies together with structure-function relationships from neuroscience, suggest that tests of working memory, spatial navigation/memory, and egocentric navigation/memory should be added to guideline studies. Collectively, the above suggest that EPA and EU DNT studies would better reflect human findings and be more relevant to children by aligning L&M tests to the same domains that are affected in children, removing less useful methods (FOB), and using newer statistical models to better account for random factors of litter and litter × sex. Common issues in study design and data analyses are discussed: sample size, random group assignment, blinding, elimination of subjective rating methods, avoiding confirmation bias, more complete reporting of species, housing, test protocols, age, test order, and litter effects. Litter in DNT studies should at least be included as a random factor in ANOVA models and may benefit from inclusion of litter × sex as random factors.