Methodological Approaches to Preclinical Evaluation of General Toxicity of Therapeutic Radiopharmaceuticals

Radiopharmaceuticals differ from other medicines mainly by the specific nature of their pharmacological action based on radioactivity, by absence of multiple-dose regimens, and by miniscule concentrations of the active pharmaceutical ingredient (10-9–10-12 mol/L). However, the official regulations and standards do not contain specific requirements for preclinical evaluation of radiopharmaceuticals. The aim of the study was to summarise preclinical data on general toxicity of therapeutic radiopharmaceuticals, which were obtained by the Laboratory of Preclinical and Clinical Studies of Radiopharmaceuticals of the Federal State Budgetary Institution ‘State Research Center of the Russian Federation¾Burnasyan Federal Medical Biophysical Center’ of the Federal Medical Biological Agency. Results: the authors analysed criteria for: selection of the study design based on physico-chemical nature of the active pharmaceutical ingredient; calculation of doses and selection of dosage regimens for radiopharmaceuticals with different administration routes; radiation safety for personnel performing the study. The authors used the data on three therapeutic radiopharmaceuticals to formulate methodological approaches to preclinical evaluation of their general toxic properties. The analysis of long-term experience in preclinical studies of therapeutic radiopharmaceuticals demonstrated the need for a judicious individual approach to the development of a preclinical study design. The authors proposed methodological approaches to preclinical evaluation of general toxic properties of therapeutic radiopharmaceuticals, which make it possible to adequately assess potential toxic effects and obtain meaningful study results. Conclusions: there is a need for a specific guideline for preclinical studies of therapeutic radiopharmaceuticals, which would take into account specific pharmacological action and nuclear-physical characteristics of radionuclides.

The effect of aging on the repeated-dose liver micronucleus assay

Background The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds. In particular, the repeated-dose liver MN (RDLMN) assay which greatly facilitates incorporation of the liver MN assay into the general toxicity study has been developed. Usefulness of the RDLMN assay was appraised highly in the 7th International Workshops on Genotoxicity Testing (2017 in Tokyo) in that sufficient numbers and types of chemicals were studied and easy integration into the general toxicity study is preferred from the 3R’s point of view. However, it was pointed out that it is necessary to evaluate the effect of age at the start of 4-week repeated administration, since there are limited data, where only those of rats of 6 week of age at the start of administration are available. In this study, we conducted the 4-week RDLMN assay using rats of 6 and 8 weeks of age (at the start of administration) to investigate the effect of age on the liver MN inducibility. Clofibrate, a weak inducer of liver MN, was used in this study to detect the slight difference in the liver MN induction. Results The liver MN induced by clofibrate was detected in both rats of 6 and 8 weeks of age at the start of administration. However, the liver MN induction was lower in rats of 8 weeks of age compared to rats of 6 weeks of age at the start of administration. Conclusion These results suggest that the liver MN inducibility decreases with age. Therefore, we recommend the use of rats of 6 weeks of age at start of administration to reliably detect the liver MN induction in the RDLMN assay.

Nervous System Sampling for General Toxicity and Neurotoxicity Studies in the Laboratory Minipig With Emphasis on the Göttingen Minipig

The use of minipigs as an alternative nonclinical species has increased in the last 20 years. The Society of Toxicologic Pathology (STP) has produced generic “best practice” recommendations for nervous system sampling in nonrodents during general toxicity studies ( Toxicol Pathol 41[7]: 1028–1048, 2013), but their adaptation to the minipig has not been attempted. Here, we describe 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the minipig brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen agents with unknown or no anticipated neurotoxic potential; this approach using 7 coronal hemisections accords with the published STP “best practice” recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses 14 coronal hemisections and 2 full coronal sections to investigate toxicants where the nervous system is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves from minipigs during nonclinical studies should follow published STP “best practice” recommendations for sampling the central (CNS, Toxicol Pathol 41[7]: 1028–1048, 2013) and peripheral (PNS, Toxicol Pathol 46[4]: 372–402, 2018) nervous systems.

Motor dysfunction in Drosophila melanogaster as a biomarker for developmental neurotoxicity

Humans interact with numerous chemical compounds with direct health implications, with several able to induce developmental neurotoxicity (DNT), which bear developmental, behavioral, and cognitive consequences from a young age. Current guidelines for DNT testing are notably costly, time consuming, and unsuitable for testing large numbers of chemicals. Therefore, there is a need for adequate alternatives to conventional animal testing for neurotoxicity and DNT. Here we show that detailed kinematic analysis can provide a strong indicator for DNT, using known (chlorpyrifos, CPS) or putative (β–N–methylamino–L–alanine, BMAA) neurotoxic compounds. Here we Drosophila melanogaster exposed to these compounds during development and evaluated for common general toxicity – notably developmental survival and pupal positioning, together with the FlyWalker system, a detailed adult kinematics evaluation method. At concentrations that do not induce general toxicity, the solvent DMSO had a significant effect on kinematic parameters. Nonetheless, CPS not only induced developmental lethality but also significantly impaired coordination in comparison to DMSO, altering 16 motor parameters, validating the usefulness of our kinematic approach. Interestingly, BMAA, although not lethal during development, induced a dose–dependent motor decay, targeting most parameters in young adult animals, phenotypically resembling normally aged, non-exposed flies. This effect was subsequently attenuated during ageing, indicating an adaptive response. Furthermore, BMAA induced an abnormal terminal differentiation of leg motor neurons, without inducing degeneration, underpinning the observed altered mobility phenotype. Overall, our results support our kinematic approach as a novel, highly sensitive and reliable tool to assess potential DNT of chemical compounds.

General Toxicity screening of Royleanone derivatives using an Artemia salina model

Cancer is the second leading cause of death globally. World Health Organization recorded an estimated 9.6 million deaths attributed to cancer in 2018. There is an urgent need for new anticancer drugs with novel modes of action. Natural products remain a valuable source for the identification and development of novel treatment options for cancer. Plectranthus species are well-known medicinal species used extensively for the treatment of different illnesses. These species are rich in diterpenoids which are reported to be responsible for various pharmacological activities including cytotoxic activities. Brine shrimp (Artemia salina) is broadly used in lethality studies and this model is a convenient starting point for cytotoxicity study when screening for general toxicity of natural products. This assay is based on their ability to kill a laboratory cultured nauplii. In this work, we report the general toxicity of some derivatives from 6,7-dehydroroyleanone (1) and 7α-acetoxy-6β-hydroxyroyleanone (2) compounds. The A. salina bioassay is a simple, rapid, and low-cost test, and is very useful for preliminary assessment of general toxicity in natural products thereby guiding the determination of possible biological activities.

Assessment of General Toxicity of the Glycyrrhiza New Variety Extract in Rats

The Glycyrrhiza radix (Licorice) is one of the most commonly used medicinal plants in Asian countries, such as China, India, and Korea. It has been traditionally used to treat many diseases, including cough, cold, asthma, fatigue, gastritis, and respiratory tract infections. A Glycyrrhiza new variety, Wongam (WG), has been developed by the Korea Rural Development Administration and revealed pharmacological effects. However, the potential adverse effects of WG have not been revealed yet. This study evaluates the general toxicity of the WG extract through a single and repeated oral dose toxicity study in Sprague-Dawley rats. After single oral dose administration, no significant toxicological changes or mortality was observed up to 5000 mg/kg. Over a 4-week repeated oral dose toxicity study, no adverse effects and target organs were observed up to 5000 mg/kg/day. Over a 13-week repeated oral dose toxicity study, no mortality or toxicological changes involving ophthalmology, water consumption, or hematology were observed up to 5000 mg/kg/day. Although other parameters were changed, the alterations in question were not considered toxicologically significant, since responses remained within normal ranges and were not dose-dependent. In conclusion, the no-observed-adverse-effect level (NOAEL) of WG was higher than 5000 mg/kg/day, and no target organs were identified in rats.

Monitoring of compliance of quality and safety of cooked smoked sausages “Servelat” with the requirements of the national standard

Meat products, in particular sausages, play a significant role in the structure of retail trade among other product groups. Sausages are a traditional product of the food industry in Ukraine, and the share of cooked and smoked sausages is up to 51 % of total production. Today, fierce competition encourages producers to introduce new recipes into production, which, unfortunately, has led to a sharp increase in cases of falsification of sausages. In addition, a significant number of low-capacity enterprises are involved in production, which often leads to a decrease in the sanitary quality of manufactured products. Therefore, the purpose of our study was to analyze the compliance of quality and safety indicators of cooked and smoked sausage of the highest grade of different manufacturers to DSTU 4591:2006 “Cooked smoked sausages. General specifications”. The material of our research were samples of cooked smoked sausage of the highest grade “Servelat” of several domestic producers. The research was conducted on the basis of Multidisciplinary Laboratory of Veterinary Medicine (Faculty of Veterinary Medicine and Biotechnologies of Odesa State Agrarian University) and on the basis of the laboratory of Department of Veterinary-Sanitary Inspection (Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies, Lviv). Sampling, organoleptic analysis of sausages and determination of microbiological parameters were performed in accordance with current regulations, determination of chemical composition – using an express analyzer FoodScan, general toxicity – express method using the infusoria Colpoda steinii. According to the results of the analysis of the labeling, it is established that the packaging of all investigated products contains the basic production and consumer information in accordance with Article 6 of the Law of Ukraine “On information for consumers about food”, but on the labels of samples № 2 and 3 there is no data provided for in Article 23 of this Law – the content of unsaturated fats, sugars and salt. The study of organoleptic and microbiological parameters of sausage samples demonstrates their compliance with the requirements of the regulated DSTU 4591:2006 “Cooked smoked sausages. General specifications”. The general toxicity of sausages was also not detected. The analysis of physicochemical parameters revealed non-compliance of sample № 3 with the requirements of the national standard in terms of mass content of moisture – excess by 5.72 %. In addition, a sufficiently high collagen content of 2.3 ± 0.04 was found in sausage loaves of sample № 1.

Chromatic Toxicity Bioassay of Water through Bacterial Ferricyanide Reduction

Water quality assessment demands a precise anatomization of specimens that comply with acclaimed water purity standards. Today, the growing number of toxicants and their synergistic consequences make it necessary to develop general toxicity assays able to examine and determine water pollution. Contemporary general toxicity methods hinder specimen analysis due to their prolonged operation protocols. Also, the equipment involved is very expensive that not everyone can afford it. In an effort to resolve these drawbacks, a quick and cost effective toxicity bioassay based on chromatic changes related to bacterial ferricyanide reduction is introduced here. E.coli cells (Model Bacteria) were stably confined on four supports: Cellulose-based Paper Discs, Silica 60, Polystyrene, and Acrylic Beads, which remained useful for a long period at -20ºC. Copper was used as a model toxic agent to perform Bioassay Assessment. Chromatic changes related to bacterial ferricyanide reduction were determined by visual inspection. Cellulose Paper Discs, Polystyrene, and Acrylic beads showed good results and better viability, while Silica 60 proved itself as a weak support and resulted in poor viability.