Introduction: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) initiated a process in 2012 to revise the S1B Guideline \"Testing for Carcinogenicity of Pharmaceuticals\". Previous retrospective analysis indicated the importance of histopathological risk factors in chronic toxicity studies, evidence of endocrine perturbation, and positive genetic toxicology results as potentially predictive indicators of carcinogenic risk. In addition, a relationship between pharmacodynamic activity and carcinogenicity outcome in long-term rodent studies has been reported. It was postulated that these factors could be evaluated in a Weight-of-Evidence (WoE) approach to predict the outcome of a 2-year rat study. Methods: The ICH S1B(R1) Expert Working Group (EWG) conducted a Prospective Evaluation Study (PES) to determine the regulatory feasibility of this WoE approach. Drug Regulatory Authorities (DRAs) evaluated 49 Carcinogenicity Assessment Documents (CADs), which describe the WoE for submitted pharmaceutical compounds. Each compound was categorized into a carcinogenic risk category including a statement of the value of the 2-year rat study. The outcome of the completed 2-year rat studies was evaluated in relation to the prospective CAD to determine the accuracy of predictions. Results: Based on the results of the PES, the EWG concluded that the evaluation process for assessing human carcinogenic risk of pharmaceuticals described in ICH S1B could be expanded to include a WoE approach. Approximately 27% of 2-year rat studies could be avoided in cases where DRAs and sponsors unanimously agreed that such a study would not add value. Discussion: Key factors supporting a WoE assessment were identified: data that inform carcinogenic potential based on drug target biology and the primary pharmacologic mechanism of the parent compound and major human metabolites; results from secondary pharmacology screens for this compound and major human metabolites that inform carcinogenic risk; histopathology data from repeated-dose toxicity studies; evidence for hormonal perturbation; genotoxicity data; and evidence of immune modulation. The outcome of the PES indicates that a WoE approach can be used in place of conducting a 2-year rat study for some pharmaceuticals. These data were used by the ICH S1B(R1) EWG to write the R1 Addendum to the S1B Guideline published in August 2022.

The realm of diazinon reduction from polluted water has witnessed a surge in the significance of advanced oxidation processes (AOPs) in recent times. However, there is a dearth of research focusing on the mitigation of its toxicity through AOPs. Thus, the primary objective of this study was to evaluate the effectiveness of the Electro-Fenton process (EFP) in the eradication and detoxification of diazinon in aqueous solutions. Synthetic wastewater samples with concentrations of 2, 2.5 and 3 mg/L were prepared. A total of 27 samples were determined using Box Behnken Design. Reaction time, pH and iron to hydrogen peroxide ratio (Fe2+/H2O2) were examined as operational parameters under a constant current of 5.4 amps. The quantification of diazinon concentration was performed using High-Performance Liquid Chromatography (HPLC). To evaluate the detoxification of diazinon, the Daphnia magna bioassay was employed as a methodology in this study. According to the results, the EFP could reduce the diazinon to zero and the LC50 values are increased by applying the process. The LC50 values for diazinon were determined using the Daphnia magna bioassay, considering initial concentrations of 2, 2.5, and 3 mg/L at a pH of 5, a reaction time of 15 min, and an iron to hydrogen peroxide molar ratio of 2. The recorded LC50 values were 3.039, 3.076, and 3.106, respectively, indicating the lowest frequency of cumulative death in Daphnia magna. In this case, after 96 h, only 3 cases (30%) of Daphnia magna death were observed. However, for all the mentioned concentrations of diazinon, after 96 h of exposure to samples without applying the Daphnia Magna death process, it was observed between 60 and 100%. Reducing the diazinon concentration and increasing the 96-h LC50 showed that the EFP can reduce the toxicity of diazinon on Daphnia Magna at the same time. Therefore, EFP can be considered a superior method with low ecotoxicity.

PFAS, or per- and polyfluoroalkyl substances, are a family of man-made chemicals found in a variety of products from non-stick cookware and food wrappers to firefighting foams. PFAS are persistent and widely distributed in the environment, including aquatic environments. In this study we examined the impact of PFAS chemicals on the physiological and behavioral endpoints of Lumbriculus variegatus (i.e., blackworms). Lumbriculus variegatus is a species of freshwater annelid worm that plays key roles in shallow freshwater ecosystems. At an environmentally relevant concentration of 1 μg/L, 12-day aqueous exposure to long chain PFAS, including PFOA, PFOS and PFDA, each markedly slowed the pulse rate of the dorsal blood vessel in L. variegatus, indicating a suppressive effect on blood circulation. The mean pulse rate was reduced from 9.6 beats/minute to 6.2 and 7.0 beats/min in PFOA and PFOS, respectively (P < 0.0001). Further, PFOA, PFOS and PFDA reduced the escape responsiveness of L. variegatus to physical stimulation. The percentage of worms showing normal escape behavior was reduced from 99.0% in control to 90.6% in the PFOS exposed group (P < 0.01). In a chronic (4 week) growth study, exposure to overlying water and sediment spiked with PFOA, PFOS or PFDA reduced the total biomass and the number of worms, indicating a suppressive effect on worm population growth. For instance, PFOA and PFDA reduced the total dry biomass by 26.3% and 28.5%, respectively, compared to the control (P < 0.05). The impact of PFAS on blackworm physiology is accompanied by an increase in lipid peroxidation. The level of malondialdehyde (MDA), an indicator of lipid peroxidation, and catalase, a major antioxidant enzyme, were markedly increased in PFOA, PFOS and PFDA exposed groups. Interestingly, exposure to PFHxA, a short chain PFAS, had no detectable effect on any of the measured endpoints. Our results demonstrate that L. variegatus is highly sensitive to the toxic impact of long chain PFAS chemicals as measured by multiple endpoints including blood circulation, behavior, and population growth. Such toxicity may have a detrimental impact on L. variegatus and the freshwater ecosystems where it resides.

BACKGROUND: Deer (Cervus elaphus Linnaeus) antler extract has been traditionally used in many Asian countries to prevent and treat various diseases. Deer antler extract is generally considered to be safe because humans have been exposed to it for a long time and it has been used as a tonic medicine originating from naturally occurring product. However, the comprehensive toxicological potential of deer antler extract during the juvenile period has not been investigated and its safety for juveniles remains unclear.
OBJECTIVE: The aim of the present study was to comprehensively investigate the potential toxicological effects of deer antler extract during the juvenile period.
METHODS: As part of a safety assessment of the juvenile period, two separate studies, a juvenile toxicity study and a uterotrophic bioassay, were conducted in accordance with Good Laboratory Practice regulations and test guidelines. In the juvenile toxicity study, deer antler extract was administered daily by oral gavage to Sprague-Dawley rats at doses of 0, 500, 1000, and 2000 mg/kg during the juvenile period to investigate potential toxicities on general systemic, developmental, and reproductive functions. For the uterotrophic bioassay, deer antler extract was administered daily by oral gavage to Sprague-Dawley rats at doses of 0, 1000, and 2000 mg/kg on postnatal days 19-21 to investigate the estrogen-mimicking effects.
RESULTS: The results showed that deer antler extract was non-toxic and no observable adverse effects on general systemic function, developmental and reproductive function, and estrogen-mimetic effects were observed with dosing up to 2000 mg/kg during the juvenile period.
CONCLUSIONS: The safety of deer antler extracts was demonstrated in these studies and the results of this study can be used to evaluate human risk or determine the maximum recommended starting dose of deer antler extract for further clinical trials.

Chemical index components, especially those defined as quality control (QC) markers through spectrum-effect relationship approach, are commonly suggested and adopted as indicator for quality control of Traditional Chinese Medicines (TCMs). However, are chemical index components and quality control of TCMs \"never change a winning team\"? In this study, under the ponderation of the applicability of QC markers strategy, spectrum-effect relationship and OPLS-DA between GC×GC-MS fingerprint and inhibitory effect on the expression of extracellular secretory TNF-α of volatile oil from Bupleuri radix (BVO) was studied with the purpose of discovery of QC markers and establish a bioactive compounds-based QC method. 290 compounds of BVO were identified by GC×GC-MS. Besides, BVO had significant inhibitory effects on the expression of extracellular secretory TNF-α in a dose-dependent manner. The potency of different batches of BVOs could be distinguished with this bioassay-based method, which has been validated in terms of intermediate precision, repeatability, linearity, range and credibility tests. The QC markers of BVO were investigated by Spearman\'s correlation test and OPLS-DA. It is regrettable that there were no ideal QC markers of BVO could be found. In conclusion, quality control method relayed on chemical QC markers is not feasible for TCMs with complex composition but lack of ingredients that dominate in content, just like BVO. Alternatively, a bioassay-based method established in our study is suitable for quality control of BVO.

To evaluate the effects of nitrofurazone on functional processes in marine ecosystems, periphytic protozoan communities were exposed to different concentrations of the antibiotic for a 10-day duration. Species trait distributions in the tested communities were observed during exposure to five concentrations of nitrofurazone. A fuzzy coding system with seven traits and seventeen categories was used to summarize the changes in functional patterns of the test organisms. Nitrofurazone had a significant influence on the function process of the periphytic ciliate communities. Bacterivores with flattened bodies were sensitive to the toxicant whereas sessile and cylindrical raptors showed a high tolerance to nitrofurazone, invariably dominating communities exposed to high concentrations. Bootstrapped-average analysis demonstrated a significant change in functional patterns at highest nitrofurazone concentrations (8 mg l-1). Based on these findings, it is suggested that nitrofurazone may negatively influence ecosystem function in marine environments.

The slow pace of discovery of bioactive natural products can be attributed to the difficulty in rapidly identifying them in complex mixtures such as plant extracts. To overcome these hurdles, we explored the utility of two machine learning techniques, i.e., Elastic Net and Random Forests, for identifying the individual anti-inflammatory principle(s) of an extract of the inflorescences of the hops (Humulus lupulus) containing hundreds of natural products. We fractionated a hop extract by column chromatography to obtain 40 impure fractions, determined their anti-inflammatory activity using a macrophage-based bioassay that measures inhibition of iNOS-mediated formation of nitric oxide, and characterized the chemical composition of the fractions by flow-injection HRAM mass spectrometry and LC-MS/MS. Among the top 10 predictors of bioactivity were prenylated flavonoids and humulones. The top Random Forests predictor of bioactivity, xanthohumol, was tested in pure form in the same bioassay to validate the predicted result (IC50 7 µM). Other predictors of bioactivity were identified by spectral similarity with known hop natural products using the Global Natural Products Social Networking (GNPS) algorithm. Our machine learning approach demonstrated that individual bioactive natural products can be identified without the need for extensive and repetitive bioassay-guided fractionation of a plant extract.

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, \'animal friendly\' Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The \"known unknown\" suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

Thorium is one of the most widespread radioactive elements in natural ecosystems, along with uranium, it is the most important source of nuclear energy. However, the effects of thorium on living organisms have not been thoroughly studied. Marine luminescent bacteria and their enzymes are optimal bioassays for studying low-dose thorium exposures. Luminescent bioassays provide a quantitative measure of toxicity and are characterized by high rates, sensitivity, and simplicity. It is known that the metabolic activity of bacteria is associated with the production of reactive oxygen species (ROS). We studied the effects of thorium-232 (10-11-10-3 M) on Photobacterium phosphoreum and bacterial enzymatic reactions; kinetics of bacterial bioluminescence and ROS content were investigated in both systems. Bioluminescence activation was revealed under low-dose exposures (<0.1 Gy) and discussed in terms of \"radiation hormesis\". The activation was accompanied by an intensification of the oxidation of a low-molecular reducer, NADH, during the enzymatic processes. Negative correlations were found between the intensity of bioluminescence and the content of ROS in bacteria and enzyme systems; an active role of ROS in the low-dose activation by thorium was discussed. The results contribute to radioecological potential of bioluminescence techniques adapted to study low-intensity radioactive exposures.

Biological indexing is based upon the ability of certain plants, referred to as indicator plants or indicators, to produce specific symptoms when inoculated with a pathogen using mechanical means or grafting. In the case of citrus viroids, clonal indicators are grafted on to vigorous rootstocks such as rough lemon (Citrus × granulata Raf.). The \'Arizona-861-S-1\' citron clonal indicator (C. medica L.) can detect and bioamplify all citrus viroids; however, for specific citrus variants of the hop stunt viroid (i.e., CVd-IIb and CVd-IIc), the clonal indicator \'Parson\'s special # 9\' mandarin (C. reticulata Blanco) is preferred. Inoculation techniques and symptom expression are described in detail. Other supporting elements, such as greenhouse conditions and propagation techniques, are also presented.