Test Substances Research Articles

Investigation of explosives traces in pre-blast contamination scenarios using optimised wet swab sampling

Explosives trace residues on surfaces are utilized for forensic investigations and to check suspicious objects using technical and non-technical detection procedures. Data about expected trace contaminations after the handling of explosives and how they relate to a potential chemical background help to determine requirements and limitations of detection procedures. Often swab sampling is used for the analysis of surface contaminations. A wet swab sampling procedure was optimized for PETN, TNT and ammonium nitrate. Good results were achieved using PU-foam swabs wetted with a mixture of acetonitrile/water (90/10). The swabs were subsequently solvent extracted and analysed using LC-triple-quad-MS and ion-chromatography. The swabs were applied for the determination of explosives residues in a mock-up car scenario simulating one person loading the car with explosives followed by a short car travel by the same person. As test substances an ANFO, TNT and plasticized PETN were used. Additional samples were taken from indoor installations regularly used for the training of explosive detection dogs as well as other objects and buildings close by. The surface contaminations found in the investigated scenario ranged from the ng to the µg scale. The results indicate that simple cleaning procedures might not sufficient to remove explosives contaminations in training settings for explosives detection dogs. A significant spreading of explosive traces beyond direct contact with the person handling the explosive or with the explosive itself was not observed.

Вплив бета-цифлутрину на репродуктивну функцію самиць щурів Wistar Hannover

Beta-cyfluthrin is the 2nd generation pirethroid, that is used as a broad-spectrum insecticide. The effect of the original beta-cyfluthrin molecule on reproductive function has not been studied. The assessment of possible reproductive toxicity of betacyfluthrin was based on the results of themulti-generational rat studyof cyfluthrin. Aim. To investigate the effect of beta-cyfluthrin on the reproductive function of femaleWistar Hannover rats during gametogenesis. Materials and Methods. Beta-cyfluthrin was administered per os 5 times a week by gavage in the form of an aqueous emulsion to two groups of females (20 animals each) in doses 3 and 30 mg/kg bw/day during 9 weeks. Control animals received distilled water with the addition of an emulsifier. In parallel with the control and experimental females, intact males were contained for mating. The estrous cycle was studied in female rats in the last 2 weeks of exposure to beta-cyfluthrin. After the period of exposure, the experimental females were mated with intact males for no longer than 3 weeks. On the 20th day of gestation, the experimental rats were euthanized to assess reproductive parameters. Results. The decrease in the body weight gain at a dose of 30 mg/kg establishes the general toxic effect of beta-cyfluthrin on female rats. It was also installed, that the test substance affects the estrous cycle of females, namely the duration of the proestrus stage, which received the maximum dose. The dose of 3 mg/kg bw/day had no effect on the reproductive function of females and no statistically significant changes were found between the experimental and control groups. Conclusion. Based on the results of the gonadotoxicity study, it can be concluded that beta-cyfluthrin has a general toxic effect and reproductive toxicity at a dose of 30 mg/kg bw/day. The no-observed-effect level (NOEL) was set at 3 mg/kg bw/day. Keywords: beta-cyfluthrin, pirethroid, reproductive function, estrous cycle, rats Wistar Han.

Assessing the impact of different solvents in the bacterial reverse mutation test.

The bacterial reverse mutation test is essential for identifying the mutagenic potential of chemicals. The solubility of the test substance is vital for achieving the recommended assay concentration. Preferred solvents like dimethyl sulfoxide and water are chosen for their compatibility and historical data. Selecting a compatible solvent with Salmonella typhimurium and Escherichia coli WP2 uvrA strains, considering a maximum cytotoxic concentration or the limit of 5 mg/plate, can be challenging. This study assessed various solvents, including N,N-dimethylformamide, acetone, acetonitrile, ethyl acetate, 95% ethanol, diethylene glycol monomethyl ether, methanol, P-dioxane, tetrahydrofuran, and dimethylacetamide, as alternative solvents in the AMES test. Results showed all solvents, except tetrahydrofuran, were compatible at concentrations up to 100 μL/plate or more, as they did not inhibit S9 enzymes, bacterial growth, or alter bacterial revertant colony counts, making them suitable for the bacterial reverse mutation test.

Comparative bioactivity analysis of latex and water extract of Euphorbia gaillardotii: chemical profile, antioxidant capacity, effects on water quality, in vitro antimicrobial-cytotoxic activities, and in silico molecular docking studies.

The genus Euphorbia, belonging to the family Euphorbiaceae, represents a significant ethnobotanical heritage due to the diverse bioactive properties exhibited. In this study, the phytochemical composition and biological activities of latex and aerial parts of the water extract of Euphorbia gaillardotii were investigated. Phytochemical analyses were performed using gas chromatography-mass spectrometry and high-performance liquid chromatography techniques and total antioxidants, phenolics, sugars, organic acids, and aroma components were quantitatively determined. The effects of the test compounds on physicochemical parameters in aqueous media were evaluated by electrical conductivity, dissolved oxygen concentration, and pH measurements. Antimicrobial activity was evaluated on Gram-positive and Gram-negative bacteria and yeast strains using the disk diffusion method. The cytotoxic activity on the MCF-7 human breast cancer cell line was measured spectrophotometrically using the Cell Counting Kit-8 proliferation/apoptosis detection kit. The results showed that E. gaillardotii latex and aerial parts water extract significantly affected the physicochemical parameters of aqueous media, especially at high concentrations. The test substances displayed antimicrobial activity, with the latex-impregnated disks demonstrating larger inhibition zones than the aerial parts extract. The results showed that both the latex and extract treatments exhibited concentration-dependent effects on MCF-7 cell viability (P < 0.001). Furthermore, in silico docking analyses revealed a robust binding affinity of succinic acid, the most prevalent bioactive compound in the extract, towards the B-cell lymphoma 2 (Bcl-2) molecule, with a binding energy of -6.16 kcal/mol. This may be associated with the observed cytotoxicity. These results suggest that E. gaillardotii may be a valuable source for potential pharmacological applications.

Enhanced tolerance to narcosis in starved Daphnia magna neonates

Abstract Guidelines for ecotoxicity testing with Daphnia magna specify particular feeding protocols during the exposure, yet standardization for preexposure feeding remains ambiguous despite its recognized significance in affecting organismal metabolic capacity and tolerance. This ambiguity may contribute to disparate responses and heightened uncertainty in determining the effect concentrations of test chemicals, particularly those inducing metabolic effects through narcosis. Here, we address this gap through a three-step dose-response experiment with neonates of D. magna subjected to two alternative feeding regimes in the preexposure phase: starved and moderately fed during the first 24 hr after birth. Following this treatment, the daphnids were exposed to narcosis-inducing substances (polycyclic aromatic hydrocarbons; PAHs) for 72 hr before being transferred to clean media with algal food ad libitum for a 48 hr recovery phase. Daphnid survivorship, individual protein content, and body size at the end of each experiment phase—pre-exposure, postexposure and postrecovery—were compared between the treatments. Significant treatment effects were observed, including lower and less variable protein content in the starved daphnids entering the PAH exposure phase, yet higher survivorship and greater recovery potential in these daphnids compared with the fed individuals. Our findings underscore the importance of early-life food access and advocate for mandatory reporting of pre-exposure feeding regimes, particularly when testing substances acting via nonpolar narcosis.

The transgenic MutaMouse hepatocyte mutation assay in vitro: mutagenicity and mutation spectra of six substances with different mutagenic mechanisms

Mutagenicity testing is a component of the hazard assessment of industrial chemicals, biocides, and pesticides. Mutations induced by test substances can be detected by in vitro and in vivo methods that have been adopted as OECD Test Guidelines. One of these in vivo methods is the Transgenic Rodent Assay (TGRA), OECD test guideline no. 488. An analogous in vitro TGRA has been described, but experience with this test method is limited. In this study, six in vivo TGRA positive mutagens were tested in the in vitro TGRA based on primary MutaMouse hepatocytes. In addition to the functional read-out of the lacZ reporter gene, induced mutations were analysed by next-generation sequencing (NGS). Five of the six in vivo TGRA positive mutagens (N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), mitomycin C (MMC), benzo[a]pyrene (B[a]P), and azathioprine (AZA), but not cyproterone acetate) mutated the lacZ gene in vitro. NGS identified mutations which matched the mutagenic mechanisms described in the literature. The alkylating agent ENU induced a greater proportion of A:T to T:A transversions than did the other alkylating agent, EMS, whereas EMS increased smaller deletions (1-4 bp). G:C to T:A transversions accounted for the majority of mutations identified after treatments with MMC and B[a]P, both of which form monoadducts at the guanine N2 position. AZA induced mainly G:C to A:T transitions, explained by the structural similarity of one of its metabolites to guanine. An increased proportion of mid-size changes (0.3-2.5 kb) was detected only for the crosslinking mutagen MMC. The in vitro TGRA based on primary MutaMouse hepatocytes is a promising in vitro assay for the assessment of mutation induction, reflecting many aspects of the corresponding in vivo TGRA and allowing for mutation spectra analysis to evaluate the induced mutations.

Comprehensive digital documentation of teratology studies.

One of the main endpoints for the evaluation of Developmental and Reproductive Toxicology (DART) studies is the determination of potential effects of a test substance on the skeleton during foetal development. In the course of a DART study according to the OECD 414 guideline, 400-500 gestational day 20-old (GD20), alizarin red and alcian blue-stained (ARAB) rat foetuses have to be assessed by a teratology expert, which is a time consuming and sub-optimally documented process. We have developed a method which allows for a standardised, comprehensive, quick and easy to perform, head-to-toe digital documentation of ARAB-stained GD20 rat foetuses. The method consists of the acquiring a video-stream or a time lapse-recording of foetuses, which for this purpose are axially rotated in a custom-made device. The obtained digital material is suitable for the complete visual inspection of stained foetal specimen from all sides in great detail, and thus can replace the conventional inspection under a binocular microscope, and might render the archiving of such specimen unnecessary.

Single oral toxicity assessment and phytochemical analysis of the Ficus erecta Thunb. Leaves extract.

Ficus erecta Thunb. is a traditional medicinal plant used to treat various diseases in Asian countries. We recently reported the potential of the ethanol extract of F. erecta Thunb. leaves (EEFE) as a source of functional foods for cognitive improvement. However, there is insufficient scientific evidence supporting the safety of EEFE. In this study, we performed an acute oral toxicity test using Sprague-Dawley rats following Good Laboratory Practice regulations to assess the possible toxicity of EEFE. After single oral administration of EEFE at a maximum dose of 5000mg/kg, the mortality, clinical signs, body weight changes, and gross findings were monitored for 2 weeks. No mortality was observed, and EEFE had no adverse effects on body weight or gross findings in all EEFE-administered rats. On the first day after administration, EEFE-colored stool was observed in both male and female groups. However, these symptoms were considered to be transient, caused by the color of the test substance. Overall, the approximate lethal dose of EEFE was determined to be over 5000mg/kg in the acute toxicity test. Additionally, we established and validated a high-performance liquid chromatography-diode array detector-based analytical method for the quality control of EEFE using rutin as a marker compound. The method showed excellent linearity, accuracy, repeatability, and precision. The findings of this study could serve as fundamental data for ensuring the safety and standardization of EEFE.

Irritation Test of Cream Preparations Made from Purple Cabbage Extract Against Healthy Skin Using The Path Test Method

Testing for skin irritation caused by new substances is a critical component of safety evaluation procedures. Pharmaceutical formulations and active ingredients intended for topical application have the potential to cause skin irritation, necessitating thorough safety assessments prior to public distribution. This study aimed to evaluate the primary irritation potential of a purple cabbage extract cream using the patch test method on healthy older adults. The experimental research involved 18 volunteers, who were administered 0.625% purple cabbage extract cream and a placebo preparation without the test substance. The test materials were applied twice daily over a period of 72 hours. Observations were conducted at baseline (0 hours) and at 24, 48, and 72 hours post-application. The degree of skin irritation was then calculated based on the recorded data. The results indicated a primary irritation index of 0.1, suggesting that the purple cabbage extract cream did not cause skin irritation. It can be concluded that the purple cabbage extract cream is safe for skin application in the tested population. Future research should explore the effects of purple cabbage cream on different skin locations, as skin sensitivity can vary across different areas of the body.

A study for the genotoxicity assessment of substances containing probiotic candidates in the in vitro TK6 cell micronucleus test: Influence of low pH conditions and antibiotic supplementation on the test results

BackgroundWhen assessing the genotoxicity of substances containing probiotic candidates, such as lactic acid-producing bacteria, using the in vitro micronucleus test (MNT), bacterial growth in the test medium may reduce the pH of the medium. The low medium pH is known to induce cytotoxicity and false-positive results. In the TK6 cell system, it is difficult to completely remove the bacteria from the medium by washing post-treatment, leading to bacterial growth during the recovery period in the short-term treatment. In the present study, the low pH range yielding false positives in the TK6 cell MNT was investigated using media supplemented with acetic, lactic, or formic acids, which are non-genotoxic bacterial metabolites. Additionally, to suppress the bacterial growth during the recovery period using antibiotics, i.e., penicillin/streptomycin (P/S), gentamicin sulfate (GM), and amphotericin B (AP), the maximum applicable concentrations of them that did not affect TK6 cell growth or micronucleus induction were determined. Then, we conducted an MNT using a substance containing live lactic acid-producing bacteria to verify the effectiveness of the antibiotics.ResultsAcetic, lactic, and formic acids induced micronuclei in TK6 cells (false positive) at an initial pH of ≤ 6.2 and ≤ 6.0 in 3 h treatment with and without S9 mix, respectively, and of ≤ 6.7 in the continuous treatment. Media supplemented with P/S, GM, and AP did not affect TK6 cell growth or micronucleated cell frequencies in the negative and positive controls ≤ 400 unit/mL-400 µg/mL, ≤ 250, and ≤ 20 µg/mL, respectively. In an MNT with fermented milk containing live lactic acid-producing bacteria, supplementation with P/S or GM to media for the recovery cultures suppressed the bacterial growth, decreasing pH, and cytotoxicity.ConclusionThis study revealed the low pH ranges yielding false positives in the TK6 cell MNT under short-term and continuous treatment conditions. These values will serve as references for interpreting the biological relevance of results. Under short-term treatment, optimal antibiotic supplementation in recovery cultures suppressed bacterial growth in the test substance and prevented the decrease in pH that could yield false positives. This approach might be useful for evaluating the genotoxicity of test substances containing probiotic candidates using the MNT.

Development of a Class A/B Hybrid GPCR System for the Proximity-Assisted Screening of GPCR Ligands.

Class A G protein-coupled receptors (GPCRs) are key mediators in numerous signaling pathways and important drug targets for several diseases. A major shortcoming in GPCR ligand screening is the detection limit for weak binding molecules, which is especially critical for poorly druggable GPCRs. Here, we present a proximity-based screening system for class A GPCRs, which adopts the natural two-step activation mechanism of class B GPCRs. In this approach, class A/B chimeras with the extracellular domain of the class B receptor CRF1R grafted to the transmembrane domain of target class A receptors are stimulated with hybrid ligands. These ligands contain a high-affinity peptide derived from CRF, which recruits the hybrid ligands to the engineered target GPCR, dramatically increasing the local concentration of the test substances. We exemplified this method for neurotensin receptor 1 (NTR1) and endothelin receptor B (ETB), two important class A GPCR drug targets for pulmonary arterial hypertension or psychological disorders and neurodegenerative diseases. We observed >20× activity enhancement by the directed proximity approach, enabling the detection of weakly activating sequences that would have otherwise remained undetected. Our approach allows to probe GPCR activation in the membrane of living cells and may be especially useful for GPCRs for which it has been difficult to generate small drug-like molecules.

The role of substance assessment tools in treatment planning and monitoring: a narrative review

Psychoactive drugs including alcohol and tobacco products can be considered as substances that lead to various negative physical and psychological consequences. On the other hand, socioeconomic deterioration also comes along with harmful substance usage. Substance addiction is a complex condition characterised by a compulsive and harmful pattern of substance use despite the adverse consequences it may comprise, and the risk of developing substance addiction is influenced by a combination of genetic, environmental, and psychological factors and relapse is the recurrence of substance use after a period of abstinence or successful treatment. Hence, healthcare professionals, educational experts, and legal officers are required to get a broader idea regarding the factors associated with substance use. Assessment tools are the most efficient way of identifying the associated factors of substance use, customising specific treatment plans, and monitoring the progress of addiction intervention. By implementing modifications and enhancements to the pre-existing tools, researchers and clinicians able to improve the accuracy of participant responses and findings, leading to a more precise identification of causal links within the context of substance use disorders. The primary focus of this study lies in the comprehensive review of the Alcohol Use Disorder Identification test, Michigan Alcohol Screening Test, Alcohol Dependency Scale, Self-administered Alcoholism Screening Test, Drug Abuse Screening Test, Tobacco, Alcohol, Prescription Medication, and Other Substance Use Tool, Alcohol, Smoking, and Substance Involvement Screening Test, Substance and Choice Scale, Screening to Brief Intervention, Adolescent Alcohol and Drug Involvement Scale, and Severity of Dependence Scale play crucial roles in identifying substance use disorders, assessing severity, guiding treatment decisions, and facilitating interventions to promote recovery and well-being.

DEVELOPMENT OF A PROGRAM FOR CALCULATING THE PREDICTED OPERATION TIME OF GAS FILTERS

Objective: Development of software to calculate the predicted duration of protective action of gas filters for gas masks and respirators under specific operating conditions. Methods: The program for determining the predicted breakthrough time of gas filters was created using the integrated development environment Visual Studio Code. The algorithm embedded in the program is based on the mathematical equations of Wheeler-Jonas and the conversion of concentrations of chemical compounds into different dimensions. The real breakthrough time of gas filters was established on a laboratory stand for determining the absorption capacity, which meets the requirements of DSTU EN 14387:2021, and also provides the opportunity to use four test substances during testing, such as cyclohexane, sulfur dioxide, hydrogen sulfide or ammonia. Results: A software package was developed to calculate the predicted breakthrough time of gas filters for gas masks and respirators under specific operating conditions. It consists of four modules: initial data input, memory, output and visualization of information, and calculation of breakthrough time of gas filters. An algorithm for calculating the predicted level of filters breakthrough time was developed, which includes five steps: determining the hazard, concentration of harmful substances, required respirator protection class, humidity and air temperature, mode of operation; entering initial data for calculation; preliminary selection of a filter from the specified catalog located in the memory module; calculation of filter capacity and analysis with permissible values of the chemical substance with the calculation of breakthrough time; visualization of results and their storage in the memory module. Novelty: Establishing the dependency of the predicted breakthrough time of FRPA-G A1, B1, K1 filters on varying concentrations of chemicals in the air and the type of work performed, which allowed identifying a five-fold reduction in filter operation time during heavy work compared to light work. Practical Value: Results obtained by comparing the predicted breakthrough time using the developed software product with the programs of personal protective equipment manufacturers showed an error not exceeding 10%, due to the lack of accounting for uneven wear of the filter filler across its surface. Keywords: adsorption, gas filter, software, breakthrough time, substance concentration.

Unveiling the anxiolytic and analgesic effects of citronellal in Swiss mice: in vivo and in silico insights into COX and GABAA receptor pathways.

This study aims to evaluate the analgesic and anxiolytic effects of citronellal (CTL) in Swiss mice using two new sensitive and economic mouse models along with possible molecular mechanisms through in silico studies. For this, we employed marble displacement and spiked-field apparatus to check the anxiolytic and analgesic effects, respectively. In silico studies were done against cyclooxygenase (COX) enzymes and GABAA receptor subunits. Findings suggest that the test sample CTL significantly reduced the number of marbles displaces (NMD), square crosses (NSC), paw massages (NPM), and escaping attempts (NEA) in animals than the control group. CTL exhibited better effects in the perforated-field study compared to the reference drug diclofenac sodium. In both cases, CTL (200mg/kg) significantly reduced the test parameters when combined with the standard drugs (diazepam or diclofenac sodium). In in silico studies, CTL showed binding affinities of - 5.5, - 5.2, - 4.8, and - 4.8kcal/mol with the COX1, COX2, and GABAA receptors (α2 and α3 subunits), respectively. Taken together, CTL may exert anxiolytic- analgesic-like effects on mice, possibly through the GABAA receptor and COXs interaction pathways. These new tools might be used to check the anxiolytic and analgesic properties of a wide variety of test substances.

Antifungal activity of thiadiazole derivative in experimental candidiasis in mice

Introduction. In the modern world, more attention is paid to the problem of increasing cases of mycosis. In October 2022, the World Health Organization (WHO) published the first fungal priority pathogens list which constitute the greatest danger to human health. Among mycotic infections, one of the leading positions in frequency is occupied by various forms of candidiasis. The WHO has classified Candida albicans as a critical priority group. Therefore, the search for new effective antimycotics is relevant. A new derivative of 1,3,4-thiadiazole was synthesized at Saint-Petersburg State Chemical and Pharmaceutical University. In vitro experiments, it was shown that it has a wide spectrum of antifungal activity in effectiveness comparable to voriconazole.Aim. To evaluate the antifungal activity of 2-[(1Z)-1-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)methyl]-3,5-diphenyl-1,3,4-thiadiazole-3-ium (thiadiazole derivative, TD) chloride drug substance in non-invasive bowel candidiasis and acute septicemia (sepsis) models caused by C. albicans.Materials and methods. Simulations of non-invasive intestinal candidiasis were performed by infecting BALB/c mice with candida with free access to a drinking bottle containing C. albicans yeast cells suspension. Previously, dysbiosis was caused in animals by adding a solution of antibiotics (vancomycin, clindamycin, gentamicin) in drinking water. Simulations of C. albicans-induced acute septicemia were performed by infecting BALB/c mice with intravenous yeast culture suspension. After the mice were infected, the test substance and the comparators Voriconazole and Fluconazole were administered intragastrically once daily. At the end of the experiment, the number of fallen animals were estimated and microbiological examination of internal organs was carried out.Results and discussion. It was shown that the number of yeast cells secreted from the large and small intestines in mice with non-invasive intestinal candidiasis after administration of a new thiadiazole derivative at all doses tested (1, 5, 10 mg/kg) were significantly reduced compared with control group No. 6 without treatment. At 10 mg/kg, the test substance demonstrated anticandidosis effects at the level of comparators drugs, Voriconazole and Fluconazole. In mice with septicemia, mortality also depended from the dose of the thiadiazole derivative, but only 10 mg/kg proved to be an effective dose.Conclusion. The study revealed that the new thiadiazol derivative 2-[(1Z)-1-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene) methyl]-3,5-diphenyl-1,3,4-thiadiazole-3-ium showed antifungal activity against Candida albicans in the model of non-invasive intestinal candidiasis and the model of septicemia (sepsis). At 10 mg/kg, TD was found to be active at the level of comparators, Voriconazole and Fluconazole.

A suitable solvent for adsorption of poorly water-soluble substances onto silica gel in a ready biodegradability test.

When a test substance is poorly water-soluble, it can be adsorbed onto silica gel to facilitate dispersibility in a ready biodegradability test. To uniformly adsorb the test substance onto silica gel, the substance is dissolved in a solvent and then mixed with the silica gel. It is desirable for the solvent to completely evaporate afterward. In this study, we identified n-hexane as a suitable solvent for this purpose. Furthermore, through fluorescence observation, we revealed that the test substance adsorbed onto the silica gel adhered to activated sludge flocs. This is thought to improve contact between the test substance and microorganisms, thereby accelerating biodegradation.

Comparative evaluation of trimethylated α-, β-, and γ-cyclodextrins as optimal dispersants for ready biodegradability testing of poorly water-soluble substances.

We investigated whether various modified cyclodextrins (CDs) and emulsifiers could be applied as dispersing agents in ready biodegradability tests of poorly water-soluble substances. Trimethylated α-, β-, and γ-CDs and partially methylated β-CD were not biodegraded in the test period but accelerated the biodegradation of octabenzone and anthraquinone. The process by which trimethylated α-, β-, and γ-CDs enhance the biodegradation of test substances has been partially uncovered. These CDs create inclusion complexes with the substances, which then coalesce into larger aggregates. These aggregates disperse throughout the testing medium and attach to clusters of activated sludge, known as flocs. This close contact with the sludge speeds up the breakdown of the hydrophobic substances being tested.

Design of optimal concentrations for in vitro cytotoxicity experiments

Concentration-dependent cytotoxicity experiments are frequently used in toxicology. Although it has been reported that an adequate choice of concentrations improves the quality of the statistical inference substantially, a recent literature review of three major toxicological journals has shown that the corresponding methods are rarely used in toxicological practice. In this study the performance of different sets of concentrations, also called designs, are analyzed, while the overall goal is to promote the advantages of optimal design procedures and to present a user-friendly guideline for planning new cytotoxicity concentration-response experiments. We compare the frequently used log-equidistant design to a Bayesian design, which is constructed by methods of optimum design theory. Using both a dense data set of concentration-cytotoxicity data of valproic acid (VPA) and regular assay data of 104 substances, the performance of the different designs is analyzed in two scenarios, where detailed previous knowledge on VPA is available or not. The results show that it is critical to apply a specific design strategy to determine optimal concentrations for cytotoxicity testing. In particular, the Bayesian design technique with and without incorporating pre-existing knowledge of a specific test substance resulted in a more precise statistical inference than the other used designs. Finally, we present a guideline for upcoming experiments and an accessible user-friendly Shiny app (see http://shiny.statistik.tu-dortmund.de:8080/app/occe).

Hypotensive Effect of an Aqueous Extract of Leaves of Senna alata (L.) ROXB (Caesalpiniaceae), a Plant Known to be Antihypertensive, in Rabbits

The leaves of Senna alata (L.) ROXB (Caesalpiniaceae) are used in traditional African medicine mainly to treat arterial hypertension. The aim of the present study was to evaluate the hypotensive effects of an aqueous extract of leaves of Senna alata on arterial hypertension in rabbits. A qualitative phytochemical study was conducted to determine the various secondary compounds contained in this extract. Using a Ludwig manometer, consisting of a U-shaped tube containing mercury surmounted by a writing stylus, the blood pressure of rabbits was recorded on smoked paper wound around a cylinder rotating at constant speed. The doses of test substances are administered to the rabbits via its exposed saphenous vein. At doses ranging from 10 to 25 mg/kg wb, aqueous extract of Senna alata leaves (EAqSa) induced a dose-dependent hypotension comparable to that of acetylcholine. The inhibition of this hypotension in the presence of doses of atropine, an antagonist of muscarinic cholinomimetic substances, of between 10-6 and 10-3mg/kg bw suggests that EAqSa contains muscarinic cholinomimetic substances. These substances would be responsible for its hypotensive effect and could be alkaloids and/or flavonoids found in this extract. These results support the use of Senna alata in the treatment of hypertension in traditional medicine.

Substance depletion of volatile monoterpenes – A confounding factor for toxicity testing in the Ames fluctuation test

In in vitro toxicology, reported test results are typically based on nominal concentrations, i.e., the calculated amounts of a substance added to a defined volume of the test system. Consequently, if a test system does not respond to a certain endpoint, the assay is interpreted as negative and the test substance is deemed to exert no toxicity at the tested nominal concentration. However, depending on the physicochemical properties of the test substance and assay setup, the actual exposure may differ widely from nominal concentrations due to different depletion processes. (R)-(+)-Limonene (RLIM), β-myrcene (βMYR) and linalool (LIN) are naturally occurring terpenes that are permitted as flavoring agents in the European Union without limitations based on their low toxicity. Nevertheless, their hydrophobicity and high volatility classifies them as difficult to test chemicals, which has not been considered in previous in vitro tests. To exclude possible false negative results, in the present study, we assessed the cytotoxic and mutagenic potential of the latter substances toward Salmonella Typhimurium in the Ames fluctuation test using different incubation setups to minimize possible substance losses due to sorption or volatilization. Actual substance concentrations during incubation were verified analytically at different time points via headspace gas chromatography-mass spectrometry (HS-GC-MS). Possible substance depletion due to sorption to well-plate material or volatilization was minimized using a polystyrene-free and headspace-free incubation setup, respectively. The results showed complete volatilization of the monoterpenes RLIM and βMYR in the conventional Ames fluctuation test, which may confound mutagenicity testing. The headspace-free incubation setup greatly improved substance exposure and showed cytotoxicity in low micromolar concentrations, but no signs of mutagenicity were observed.