Considerations for the development of guidance on dose level selection for developmental and reproductive toxicity studies.

In 2022, the European Chemicals Agency issued advice on the selection of high dose levels for developmental and reproductive toxicity (DART) studies indicating that the highest dose tested should aim to induce clear evidence of reproductive toxicity without excessive toxicity and severe suffering in parental animals. In addition, a recent publication advocated that a 10% decrease in body weight gain should be replaced with a 10% decrease in bodyweight as a criterion for dose adequacy. Experts from the European Centre for Ecotoxicology and Toxicology of Chemicals evaluated these recent developments and their potential impact on study outcomes and interpretation and identified that the advice was not aligned with OECD test guidelines or with humane endpoints guidance. Furthermore, data analysis from DART studies indicated that a 10% decrease in maternal body weight during gestation equates to a 25% decrease in body weight gain, which differs from the consensus of experts at a 2010 ILSI/HESI workshop. Dose selection should be based on a biological approach that considers a range of other factors. Excessive dose levels that cause frank toxicity and overwhelm homeostasis should be avoided as they can give rise to effects that are not relevant to human health assessments.

Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos-oxon.

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF-oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2 mg/kg CPF for brain and 0.5 mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12 h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1 mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1 mg/kg (acute) and 0.01 mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10 mg/kg (adult) for acute dosing or 0.5 mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

[Flow cytometry evaluation of the rhesus monkey cellular immunity following the Zaire ebolavirus (Filoviridae; Ebolavirus: Zaire ebolavirus) experimental infection].

INTRODUCTION: The outbreaks of the Zaire ebolavirus (ZE) disease (ZED) that have arisen in the last decade determine the need to study the infection pathogenesis, the formation of specific immunity forming as well as the development of effective preventive and therapeutic means. All stages of fight against the ZED spread require the experimental infection in sensitive laboratory animals, which are rhesus monkeys in case of this disease .The aim of the study is to evaluate the rhesus monkey cellular immunity following the ZE experimental infection by the means of flow cytometry (cytofluorimetry). MATERIAL AND METHODS: Male rhesus monkeys were intramuscularly infected by the dose of 15 LD50 (dose of the pathogen that causes 50% mortality of infected animals) of the ZE, the Zaire strain (ZEBOV). Levels of 18 peripheral blood lymphocyte populations of the animals before the ZE experimental infection and at the terminal stage of the disease were assessed using flow cytometry. RESULTS AND DISCUSSION: The certain changes in the levels of the lymphocyte populations were observed following infection, indicating simultaneous activation and suppression of the immune system during ZED. The increase in content was observed for T-lymphocytes, T-helper and cytotoxic T-lymphocytes expressing the corresponding markers of early activation. The decrease was recorded for T-lymphocytes and double-positive T-lymphocytes expressing corresponding markers of late activation, as well as natural killer cells expressing CD8 (p < 0.05). CONCLUSION: For the first time in the Russian Federation, the rhesus monkey cellular immunity before and after the ZE experimental infection was assessed using flow cytometry.

Evaluation of the safety and nutritional equivalence of a genetically modified cottonseed meal in a 90-day dietary toxicity study in rats.

Meal prepared from Cry1F/Cry1Ac transgenic/genetically modified cottonseed (WIDESTRIKE Insect Protection, hereafter referred to as WIDESTRIKE) was compared to cottonseed meal prepared from four conventionally bred lines of cotton (three commercial non-transgenic line controls (PHY72, PHY78 and 98M-2983), and a near isoline non-transgenic control (PSC355) in a 90-day dietary study to evaluate safety and nutritional equivalence. Diets were formulated with 10% WIDESTRIKE cottonseed meal equivalent to 7,235 mg/kg/day for males and 7,935 mg/kg/day for females. Animals were evaluated by cage-side and hand-held detailed clinical observations, body weight, and feed consumption. Functional tests, motor activity and ophthalmic examinations were conducted pre-exposure and prior to study termination. Standard hematology, clinical chemistry, prothrombin time and urinalysis parameters were evaluated. All rats had a complete necropsy and selected organs were weighed. Histopathologic examinations were performed on all rats fed the diets containing the near isoline non-transgenic control or WIDESTRIKE. Following 90 days of feeding, no adverse effects were observed during the conduct of clinical observations or in any of the parameters measured in this study. This study demonstrated that rodent diets prepared with 10% cottonseed meal from WIDESTRIKE cottonseeds do not produce any untoward effects and are nutritionally equivalent to cottonseed meals prepared from other, non-transgenic cottonseeds.

Safety and immunogenicity of GamEvac-Combi, a heterologous VSV- and Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia.

Ebola hemorrhagic fever, also known as Ebola virus disease or EVD, is one of the most dangerous viral diseases in humans and animals. In this open-label, dose-escalation clinical trial, we assessed the safety, side effects, and immunogenicity of a novel, heterologous prime-boost vaccine against Ebola, which was administered in 2 doses to 84 healthy adults of both sexes between 18 and 55 years. The vaccine consists of live-attenuated recombinant vesicular stomatitis virus (VSV) and adenovirus serotype-5 (Ad5) expressing Ebola envelope glycoprotein. The most common adverse event was pain at the injection site, although no serious adverse events were reported. The vaccine did not significantly impact blood, urine, and immune indices. Seroconversion rate was 100 %. Antigen-specific IgG geometric mean titer at day 42 was 3,277 (95 % confidence interval 2,401-4,473) in volunteers immunized at full dose. Neutralizing antibodies were detected in 93.1 % of volunteers immunized at full dose, with geometric mean titer 20. Antigen-specific response in peripheral blood mononuclear cells was also detected in 100 % of participants, as well as in CD4+ and CD8+ T cells in 82.8 % and 58.6 % of participants vaccinated at full dose, respectively. The data indicate that the vaccine is safe and induces strong humoral and cellular immune response in up to 100 % of healthy adult volunteers, and provide a rationale for testing efficacy in Phase III trials. Indeed, the strong immune response to the vaccine may elicit long-term protection. This trial was registered with grls.rosminzdrav.ru (No. 495*), and with zakupki.gov.ru (No. 0373100043215000055).

Acute toxicologic and neurotoxic effects of inhaled 1,2-dichloroethane in adult Fischer 344 rats.

Acute toxicologic and neurotoxic effects were evaluated in Fischer 344 rats exposed to 0, 50, 200, 600, or 2000 ppm 1,2-dichloroethane (ethylene dichloride; EDC) for 4 h or 0, 50, 100 or 150 ppm for 8 h. Neurobehavioral and neuropathologic effects were assessed using a functional observational battery (FOB; baseline, days 1, 8, and 15), and by light microscopy, respectively. Acute toxicologic effects were assessed by bronchoalveolar lavage (BAL) and histopathology of the respiratory tract and selected target organs. Neurobehavioral effects consistent with central nervous system (CNS) depression were present at concentrations >200 ppm and were restricted to day 1. There were no neuropathologic changes in the CNS, however, olfactory epithelial regeneration 15 days after exposure to > or = 200 ppm was observed. The no-observed-effect concentration (NOEC) for behavioral neurotoxicity was 200 ppm EDC for 4 h. There were no effects on BAL parameters in any exposure group. Exposure to 2000 ppm EDC altered adrenal gland, kidney, and liver weights, and resulted in morphologic alterations in the kidney and liver. Degeneration/necrosis of the olfactory epithelium was observed at > or = 200 ppm for 4 h and > or = 100 ppm for 8 h. Based on olfactory epithelial degeneration/necrosis, the most sensitive indicator of toxicity in this study, the overall NOEC was 50 ppm EDC for up to 8 h in rats.

Double-hadron leptoproduction in the nuclear medium.

The first measurements of double-hadron production in deep-inelastic scattering within the nuclear medium were made with the HERMES spectrometer at DESY HERA using a 27.6 GeV positron beam. By comparing data for deuterium, nitrogen, krypton, and xenon nuclei, the influence of the nuclear medium on the ratio of double-hadron to single-hadron yields was investigated. Nuclear effects on the additional hadron are clearly observed, but with little or no difference among nitrogen, krypton, or xenon, and with smaller magnitude than effects seen on previously measured single-hadron multiplicities. The data are compared with models based on partonic energy loss or prehadronic scattering and with a model based on a purely absorptive treatment of the final-state interactions. Thus, the double-hadron ratio provides an additional tool for studying modifications of hadronization in nuclear matter.

Measurement of the tensor structure function b1 of the deuteron.

The Hermes experiment has investigated the tensor spin structure of the deuteron using the 27.6 GeV/c positron beam of DESY HERA. The use of a tensor-polarized deuteron gas target with only a negligible residual vector polarization enabled the first measurement of the tensor asymmetry A(d)zz and the tensor structure function b(d)1 for average values of the Bjorken variable 0.01< <0.45 and of the negative of the squared four-momentum transfer 0.5 GeV2 < <5 GeV2. The quantities A(d)zz and b(d)1 are found to be nonzero. The rise of b(d)1 for decreasing values of x can be interpreted to originate from the same mechanism that leads to nuclear shadowing in unpolarized scattering.

Single-spin asymmetries in semi-inclusive deep-inelastic scattering on a transversely polarized hydrogen target.

Single-spin asymmetries for semi-inclusive electroproduction of charged pions in deep-inelastic scattering of positrons are measured for the first time with transverse target polarization. The asymmetry depends on the azimuthal angles of both the pion (phi) and the target spin axis (phi(S)) about the virtual-photon direction and relative to the lepton scattering plane. The extracted Fourier component sin((phi+phi(S))(pi)(UT) is a signal of the previously unmeasured quark transversity distribution, in conjunction with the Collins fragmentation function, also unknown. The component sin((phi-phi(S)(pi)(UT) arises from a correlation between the transverse polarization of the target nucleon and the intrinsic transverse momentum of quarks, as represented by the previously unmeasured Sivers distribution function. Evidence for both signals is observed, but the Sivers asymmetry may be affected by exclusive vector meson production.

Rapid desilylation of oligoribonucleotides at elevated temperatures: cleavage activity in ribozyme-substrate assays.

Treatment of 2'-O-silyl-oligoribonucleotides with triethylamine trihydrofluoride in DMF at 55 degrees C for 1 h effected complete desilylation. The product was isolated by a single addition of 1-butanol to the reaction mixture. The resulting RNA was found to be identical with that obtained by traditional desilylation methods as analyzed by HPLC, enzyme digest and ribozyme-substrate assays.

Fluorescent dye phosphoramidite labelling of oligonucleotides.

A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.

Overview of purification and analysis of synthetic nucleic acids.

Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

Polyacrylamide gel electrophoresis (PAGE) of synthetic nucleic acids.

Protocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.

Analysis and purification of synthetic nucleic acids using HPLC.

HPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed-phase HPLC allows a high-capacity method for purification, and uses volatile buffer systems that simplify product recovery. Anion-exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed-phase and anion-exchange HPLC, it can be used for oligonucleotides of up to approximately 50 nt in length.

Base composition analysis of nucleosides using HPLC.

In this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed-phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.

Cartridge methods for oligonucleotide purification.

Protocols are given for purification of oligonucleotides by dimethoxytrityl-sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean-up by selective adsorption, and elution on solid-phase media. Many of these products are prepackaged, single-use cartridges or columns filled with affinity or size-exclusion media.

Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay.

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.

Dialkylformamidine protected deoxyadenosine phosphoramidites in oligonucleotide synthesis.

A series of dialkylformamidine protected deoxyadenosine phosphoramidites were prepared for automated, solid-support DNA synthesis. The set of Abz, Gdmf, Cbz, T phosphoramidites gave high purity and high yield oligonucleotides, with rapid and complete deprotection at 65 degrees C in one hour. Different times and temperatures of exposure to concentrated ammonium hydroxide were examined to establish the optimum conditions for deprotection of oligonucleotides.