Good Laboratory Practice Research Articles

The impact of COVID-19 on human reproduction and directions for fertility treatment during the pandemic.

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly, resulting in a pandemic. The virus enters host cells through angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine subtype 2 (TMPRSS2). These enzymes are widely expressed in reproductive organs; hence, coronavirus disease 2019 (COVID-19) could also impact human reproduction. Current evidence suggests that sperm cells may provide an inadequate environment for the virus to penetrate and spread. Oocytes within antral follicles are surrounded by cumulus cells, which rarely express ACE2 and TMPRSS2. Thus, the possibility of transmission of the virus through sexual intercourse and assisted reproductive techniques seems unlikely. Early human embryos express coronavirus entry receptors and proteases, implying that human embryos are potentially vulnerable to SARS-CoV-2 in the early stages of development. Data on the expression of ACE2 and TMPRSS2 in the human endometrium are sparse. Moreover, it remains unclear whether SARS-CoV-2 directly affects the embryo and its implantation. A study of the effect of SARS-CoV-2 on pregnancy showed an increase in preterm delivery. Thus, vertical transmission of the virus from mother to fetus in the third trimester is possible, and further data on human reproduction are required to establish this possibility. Based on analyses of existing data, major organizations in this field have published guidelines on the treatment of infertility. Regarding these guidelines, despite the COVID-19 pandemic, reproductive treatment is crucial for the well-being of society and must be continued under suitable regulations and good standard laboratory practice protocols.

Chitosan-Platelet-Rich Plasma Implants Improve Rotator Cuff Repair in a Large Animal Model: Pivotal Study.

The purpose of this study was to assess the safety and efficacy of chitosan–platelet-rich plasma (PRP) hybrid implants used as an adjunct to surgical rotator cuff repair in a pivotal Good Laboratory Practice (GLP)-compliant study. The infraspinatus tendon was transected in 48 skeletally mature ewes and repaired with a transosseous-equivalent (TOE) technique. In the two treatment groups, a chitosan–PRP solution was injected at the footprint between the tendon and the bone and on top of the repaired site (2 mL or 3 mL doses, n = 12 per group). To further assess chitosan safety, a chitosan–water solution was injected at the same sites (3 mL, n = 12). Outcome measures included Magnetic Resonance Imaging (MRI) assessment and clinical pathology at 3 months and 6 months and histopathology at 6 months. The tendon gap was decreased at 3 months on MRI images and certain histopathological features were improved at 6 months by chitosan–PRP treatment compared to controls. The group treated with chitosan–water was not different from controls. Chitosan–PRP treatment induced no negative effects in the sheep, which suggests high safety. This study provides further evidence on the safety and efficacy of chitosan–PRP for rotator cuff repair augmentation, which could eventually be used in a clinical setting.

Acute and subacute repeated oral toxicity study of fragmented microplastics in Sprague-Dawley rats

Polypropylene (PP) is the second most highly produced plastic worldwide, and its microplastic forms are found in water and food matrices. However, the effects of PP microplastics on human health remain largely unknown. Here, we prepared 85.2 µm-sized weathered PP (w-PP) microplastics by sieving the microplastic particles after fragmentation and accelerated weathering processes. The prepared particles are irregular in shape and no chemical additives including phthalates and bisphenol A were not released in simulated body fluids. Then, the w-PP samples were gavaged to rats for acute and subacute toxicity testing in accordance to the Organization for Economic Co-operation and Development (OECD) test guidelines under good laboratory practice regulations. The highest dose for gavaging to rats was 25 mg/kg bw/day, which was the maximum feasible dose based on the dispersibility of microplastics. Both toxicity testings for w-PP microplastics showed no adverse effects and mutagenicity. Thus, the no observed adverse effect level (NOAEL) of w-PP microplastics is higher than 25 mg/kg bw/day. Furthermore, the w-PP microplastics did not show any skin or eye irritation potentials in the 3-dimensional reconstructed human skin or corneal culture model. The dose of 25 mg/kg of w-PP microplastics is roughly equal to 2.82 × 105 particles/kg, which suggests that human exposure to w-PP microplastics in a real-life situation may not have any adverse effects.

Investigation of Ifosfamide Toxicity Induces Common Upstream Regulator in Liver and Kidney.

Ifosfamide is an alkylating agent, a synthetic analogue of cyclophosphamide, used to treat various solid cancers. In this study, the toxicity of ifosfamide was evaluated using single-and multiple-dose intraperitoneal administration in rats under Good Laboratory Practice guidelines, and an additional microarray experiment was followed to support toxicological findings. A single dose of ifosfamide (50 mg/kg) did not induce any pathological changes. Meanwhile, severe renal toxicity was observed in the 7 and 28 days consecutively administered groups, with significant increases in blood urea nitrogen and creatinine levels. In the tox-list analysis, cholesterol synthesis-related genes were mostly affected in the liver and renal failure-related genes were affected in the kidney after ifosfamide administration. Moreover, interferon regulatory factor 7 was selected as the main upstream regulator that changed in both the liver and kidney, and was found to interact with other target genes, such as ubiquitin specific peptidase 18, radical S-adenosyl methionine domain containing 2, and interferon-stimulated gene 15, which was further confirmed by real-time RT-PCR analysis. In conclusion, we confirmed kidney-biased ifosfamide organ toxicity and identified identically altered genes in both the liver and kidney. Further comprehensive toxicogenomic studies are required to reveal the exact relationship between ifosfamide-induced genes and organ toxicity.

Good Laboratory Practice (GLP) in The European Union

Regulasi GLP (Good Laboratory Practice) mengatur tentang cara bekerja pada laboratorium, pelatihan personel, standar operasional prosedur, software untuk pencatatan data, dokumentasi, dsb. Tujuan utama dari prinsip GLP adalah mengembangkan kualitas dari data pengujian sehingga tiap negara tersebut dapat menghasilkan data pengujian yang andal, menghindari pengujian berulang, serta menghemat waktu. Anggota OECD (Organization for Economic Co-operation and Development) sebanyak 29 negara di seluruh dunia, mencakup Asia-Pasifik, Amerika, serta Eropa. Patuh terhadap GLP dapat meniadakan barier perdagangan antar negara anggota. Prinsip GLP yang diaplikasikan di Uni Eropa sejalan dengan OECD, yakni untuk meningkatkan kualitas data pengujian yang valid. Anggota negara Uni Eropa adalah sebanyak 27 negara. Uni Eropa mengadopsi peraturan GLP pada tahun 1987. Amandemen terkait GLP dilakukan mengikuti OECD pada tahun 1999. Peraturan perundangan Uni Eropa tentang GLP ‘Directive 2004/9/EC’ kemudian menggantikan ‘Directive 88/320/EEC’ menjelaskan tentang tanggungjawab yang diberikan kepada anggota negara Uni Eropa untuk menetapkan otoritas inspeksi GLP pada masing-masing wilayah dan persyaratan Mutual Acceptance of Data (MAD). Pengujian non-klinik pada produk obat sesuai dengan ketentuan GLP mengacu pada OECD serta peraturan perundangan Uni Eropa yang terdiri dari ‘Directive 2004/10/EC’ dan ‘Directive 2004/9/EC’. Meskipun Inggris sudah keluar dari Uni Eropa, namun Mutual Acceptance of Data (MAD) tetap berlaku pada Inggris terhadap negara Eropa lainnya yang sama-sama anggota dari OECD. Otoritas terhadap inspeksi serta audit studi diserahkan pada negara masing-masing anggota.

Scientific and Regulatory Policy Committee Points to Consider: Biological Sample Retention From Nonclinical Toxicity Studies.

Samples of biologic specimens and their derivatives (eg, wet tissues, paraffin-embedded tissue blocks, histology slides, frozen tissues, whole blood, serum/plasma, and urine) are routinely collected during the course of nonclinical toxicity studies. Good Laboratory Practice regulationsand/or guidance specify minimum requirements for specimen retention duration, with the caveat that retention of biologic specimens need not extend beyond the duration of sample stability. However, limited availability of published data regarding stability for various purposes following storage of each specimen type has resulted in confusion, uncertainty, and inconsistency as to the appropriate duration for storage of these specimens. To address these issues, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to review published information, regulations, and guidance pertinent to this topic and to summarize the current practices and rationales for retention duration through a survey-based approach. Information regarding experiences reaccessing biologic specimens and performing sample stability investigations was also collected. Based on this combined information, the working group developed several points to consider that may be referenced when developing or revising sample retention practices. [Box: see text].

A reproductive and developmental toxicity screening study of 1,3-butadiene in Sprague-Dawley rats

1,3 Butadiene (BD) is an industrial intermediate used primarily in product manufacturing with the greatest exposure potential via inhalation. BD was evaluated for reproductive and developmental effects in a Good Laboratory Practice (GLP)-compliant, extended OECD 421 guideline study (completed 2003). Twelve-week old rats (12/sex/dose) were exposed via whole-body inhalation to BD vapor (0, 300, 1500, 6000 ppm) for 6 h/day, 7 days/week, starting 14 days prior to mating through the day prior to euthanasia (total exposures: 83–84 days for F0 males 60–70 days for F0 females). Select F1 offspring (1/sex/litter) were dosed 7 days (postnatal days 21–27 or 28–34), then necropsied. At 1500 and 6000 ppm, treatment-related facial soiling was seen in F0 males and females with decreased body weights/gains in F0 males. F1 males and females exhibited similar effects at 1500 and 6000 ppm. Importantly, the F0 generation had no evidence of altered sperm production, testicular effects, or ovarian atrophy, which were sensitive responses in mice. The no-observed-adverse-effect-level (NOAEL) is 300 ppm due to decreased body weight/gain and facial soiling at 1500 ppm, whereas 6000 ppm serves as a NOAEL for reproductive and developmental endpoints. This study contributes to the weight-of-evidence of differential BD reproductive toxicity in rats and mice.

An estrogen (17α-ethinyl estradiol-3-sulfate) reduces mortality in a swine model of multiple injuries and hemorrhagic shock.

Although 17α-ethinyl estradiol-3-sulfate (EES) reduces mortality in animal models of controlled hemorrhage, its role in a clinically relevant injury model is unknown. We assessed the impact of EES in a swine model of multiple injuries and hemorrhage. The study was performed under Good Laboratory Practice, with 30 male uncastrated swine (25-50 kg) subjected to tibial fracture, pulmonary contusion, and 30% controlled hemorrhage for an hour. Animals were randomized to one of five EES doses: 0 (control), 0.3, 1, 3, and 5 mg/kg, administered postinjury. Subjects received no resuscitation and were observed for 6 hours or until death. Survival data were analyzed using Cox-proportional hazard regression. Left ventricular pressure-volume loops were used to derive preload recruitable stroke work as a measure of cardiac inotropy. Immediate postinjury preload recruitable stroke work values were compared with values at 1 hour post-drug administration. Six-hour survival for the 0, 0.3, 1, 3, and 5 mg/kg groups was 0%, 50%, 33.3%, 16.7%, and 0%, respectively. Following Cox regression, the hazard (95% confidence interval) of death was significantly reduced in the 0.3 (0.22 [0.05-0.93]) and 1 (0.24 [0.06-0.89]) mg/kg groups but not the 3 (0.49 [0.15-1.64]) and 5 (0.46 [0.14-1.47]) mg/kg groups. Mean survival time was significantly extended in the 1 mg/kg group (246 minutes) versus the 0 mg/kg group (96 minutes) (p = 0.04, t test). At 1 hour post-drug administration, inotropy was significantly higher than postinjury values in the 0.3 and 1 mg/kg groups (p = 0.003 and p < 0.001, respectively). Inotropy was unchanged in the 3 and 5 mg/kg groups but significantly depressed in the control (p = 0.022). Administration of EES even in the absence of fluid resuscitation reduces mortality and improves cardiac inotropy in a clinically relevant swine model of multiple injuries and hemorrhage. These findings support the need for a clinical trial in human trauma patients.

Reference value of hematologic, urologic, and organ weights of 2‐ to 4‐year‐old long‐tailed macaques (Macaca fascicularis fascicularis) in the context of toxicological studies

The long-tailed macaque (Macaca fascicularis fascicularis) is an Old World species, which is one among the most commonly used monkeys for pharmaceutical research. However, most of the available background data are not suitable for good laboratory practice (GLP)-regulated drug safety tests because the current reverence value covers fewer indices than necessary. Therefore, in this study, historical data for preclinical safety test were collected and managed. Twenty-five hematology, 20 clinical chemistry, 19 urine analysis, and 16 organ weights were evaluated in a drug safety test of 228 male and 140 female 2- to 4-year-old long-tailed macaques at the Korea Institute of Toxicology under GLP regulations. The absolute and relative count of lymphocyte, basophil, and large unstained cell were higher, whereas neutrophil was lower in male than in female monkeys. In serum biochemistry, IP, GGT, ALP, and TCHO of male were higher than female. Historical data suitable for preclinical safety analysis were determined.

SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of − 3 to − 4 at a concentration of 10–100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

Reproductive toxicity assessment of alkyl dimethyl benzyl ammonium chloride and didecyl dimethyl ammonium chloride in CD® rats.

AimTo determine the potential of alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC) to induce reproductive toxicity in CD® rats in two independent 2‐generation reproduction studies conducted according to Good Laboratory Practices and standardized testing guidelines.Materials and MethodsMale and female rats (parents and offspring) were allowed continual free access to diets containing concentrations of ADBAC (0, 300, 1,000, or 2,000 ppm) or DDAC (0, 300, 750, or 1,500 ppm), beginning with F0 generation adults at 10 weeks prior to breeding.ResultsNo clinical signs of toxicity were observed in parental rats or their offspring in either study. Dietary exposure of parental rats to ADBAC or DDAC at the highest concentrations produced transient decreases in body weight and/or body weight changes with no or minimal corresponding reduction in food consumption. Offspring (F1 and F2) in the highest concentration group in each study also exhibited reduced body weights, often with a corresponding reduction in weight change, beginning on postnatal day (PND) 14 through weaning on PND 28. This reduction in pup body weight corresponded to initiation of self‐feeding.ConclusionsBased on reduced body weights, the no observed adverse effect level (NOAEL) for adult and offspring systemic toxicity was 1,000 ppm for ADBAC and 750 ppm for DDAC (equivalent to approximate daily oral doses of 59 and 45 mg/kg/day, respectively). The reproductive and developmental NOAEL for F0, F1, and F2 generation male and female rats was 2,000 ppm for ADBAC and 1,500 ppm for DDAC (equivalent to approximate daily oral doses of 118 and 91 mg/kg/day, respectively).

AbsorbaSeal™ 5.6.7F vascular closure device: A good laboratory practice chronic study evaluating safety and efficacy in a healthy porcine model.

Vascular closure devices (VCDs) are widely used for arteriotomy closure after percutaneous catheter-based procedures. In comparison to manual compression, VCDs have been associated with shorter time to hemostasis, shorter time to ambulation, and also decreased length of stay. Complexity of deployment, lack of immediate hemostasis, and residual deformity of the arterial wall remain as limitations of current VCDs. The aim of this study was to investigate the AbsorbaSeal™ 5.6.7F vascular closure device, a novel, completely bioabsorbable, intuitive, and easy to use VCD which uses a compressive, "sandwich"-type design comprising a low profile intravascular distal seal and gasket and an extravascular floating foot and proximal seal, in an open infrarenal aortic swine model. Eight fully heparinized swine at a good laboratory practices facility underwent AbsorbaSeal™ 5.6.7F VCD closure of three 6F arteriotomies each in the proximal, mid, and distal infrarenal aorta. Two swine underwent harvest at each of four time cohorts: 30, 60, 90, and 120days. Acute and chronic procedural safety and efficacy, as well as target site vascular remodeling over time, were the primary outcomes evaluated. Secondary outcome measures included local and systemic inflammatory responses, end-organ tissue analysis, and device-related complications through the follow-up periods. Histopathological evaluation was performed by a blinded pathologist. Standard statistical methods were used. In deployment of 24 AbsorbaSeal™ 5.6.7F VCDs, there were no device-related complications or mortalities. All deployments resulted in rapid arteriotomy seal (100% deployment success), with a mean time to hemostasis (cessation of arterial flow) of 21.5s (median: 6.5s) across a mean activated clotting time (ACT) of 356s. Twenty of the 24 implant sites (83%) attained complete hemostasis within 20s. Immediate post-implant and pre-termination angiographies at all time points were performed of all swine which demonstrated normal aortic appearance and tissue structure and normal downstream vascular beds. At 30days, each implant's intravascular distal seal and gasket were removed from the circulation and completely covered with a smooth neointimal layer. Minimal inflammation and no intimal or luminal thrombus were observed at any site at every time point. AbsorbaSeal™ 5.6.7F is a safe, effective, and secure VCD that demonstrates rapid hemostasis in a fully heparinized open aortic porcine model. Removal from circulation and complete coverage of the intravascular distal seal and gasket with neointima occurred within 30days post-implant. Natural transmural vessel healing from the arteriotomy itself with minimal inflammation was noted for each implant at every time point.

Safety and toxicology assessment of sodium nitrite administered by intramuscular injection

Intramuscular (IM) injection of nitrite (1-10 mg/kg) confers survival benefit and protects against lung injury after exposure to chlorine gas in preclinical models. Herein, we evaluated safety/toxicity parameters after single, and repeated (once daily for 7 days) IM injection of nitrite in male and female Sprague Dawley rats and Beagle dogs. The repeat dose studies were performed in compliance with the Federal Drug Administration's (FDA) Good Laboratory Practices Code of Federal Regulations (21 CFR Part 58). Parameters evaluated consisted of survival, clinical observations, body weights, clinical pathology, plasma drug levels, methemoglobin and macroscopic and microscopic pathology. In rats and dogs, single doses of ≥100 mg/kg and 60 mg/kg resulted in death and moribundity, while repeated administration of ≤30 or ≤ 10 mg/kg/day, respectively, was well tolerated. Therefore, the maximum tolerated dose following repeated administration in rats and dogs were determined to be 30 mg/kg/day and 10 mg/kg/day, respectively. Effects at doses below the maximum tolerated dose (MTD) were limited to emesis (in dogs only) and methemoglobinemia (in both species) with clinical signs (e.g. blue discoloration of lips) being dose-dependent, transient and reversible. These signs were not considered adverse, therefore the No Observed Adverse Effect Level (NOAEL) for both rats and dogs was 10 mg/kg/day in males (highest dose tested for dogs), and 3 mg/kg/day in females. Toxicokinetic assessment of plasma nitrite showed no difference between male and females, with Cmax occurring between 5 mins and 0.5 h (rats) or 0.25 h (dogs). In summary, IM nitrite was well tolerated in rats and dogs at doses previously shown to confer protection against chlorine gas toxicity.

Analysis of Phthalates and Alternative Plasticizers in Gloves by Gas Chromatography-Mass Spectrometry and Liquid Chromatography-UV Detection: A Comparative Study.

Gloves represent an essential feature for hand protection because it is a requirement in the professional framework to comply with both hand hygiene standards and the principles of good laboratory practice. Despite their wide use, there is a knowledge gap regarding their composition, including phthalates. The purpose of the present study was to develop two orthogonal methods, GC–MS and HPLC–DAD, for the screening of plasticizers in gloves. Performances of these two methods were compared in terms of ease of use, number of analyzed plasticizers, and sample preparation. The two methods were validated and applied for the identification and quantification of plasticizers in ten gloves made with different materials (vinyl, nitrile, latex, and neoprene). Results revealed the presence of three main ones: DEHP, DEHT, and DINP. Additionally, the contents of plasticizers were extremely variable, depending on the glove material. As expected, the results point out a predominant use of plasticizers in vinyl gloves with an amount that should be of concern. While DEHP is classified as a toxic substance for reproduction 1B, it was, however, quantified in the ten different glove samples studied. This study provides new data regarding the plasticizers’ content in protective gloves, which could be useful for risk assessment.

Opinion on Maintaining In-House GLP Status for Toxicologic Pathology in Pharmaceutical and (Agro)Chemical Development.

Many pharmaceutical companies have recently elected to stop maintaining good laboratory practices (GLP) status of their R&D sites. Similar discussions have also been engaged in the (agro)chemical industry. This opinion paper examines the pros and cons of maintaining facility GLP status for the purposes of performing the pathology interpretation or peer reviews of GLP studies internally. The toxicologic pathologist provides gross and histomorphologic evaluation and interpretation of nonclinical exploratory and regulatory studies during drug and (agro)chemical development. This assessment significantly contributes to human risk assessment by characterizing the toxicological profile and discussing the human relevance of the findings. The toxicologic pathologist is a key contributor to compound development decisions (advancement or termination) and in the development of de-risking strategies for backup compounds, thus playing a critical role in helping to reduce the late attrition of drugs and chemicals. Maintaining GLP compliance is often perceived as a costly and cumbersome process; a common and short-term strategy to reduce the costs is to outsource regulatory toxicity studies. However, there are significant advantages in maintaining the GLP status for toxicologic pathology activities in-house including the sustainable retention of internal pathology expertise that has maintained the necessary training needed to manage GLP studies. [Box: see text].

Granulometric distribution of metals in road-deposited sediments by using different sieving methods

Road-deposited sediments (RDS) contain metals that can pollute receiving waters during precipitation events. The concentration of particle-bound metals tends to increase with decreasing particle size. In terms of metal load, the highest loads were often located in the medium range of particle sizes. Many studies have used varying or undocumented sieving techniques without showing the quality of the separation process. This is important as different particle size classes are documented with different pollution contents and loads. This knowledge builds the foundation for the selection of best management practices to improve receiving water quality. Based on the knowledge that the separation of particle sizes by the commonly applied dry sieving can be methodologically biased, this study compares particle size classes obtained by dry and wet sieving methods. The aim of the study was to show the methodological influence of the sieving process on the particle-size distribution (PSD) with focus on the metal distribution within RDS. Therefore, concentrations of Cu, Zn and Pb were determined and loads were calculated per sieve fraction after wet and dry sieving. Wet sieving causes a shift of the PSD to finer particles and both metal concentrations and loads are significantly higher especially in the smallest sieve fractions. Fine, highly polluted particles remained in coarser sieve fractions after dry sieving. This leads to diverging metal contents and especially loads in the fractions > 250 µm and thus lead to varying conclusions from the results. The results of the comparison of sieving methods however confirmed the relevance of the fine particle fractions in terms of best management practices. The fraction < 40 µm showed highest concentration and load in RDS after wet sieving. The results also showed the considerable influence of the sieving method used and the need to document laboratory methods as part of good laboratory practice.

Assessment of experimental conditions affecting spontaneous mutation level of Salmonella strains used in the Ames test

Introduction. The bacterial reverse gene mutations test (the Ames test) is widely used to assess chemicals’ mutagenic activity. The spontaneous mutation level of test strains is a mandatory characteristic that has to be monitored in a laboratory performing mutagenicity studies using the Ames test. In this regard, it is important to assess the factors affecting the spontaneous mutation level in the experiment and, therefore, on the general conclusion on the test item mutagenicity. Material and methods. A plate incorporation test version was used both in the presence and absence of a metabolic activation system. Results. We summarized the historical control data obtained in the laboratory in 2016-2020, determine the fluctuation limits in the number of revertant colonies for each strain, and identify the factors affecting the negative control variability. No significant differences were found in the spontaneous background of test strains when using DMSO or water as solvents, polypropylene or polystyrene tubes, as well as Petri dishes of different types. In the case of the TA1535, TA102 and TA100 cultures, no influence of the presence of the S9 mixture on the spontaneous reversion range was revealed (p≤0.05). Statistically significant differences in the number of spontaneous revertants (at + S9 or -S9) were found for the strains that allow detecting frameshift mutations, TA97 and TA98. It has been shown that the volume of the selective medium and the brand of gelling agent in its composition are important factors leading to the variability of the historical negative control. Conclusion. To ensure the quality of experiments according to the principles of good laboratory practice and the reliability of the data obtained using the bacterial reverse mutation method, it is necessary to standardize the operations in advance of experiments.

Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis

Visceral Leishmaniasis (VL), a potentially fatal disease is caused by Leishmania donovani parasites with no vaccine available. Here we produced a dermotropic live attenuated centrin gene deleted Leishmania major (LmCen−/−) vaccine under Good Laboratory Practices and demonstrated that a single intradermal injection confers robust and durable protection against lethal VL transmitted naturally via bites of L. donovani-infected sand flies and prevents mortality. Surprisingly, immunogenicity characteristics of LmCen−/− parasites revealed activation of common immune pathways like L. major wild type parasites. Spleen cells from LmCen−/− immunized and L. donovani challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ, TNF-α, and reduced expression of the anti-inflammatory cytokines like IL-10, IL-21, compared to non-immunized challenged animals. PBMCs, isolated from healthy people from non-endemic region, upon LmCen−/− infection also induced more IFN-γ compared to IL-10, consistent with our immunogenicity data in LmCen−/− immunized hamsters. This study demonstrates that the LmCen−/− parasites are safe and efficacious against VL and is a strong candidate vaccine to be tested in a human clinical trial.

Genotoxicity test battery – An assessment of its utility in early drug development

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

The National Heart, Lung, and Blood Institute-funded Production Assistance for Cellular Therapies (PACT) program: Eighteen years of cell therapy.

The Production Assistance for Cellular Therapies (PACT) Program, is funded and supported by the US Department of Health and Human Services’ National Institutes of Health (NIH) National Heart Lung and Blood Institute (NHLBI) to advance development of somatic cell and genetically modified cell therapeutics in the areas of heart, lung, and blood diseases. The program began in 2003, continued under two competitive renewals, and ended June 2021. PACT has supported cell therapy product manufacturing, investigational new drug enabling preclinical studies, and translational services, and has provided regulatory assistance for candidate cell therapy products that may aid in the repair and regeneration of damaged/diseased cells, tissues, and organs. PACT currently supports the development of novel cell therapies through five cell processing facilities. These facilities offer manufacturing processes, analytical development, technology transfer, process scale‐up, and preclinical development expertise necessary to produce cell therapy products that are compliant with Good Laboratory Practices, current Good Manufacturing Practices, and current Good Tissue Practices regulations. The Emmes Company, LLC, serves as the Coordinating Center and assists with the management and coordination of PACT and its application submission and review process. This paper discusses the impact and accomplishments of the PACT program on the cell therapy field and its evolution over the duration of the program. It highlights the work that has been accomplished and provides a foundation to build future programs with similar goals to advance cellular therapeutics in a coordinated and centralized programmatic manner to support unmet medical needs within NHLBI purview.