A double ttrA and pduA knock-out mutant of Salmonella Typhimurium is not attenuated for mice (Mus musculus).

Despite numerous studies on Salmonella enterica subsp. enterica serovar Typhimurium, the underlying mechanisms of several aspects of its virulence are still under investigation, including the role of the pdu and ttrA genes, associated with the metabolism of 1,2-propanediol using tetrathionate as an electron acceptor respectively. Our objective was to contribute to an understanding of the role of these genes inbacterial virulence for mice (Mus musculus) using an S. Typhumirum ΔttrApduA mutant. The experiment was conducted with a group infected by the S. Typhimurium mutant and a control group infected with a wild-type strain. The mutant was not attenuated compared with the parent strain. There were no differences in the bacterial numbers recovered from the mesenteric lymph nodes and Peyer's patches but at 8-day after oral infection higher numbers were recovered from the spleen, liver, and cecum. Unlike the single pduA and ttrA mutants, the double ΔttrApduA mutation did not affect invasion and survival in mice, which highlights the need for further studies to clarify the role of these important metabolism genes under reduced redox conditions linked to Salmonella virulence.

Review of embryo-fetal developmental toxicity studies performed for pharmaceuticals approved by FDA in 2022 and 2023.

92 novel drugs were approved by the FDA in 2022-2023. 48 of these approvals were for orphan indications. Embryofetal development (EFD) studies were conducted for 79 % of approvals. Rats and rabbits were the most common species used (77 % and 62 % of studies, respectively). For the testing of biopharmaceuticals, rodents were more often used (43 % of EFD studies) than non-human primates (29 %) and rabbits (29 %). Most (75 %) biopharmaceuticals intended to treat cancer were approved without EFD studies. Amongst the 41 drugs for which both rat and rabbit EFD studies were performed, the rabbit appeared more sensitive to both maternal toxicity and developmental toxicity (61 % and 63 % of drugs, respectively). Most drugs (76 %) showed more than a 2-fold difference in the LOAEL for developmental toxicity between the rat and rabbit. EFD studies were not required for drugs with a mode of action known to pose a clear hazard for pregnancy and further EFD studies were generally not performed when clinically relevant developmental effects had already been observed in one species or in a preliminary EFD study. Many drug labels showed minor deviations from the PLLR rule: the metric used to calculate exposure margins and the presence or absence of maternal toxicity were not always specified. These omissions, however, are of little significance for the prescriber. The five reviews in this series now show compiled information on EFD studies for all small molecule pharmaceuticals approved since 2014 and for all therapeutic monoclonal antibodies approved to date.

Male premating treatment in FEED studies: Length is not everything.

The ICH S5(R3) guideline recommends that male rodents in a FEED study are treated for ≥2 weeks before mating, which has frequently been criticized as being too short for the detection of all effects on sperm maturation, mating behavior and male fertility. In a FEED study, males generally continue for ≥5 weeks after the start of cohabitation. This review determines how often a 2-week premating treatment period for males was used in FEED studies of novel drugs approved by the FDA in 2022 and 2023. The male premating treatment duration was specified for 44 drugs. Only 16 % of these had a 2-week male premating treatment period. 52 % of drugs had a 4-week period. No examples were found in the literature of drugs for which male-mediated reproductive toxicity could have been detected using a 4-week, but not a 2-week, premating treatment period. Repeat dose studies in 2 species, with a duration of treatment at least equivalent to that in patients, are generally completed before the FEED study is planned. Providing no effects on male reproductive organs are detected in the repeat dose studies, a 2-week premating treatment period appears sufficient for the detection of effects on male mating performance. If toxic effects on spermatogenesis are detected in the repeat dose studies, a male FEED study serves little regulatory purpose. Even in the absence of effects on mating performance and fertility in the FEED study, a drug-related disruption of spermatogenesis would likely be considered pertinent to the human.

Microbiological and Molecular Investigation of Antimicrobial Resistance in Staphylococcus aureus Isolates from Western Romanian Dairy Farms: An Epidemiological Approach.

Antimicrobial therapy is the most frequently used medical intervention for bovine mastitis in the dairy industry. This study aims to monitor the extent of the antimicrobial resistance (AMR) problem in Staphylococcus aureus in the dairy industry in Western Romania. Twenty farms were selected by random sampling in a transverse epidemiological study conducted across four counties in Western Romania and divided into livestock units. This study assessed the association between the resistance genes to phenotypic expression of resistance and susceptibility. Isolates of S. aureus were identified and q-PCR reactions were used to detect antibiotic resistance genes. One hundred and fifty bovine and 20 human samples were positive for S. aureus. Twenty five percent of bovine isolates (30/120) and none(0/30) of the human isolates were methicillin-resistant S. aureus (MRSA). All isolates were susceptible to fosfomycin, ciprofloxacin, netilmicin, and resistant to ampicillin and penicillin. S. aureus isolates regarded as phenotypically resistant (R) were influenced by the origin of the samples (human versus bovine, χ2 = 36.510, p = 0.013), whether they were methicillin-resistant S. aureus (χ2 = 108.891, p < 0.000), the county (χ2 = 103.282, p < 0.000) and farm of isolation (χ2 = 740.841, p < 0.000), but not by the size of the farm (χ2 = 65.036, p = 0.306). The multiple antibiotic resistance index was calculated for each sample as the number regarded as phenotypically resistant (R)/total antibiotics tested (MARI = 0.590 ± 0.023) was significantly higher (p < 0.000) inmethicillin-resistant S. aureus (0.898 ± 0.019) than non-methicillin-resistant S. aureus (0.524 ± 0.024) isolates. For the antibiotics tested, the total penetrance (P%) of the resistance genes was 59%, 83% for blaZ, 56% for cfr, 50% for erm(B), 53% for erm(C), 57% for mecA and 32% for tet(K). Penetrance can be used as a parameter for guidance towards a more accurate targeting of chemotherapy. P% in S. aureus was strongly positively correlated with the multiple antibiotic resistance index (r = +0.878, p < 0.000) with the potential to use the same limit value as an antibiotic management decision criterion. Considering cow mastitis, the penetrance value combined with the multiple antibiotic resistance index suggests that penetrance could serve as a useful parameter for more precise targeting of chemotherapy for S. aureus.

Correction: Lungu et al. Molecular Characterisation of Antimicrobial Resistance in E. coli Isolates from Piglets in the West Region of Romania. Antibiotics 2023, 12, 1544.

The authors Bianca Cornelia Lungu and Ioan Hutu did not state contributed equally [...].

Porcine Monocyte DNA Traps Formed during Infection with Pathogenic Clostridioides difficile Strains.

Clostridioides (Clostridium) difficile is an enteric pathogen of several mammalian species including man, frequently involving nosocomial resurgence, following oral administration of broad-spectrum antibiotics, but also with human-to-human infection occurring, and neonatal pigs with zoonotic transmission. To date, the immune response to C. difficile has mostly focused on neutrophils and cytokine/chemokines, particularly in human infection. The neonatal pig is now recognized as a valuable model for human infection. We show that porcine monocytes respond to C. difficile differently compared with many other bacterial infections. Infection of porcine monocytes with human C. difficile strains CD630 (Ribotype 078) or R20291 (Ribotype 027) for 3 or 24 h post-infection (pi) resulted in a lack of oxidative burst or nitrite ion production when compared to uninfected controls (p > 0.05). The survival dynamics of both CD630 and R20291 in monocytes were similar with intracellular bacterial numbers being similar at 3 h pi and 24 h pi (p > 0.05). However, we show that porcine monocytes entrap C. difficile via extracellular DNA traps. This process began as early as 3 h pi, and at 24 h pi the nuclei appeared to be depleted of DNA, although extracellular DNA was associated with the cell membrane. Our preliminary study also suggests that entrapment of C. difficile by extracellular DNA may occur via a process of monocyte etosis.

New Approaches to Tackling Intractable Issues in Infectious Disease.

Despite major progress in the last several decades in reducing the public and animal health burden of infectious disease a number of issues remain to be resolved and which have thus far been regarded as intractable. These include (i) the persistent carrier state in individuals convalescent from typhoid and typhoid-like infections, (ii) the increasing prevalence of multi-antibiotic resistance in enteric pathogens, much of which is mediated by self-transmissible plasmids, and (iii) parasite infections which are difficult to control by vaccination and where resistance to chemotherapeutics is also increasing. The author describes very recent work carried out by his group to look at resolving these problems in new and imaginative ways.

Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model.

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.

Effects of in ovo injection of bacterial peptides and CpG-ODN on Salmonella enterica serovar Heidelberg infection in specific pathogen-free (SPF) chicks.

RESEARCH HIGHLIGHTS: Peptides + CpG-ODN reduced SH in caeca at the first week post-infection.Administered formulations did not reduce SH-faecal excretion.Levels of intestinal IgA were similar between all groups.CpG-ODN improved some parameters associated with chick intestinal health.

Genome degradation promotes Salmonella pathoadaptation by remodeling fimbriae-mediated proinflammatory response.

Understanding changes in pathogen behavior (e.g. increased virulence, a shift in transmission channel) is critical for the public health management of emerging infectious diseases. Genome degradation via gene depletion or inactivation is recognized as a pathoadaptive feature of the pathogen evolving with the host. However, little is known about the exact role of genome degradation in affecting pathogenic behavior, and the underlying molecular detail has yet to be examined. Using large-scale global avian-restricted Salmonella genomes spanning more than a century, we projected the genetic diversity of Salmonella Pullorum (bvSP) by showing increasingly antimicrobial-resistant ST92 prevalent in Chinese flocks. The phylogenomic analysis identified three lineages in bvSP, with an enhancement of virulence in the two recently emerged lineages (L2/L3), as evidenced in chicken and embryo infection assays. Notably, the ancestor L1 lineage resembles the Salmonella serovars with higher metabolic flexibilities and more robust environmental tolerance, indicating stepwise evolutionary trajectories towards avian-restricted lineages. Pan-genome analysis pinpointed fimbrial degradation from a virulent lineage. The later engineered fim-deletion mutant, and all other five fimbrial systems, revealed behavior switching that restricted horizontal fecal-oral transmission but boosted virulence in chicks. By depleting fimbrial appendages, bvSP established persistent replication with less proinflammation in chick macrophages and adopted vertical transovarial transmission, accompanied by ever-increasing intensification in the poultry industry. Together, we uncovered a previously unseen paradigm for remodeling bacterial surface appendages that supplements virulence-enhanced evolution with increased vertical transmission.

Molecular Characterisation of Antimicrobial Resistance in E. coli Isolates from Piglets in the West Region of Romania.

Antibiotics are widely used for prophylaxis and therapy, reducing morbidity and mortality produced by bacterial pathogensin pigs, including infections caused by Escherichia coli. The aim of this study was to characterise antibiotic resistance phenotypes and genotypes in E. coli isolates in pigs in West Romanian grower farms. Differential phenotypic susceptibility profiles and the contribution of resistance genes to phenotypic expression of susceptibility or resistance were evaluated. A total of 76 E. coli isolates were identified and confirmed by the MicroScan Walk Away System. The occurrence of four resistance genes, ampC, blaZ, blaTEM and tetK in strains resistant to 13 antibiotics was assessed. Of the E. coli isolates, 0% showed resistance to meropenem, 3.9% to tigecycline and 10.5% to piperacillin/tazobactam, whereas, in contrast, 100% were resistant to ampicillin and mezlocillin, 76.31% to piperacillin and 59.3% to tetracycline. The prevalence of resistance genes in resistant isolates detected by q-PCR analysis was 97.0% for ampC, 96% for blaZ, 32.9% for blaTEM and 58.8% for tetK. Penetrance (the proportion of individuals carrying a particular variant of a gene that also expresses an associated trait) was 50% for ampC (32% for amoxicillin/clavulanate, 62% for cefazolin, 32% for cefepime, 100% for cefotaxime, 56% for cefuroxime and 99% for ampicillin), 65% for blaZ (32% for amoxicillin/clavulanate and 99% for ampicillin), 51% for blaTEM (81% for piperacillin) and 44% for the tetK gene (83% for tetracycline). The result of phenotypic antibiotic resistance testing may indicate the presence of plasmid-borne resistance, with a diagnostic odds ratio of a positive phenotypic resistance for tetK being 4.52. As a management decision, the maximum penetrance admitted for using a specific antibiotic for E. coli infections in pigs is recommended to be less than 20%.

Nifuratel reduces Salmonella survival in macrophages by extracellular and intracellular antibacterial activity.

Salmonella are intracellular bacterial pathogens for which, as with many of the other Enterobacteriaceae, antibiotic resistance is becoming an increasing problem. New antibiotics are being sought as recommended by the World Health Organization and other international institutions. These must be able to penetrate macrophages, and infect the major host cells and the Salmonella-containing vacuole. This study reports screening a small library of Food and Drug Administration (FDA)-approved drugs for their antibacterial effect in macrophages infected with a rapid-multiplying mutant of Salmonella Enteritidis. The most effective drug that was least toxic for macrophages was Nifuratel, a nitrofuran antibiotic already in use for parasitic infections. In mice, it provided 60% protection after oral infection with a lethal S. Enteritidis dose with reduced bacterial numbers in the tissues. It was effective against different serovars, including multidrug-resistant strains of Salmonella Typhimurium, and in macrophages from different host species and against Listeria monocytogenes and Shigella flexneri. It reduced IL-10 and STAT3 production in infected macrophages which should increase the inflammatory response against Salmonella. IMPORTANCE Salmonella can keep long-term persistence in host's macrophages to evade cellular immune defense and antibiotic attack and exit in some condition and reinfect to cause salmonellosis again. In addition to multidrug resistance, this infection circle causes Salmonella clearance difficult in the host, and so there is a great need for new antibacterial agents that reduce intramacrophage Salmonella survival to block endogenous Salmonella reinfection.

Reproductive findings in male animals exposed to selective survival of motor neuron 2 (SMN2) gene splicing-modifying agents.

Risdiplam is a daily, orally dosed, survival of motor neuron 2 (SMN2) mRNA splicing-modifying agent approved for the treatment of spinal muscular atrophy (SMA). RG7800 is a closely related SMN2 mRNA-splicing compound. Effects on secondary mRNA splice targets such as Forkhead Box M1 (FOXM1) and MAP kinase-activating death domain protein (MADD), which have been implicated in cell-cycle regulation, were observed in non-clinical studies with both risdiplam and RG7800. Potential effects of risdiplam on male fertility via FOXM1 and MADD are important as these secondary splice targets exist in humans. This publication reports the findings from 14 in vivo studies that investigated the reproductive tissues of male animals in various stages of development. Exposure to risdiplam or RG7800 induced changes within the germ cells in the testes of male cynomolgus monkeys and rats. Germ-cell changes included both cell-cycle gene changes (alteration of mRNA-splicing variants) and seminiferous tubule degeneration. In monkeys treated with RG7800, there was no evidence of damage to spermatogonia. Observed testicular changes were stage-specific with spermatocytes in the pachytene stage of meiosis and were fully reversible in monkeys following a sufficient recovery period of eight weeks following cessation of RG7800. In rats, seminiferous tubule degeneration was present, and full reversibility of germ-cell degeneration in the testes was observed among half of the rats that were exposed to risdiplam or RG7800 and then allowed to recover. With these results, coupled with histopathological findings, the effects on the male reproductive system are expected to be reversible in humans for these types of SMN2 mRNA-splicing modifiers.

The Extraperitoneal Approach to Left-Sided Colorectal Resections: A Human Cadaveric Study.

INTRODUCTION: Technical challenges during laparoscopic and robotic anterior resection include identification of key retroperitoneal structures and obtaining clear views of the inferior mesenteric artery (IMA) pedicle and total mesorectal excision (TME) plane. Steep head-down position improves surgical exposure but is associated with cerebral oedema, high intrapulmonary pressures, and rare neurological complications. In this article we describe the key steps of an anterior resection performed via the extra-peritoneal (XP) space in the supine position. METHODS: The technique of same-side lateral-to-medial XP dissection has been developed and refined in serial cadaveric workshops. A standard periumbilical port is inserted for initial laparoscopic exploration. Dissection is then performed in the left XP space via a 5 cm skin incision (later used as the extraction site) to allow for insertion of four (latterly three) working ports. The colon is mobilized along its lateral attachments, reflecting retroperitoneal structures down and away. The IMA pedicle is taken proximally, next to the duodenum. If required, TME dissection can be continued in the same plane. A short intraperitoneal phase is then required to complete the procedure. RESULTS: Eight cadavers were studied (seven males; median 78 y). Four operations were performed laparoscopically and four robotically. Excellent views of the key retroperitoneal structures were achieved early in the procedure. Anatomical identification was performed sequentially for left-sided structures-psoas tendon, gonadal vessel, ureter, common iliac artery, IMA, and duodenum before ligation of the IMA pedicle. High ligation of IMA on the aorta and splenic flexure mobilization were performed in all eight procedures. CONCLUSIONS: This novel study shows it is feasible to perform the key steps of an anterior resection using the XP space in the supine position. This will reduce the need for steep head-down positioning which may have meaningful clinical benefits. Prospective clinical studies are required to validate the technique within a patient population.

Science-Based Approach to Harmonize Contraception Recommendations in Clinical Trials and Pharmaceutical Labels.

This review presents a European Federation of Pharmaceutical Industries and Association/PreClinical Development Expert Group (EFPIA-PDEG) topic group consensus on a data-driven approach to harmonized contraception recommendations for clinical trial protocols and product labeling. There is no international agreement in pharmaceutical clinical trial protocols or product labeling on when/if female and/or male contraception is warranted and for how long after the last dose. This absence of consensus has resulted in different recommendations among regions. For most pharmaceuticals, contraception recommendations are generally based exclusively on nonclinical data and/or mechanism. For clinical trials, contraception is the default position and is maintained for women throughout clinical development, whereas appropriate information can justify removing male contraception. Conversely, contraception is only recommended in product labeling when warranted. A base case rationale is proposed for whether or not female and/or male contraception is/are warranted, using available genotoxicity and developmental toxicity data. Contraception is generally warranted for both male and female subjects treated with mutagenic pharmaceuticals. We propose as a starting point that contraception is not typically warranted when the margin is 10-fold or greater between clinical exposure at the maximum recommended human dose and exposure at the no observed adverse effect level (NOAEL) for purely aneugenic pharmaceuticals and for pharmaceuticals that induce fetal malformations or embryo-fetal lethality. Other factors are discussed, including contraception methods, pregnancy testing, drug clearance, options for managing the absence of a developmental toxicity NOAEL, drug-drug interactions, radiopharmaceuticals, and other drug modalities. Overall, we present a data-driven rationale that can serve as a basis for consistent contraception recommendations in clinical trials and in product labeling across regions.

An assessment of the reliability of 52 enhanced preliminary embryofetal development studies to detect developmental toxicity.

BACKGROUND: Preliminary embryofetal development (pEFD) data from two species are currently recommended before inclusion of women of child-bearing potential (WOCBP) in clinical trials in Europe or Japan, but not before trials in the United States. The ICH S5(R3) guideline advises an "enhanced" study design for this purpose. METHODS: The reliability of pEFD studies was assessed by comparing the outcome of 52 enhanced pEFD studies (25 rat, 23 rabbit, and 4 mouse) with the results of the definitive nonclinical EFD assessment. RESULTS: Four pEFD studies revealed severe developmental hazard without the need for a main EFD study. Only one pEFD study failed to detect drug-related teratogenicity or pregnancy failure subsequently detected in the main study. There were, however, some false positive and some equivocal pEFD study results. Of the 48 pEFD studies for which a main EFD study was performed, 16 (33%) failed to accurately predict (within two-fold) the no adverse effect level (NOAEL) for developmental toxicity subsequently defined in the main EFD study. Skeletal examination of fetuses in the pEFD study was necessary was to detect drug-induced malformations. One quarter (23%) of EFD investigations revealed malformations and/or pregnancy failure at one dose level or more. CONCLUSIONS: pEFD studies are effective for the detection of serious or irreversible effects on embryofetal development, provided that full fetal examinations are completed. They are not, however, sufficiently powered to reliably define the NOAEL for developmental toxicity. The regulatory impact of pEFD studies remains obscure since maximum pregnancy prevention precautions are required in clinical trials in all regions until the results of the main EFD studies are available.

The Loss of focA Gene Increases the Ability of Salmonella Enteritidis to Exit from Macrophages and Boosts Early Extraintestinal Spread for Systemic Infection in a Mouse Model.

Salmonella Enteritidis (SE) can spread from the intestines to cause systemic infection, mainly involving macrophages. Intramacrophage Salmonella exits and reinfects neighboring cells, leading to severe disease. Salmonella genes involved in exiting from macrophages are not well understood or fully identified. A focA::Tn5 mutant was identified by an in vitro assay, with increased ability to exit from macrophages. A defined SEΔfocA mutant and its complemented derivative strain, SEΔfocA::focA, were constructed to confirm this phenotype. Although the lethal ability of focA mutants was similar to that of the parental SE in mice, it was isolated earlier from the liver and spleen than the parental SE. focA mutants induced higher levels of proinflammatory IL-12 and TNF-α compared with the parental SE and SEΔfocA::focA. focA mutants showed higher cytotoxicity and lower formate concentrations than SE and SEΔfocA::focA, whereas there was no change in pyroptosis, apoptosis and flagella formation ability. These current data suggest that the focA gene plays an important role in regulating intramacrophage Salmonella exiting and extraintestinal spread in mice, although the specific mechanism requires further in-depth studies.

Salmonella Heidelberg side-step gene loss of respiratory requirements in chicken infection model.

Among the important recent observations involving anaerobic respiration was that an electron acceptor produced as a result of an inflammatory response to Salmonella Typhimurium generates a growth advantage over the competing microbiota in the lumen. In this regard, anaerobically, salmonellae can oxidize thiosulphate (S2O32-) converting it into tetrathionate (S4O62-), the process by which it is encoded by ttr gene cluster (ttrSRttrBCA). Another important pathway under aerobic or anaerobic conditions is the 1,2-propanediol-utilization mediated by the pdu gene cluster that promotes Salmonella expansion during colitis. Therefore, we sought to compare in this study, whether Salmonella Heidelberg strains lacking the ttrA, ttrApduA, and ttrACBSR genes experience a disadvantage during cecal colonization in broiler chicks. In contrast to expectations, we found that the gene loss in S. Heidelberg potentially confers an increase in fitness in the chicken infection model. These data argue that S. Heidelberg may trigger an alternative pathway involving the use of an alternative electron acceptor, conferring a growth advantage for S. Heidelberg in chicks.

Review of embryo-fetal developmental toxicity studies performed for pharmaceuticals approved by FDA in 2020 and 2021.

103 novel drugs were approved by the FDA in 2020-2021. Embryofetal development (EFD) studies were conducted for 76 % of these approvals. For the majority of drugs, EFD studies were conducted in rats and rabbits. Both species were equally sensitive to developmental toxicity, but the rabbit was slightly more sensitive to maternal toxicity at the same systemic exposure level. Nonetheless, 68 % of drugs showed more than a 2-fold difference in the low adverse effect level for developmental toxicity between the rat and rabbit. Previous reviews in this series compiled information on EFD studies for all small molecule pharmaceuticals approved since 2014 and for all therapeutic monoclonal antibodies approved to date. The use of non-human primates for the developmental toxicity testing of biopharmaceuticals has fallen over recent years (22 % of biologics license applications (BLAs) for 2020-2021, compared with 62 % for 2002-2015), with more biopharmaceuticals now tested in rodents (37 % of BLAs for 2020-2021). While the Pregnancy and Lactation Labeling Rule (PLLR), adopted in 2014, has brought consistency to the presentation of EFD data in drug labels, prescribers complain that the pregnancy section of current drug labels is neither concise nor clear. The FDA has pledged to address the concerns of clinicians in a future revision of the PLLR rule. The recommendations on risk assessment in the recently revised ICHS5(R3) guideline could be incorporated into the PLLR rule to remove extraneous nonclinical details from the label with the aim of facilitating rapid understanding by the practitioner.

Considerations for and against dosing rodent pups before 7 days of age in juvenile toxicology studies.

This review focuses on preweaning ontogenic and developmental processes that can influence the selection of the appropriate age at which to start dosing rodent pups in juvenile animal studies (JAS). The ICH S11 guideline on 'Nonclinical Safety Testing in Support of Development of Paediatric Medicines' highlights the need to adapt the age from which animals are dosed according to the stage of development in the target organs/tissues of concern in the youngest pediatric patients. Rodents (rat or mouse) are the most common species for JAS. Despite previous practices, based on comparative ontogeny, it is rarely necessary to dose rodents younger than one week of age since postnatal day (PND)7 is appropriate to address concern for the vast majority of organs. In exceptional cases, earlier dosing (e.g., PND4) can be appropriate to address specific concern in preterm neonates and when a tissue of concern has a particularly early developmental trajectory in the rodent compared to humans. The comparative development of the CNS is particularly complex. While exposure of rodents from PND10 covers most CNS development stages relevant to human neonates, a later dosing start (yet, not later than PND14) can sometimes be appropriate to reflect specific aspects (e.g., transformation of GABAergic transmission). An extended study design including subsets of several ages can be helpful to address multiple concerns within a preweaning JAS. Such design can allow for individual assessment of each concern, whilst minimizing (potentially irrelevant) signals from tissues exposed at a developmental stage that do not match the human situation.