Section I. Funding Opportunity Description This Funding Opportunity Announcement (FOA) invites eligible United States small business concerns (SBCs) to submit Small Business Innovation Research (SBIR)Phase I and Phase II (Fast track and Direct to phase II) grant applications. The funding opportunity will utilize a U43/U44 cooperative agreement to support small business concerns (SBCs) to propose applications for the qualification of neuromuscular junction tissue chips (TC) as an alternative approaches method (NAM) to replace the current LD50 assay (mouse lethality bioassay (MLB) as a potency assay for botulinum toxin. The NIH NCATS and FDA Center for Food Safety and Applied Nutrition (CFSAN) are partners in this FOA and will collaborate and coordinate efforts with awardees to help position neuromuscular junction tissue chips for use as a reliable and qualified potency assay for botulinum toxin. The goal of this collaboration is to establish the Botulinum Toxin Potency Assay using Tissue Chips (BoT PATCh) as a Drug Development Tool (DDT). Qualification of BoT PATCh as an alternative test method is needed not only for US federal agency acceptance, but also for international acceptance, and the subsequent commercialization of these test methods for products intended for global markets. This FOA is specifically intended to accelerate the development, qualification, acceptance, and commercialization of BoT PATCh that replace the use of animals in toxicological testing currently required or conducted by US federal agencies. Applicants are encouraged to contact staff at NCATS and FDA per Agency Contacts below to ensure that their study design, qualification plan and objectives are in line with the goals of the FOA. Applicants will be expected to work with NCATS and FDA post-award to address additional testing or standards required by these agencies. These activities will be coordinated post-award through a joint NIH and FDA Steering Committee. Background Botulinum toxin (BoT) is a neurotoxin produced from Clostridium botulinum which in low doses is clinically effective in treating numerous medical conditions (including muscle spasticity, strabismus, hyperactive urinary bladder, excessive sweating, and migraine) and is also widely used for cosmetic purposes. Acetylcholine is a neurotransmitter that is released through the SNARE protein complex (synaptobrevin, SNAP-25, and syntaxin) at neuromuscular junctions by binding to the acetylcholine receptor in muscles causing the muscle fibers to contract. BoT prevents the release of acetylcholine into the synaptic cleft by cleaving one of the three SNARE proteins involved in neurotransmitter release. Botulinum toxin can also cause the rare but life-threatening condition called botulism, characterized by weakness, blurred vision, speech impairment, muscle cramps, vomiting, diarrhea, and fever. The estimated human lethal dose of botulinum toxin is 1.3-2.1 ng/kg when administered by the intravenous or intramuscular route and 10-13 ng/kg when administered by the inhalation route. The LD50 assay (mouse lethality bioassay (MLB)) has been the standard method to determine the safety and potency of each batch of botulinum toxin manufactured for medical and cosmetic uses. Different doses of botulinum toxin are injected intraperitoneally into a large number of mice to assess mortality following respiratory failure. MLB is a laborious and an expensive procedure requiring a sophisticated animal facility and a skilled and dedicated workforce. Additionally, ethical concerns have led to bans of the sale of cosmetic products or their components which have been tested on animals, which has led to efforts to develop alternative testing methods for the safe use of botulinum toxin in humans. NCATS provides leadership and support of the Tissue Chips for Drug Screening program https://ncats.nih.gov/tissuechip/about in the implementation and adoption of this technology in the drug development process. Tissue chips have emerged as a solution toward in vitro New Approaches Methods (NAMs) that are more predictive of human response in the safety and efficacy assessment of leading therapeutics. Tissue chips (TC), specifically those that recapitulate the human neuromuscular junction, provide a useful alternative platform as a NAMs for quantitative analysis and titer evaluation of botulinum neurotoxins, and also advance medical countermeasure development. The qualification plan must include: 1. A detailed protocol for the test method and data analysis. Post review and pre-award, the Joint NCATS and FDA Steering Committee can provide further assistance in developing and/or refining a robust test method protocol for recipient review and consideration. 2. Demonstration ofNMJ markers and functions, in-vitro maintenance for longer duration,andrelevanceof microfabrication,microfluidic design, and endpoint-readoutto the BoT potency testing, as applicable. 3. Test method performance (accuracy, precision, specificity, reproducibility, ability to obtain a linear or log standard curve, etc.) criteria. 4. Plan to demonstrate test method’s performance using reference batches of BoT. All projects (other than Direct to Phase II applications) will be Fast-Track applications which include both SBIR Phase I and Phase II components. The duration of Phase I of the Fast-Track will depend on the maturity of the project at entry, however, duration is 6 months to one year is typical. Only those Phase I projects that have met specific criteria (see below) will be eligible for transition to Phase II of the Fast-Track after NIH administrative review. Phase II of the Fast-Track will support commercialization of BoT PATCh to replace the use of animals in toxicological testing currently required or conducted by US federal agencies. Projects that have already demonstrated feasibility but have not received a Phase I SBIR or STTR, can apply for a Direct to Phase II award and bypass Phase I. The SBIR U44 cooperative agreement mechanism is milestone-driven and involves significant input from NIH program staff regarding project and milestone planning, monitoring of research progress, and go/no-go decision-making.