RFQ-75F40124Q00126.docx

Offeror Name UEI Comprehensive toxicity analysis of individual ENDS flavor ingredient chemicals alone and in complex with metals using high throughput approaches.

SECTION 1 – COMBINED SYNOPSIS SOLICITATION

SECTION 2 – LINE ITEMS

SECTION 3 – STATEMENT OF WORK/DESCRIPTION

· 3.1 Background

· 3.2 Objective

· 3.3 Scope

· 3.4 Task

· 3.5 List of Deliverable

SECTION 4: CONTRACT REQUIREMENTS

· 4.1 Conflict of Interest

· 4.2 Government-Furnished Property, Material, Equipment, or Information (GFP, GFM, GFE, OR GFI)

· 4.3 Security

· 4.4 Travel

· 4.5 Special Material Requirements

· 4.6 Other Unique Requirements

· 4.7 Place of Performance

· 4.8 Period of Performance

· 4.9 Contract Type

SECTION 5: GENERAL EVALATUATION INFORMATION EVALUATION AND AWARD

SECTION 6: ADDEDUM / CONTRACT CLAUSES AND PROVISIONS / ADDITIONAL TERMS AND CONDITIONS

· 6.1 52.212-5 CONTRACT TERMS AND CONDITIONS REQUIRED TO IMPLEMENT STATUTES OR EXECUTIVE ORDERS6.2 FAR 52.204-27 PROHIBITION ON A BYTEDANCE COVERED APPLICATION

· 6.2 FAR 52.204-27 PROHIBITION ON A BYTEDANCE COVERED APPLICATION (JUN 2023)

· 6.3 FAR 52.252-2 CLAUSES INCORPORATED BY REFERENCE (FEB 1998)

· 6.4 INVOICE INSTRUCTIONS

· 6.5 DEPARTMENT OF HEALTH AND HUMAN SERVICES ACQUISITION REGULATION (HHSAR) (48 CFR CHAPTER 3) CONTRACT CLAUSES

· 6.6 FAR 52.212-1 INSTRUCTIONS TO OFFERORS—COMMERCIAL PRODUCTS AND COMMERCIAL SERVICES (MAR 2023)

· 6.7 52.212-2 Evaluation - Commercial Products and Commercial Services. (NOV 2021)

· 6.8 2.212-3 OFFEROR REPRESENTATIONS AND CERTIFICATIONS-COMMERCIAL PRODUCTS AND COMMERCIAL SERVICES. (DEC 2022)

· 6.9 352.215-70 LATE PROPOSALS AND REVISIONS. (DEC 2015)

· 6.10 RFP INQUIRIES AND QUESTIONS DUE DATE

· 6.11 PROPOSAL SUBMISSION

SECTION 7: QUOTE INSTRUCTIONS

· 7.1 Evaluation Factors

SECTION 8: BASIS FOR AWARD

ATTACHMENT 1: APPENDIX

SECTION 1 – COMBINED SYNOPSIS SOLICITATION

This is a combined synopsis/solicitation for commercial products or commercial services prepared in accordance with the format in subpart 12.6, as supplemented with additional information included in this notice. This announcement constitutes the only solicitation; proposals are being requested and a written solicitation will not be issued. The solicitation number is 75F40124Q00126 and is issued as an RFQ. This solicitation and incorporated provisions and clauses are those in effect through Federal Acquisition Circular 2024-02, effective January 22,2024. The North American Industry Classification Systems (NAICS) Code 541715 - Research and Development in the Physical, Engineering, and Life Sciences.

SECTION 2 – LINE ITEMS AND PRICES

The Government intends to issue a contract. Offerors are requested to provide a fixed price quote in accordance with the Statement of Work.

SECTION 3 - STATEMENT OF WORK (SOW): Comprehensive toxicity analysis of individual ENDS flavor ingredient chemicals alone and in complex with metals using high throughput approaches.

3.1 Background

The primary function of the Food and Drug Administration (FDA) is to promote public health associated with the use of foods, dietary supplements, cosmetics, drugs, tobacco, and other products under its jurisdiction. FDA's Center for Tobacco Products (CTP) implements PL 111-31 of the Federal Food, Drug, and Cosmetic Act (FD&C Act), the Family Smoking Prevention and Tobacco Control Act (Tobacco Control Act), which was signed into law on June 22, 2009. The Tobacco Control Act granted the FDA important new authority to regulate the manufacture, marketing, and distribution of tobacco products, such as cigarettes, cigars, and electronic nicotine delivery systems (ENDS), to protect public health.

The e-liquids in ENDS are mixtures that contain solvents, nicotine, and flavor ingredient chemicals, and may also contain leachates (e.g., that leach from structural materials), making them chemically diverse compounds with harmful effects. This proposed research investigates cytotoxicity and mutagenicity of flavor ingredient chemicals alone and in combination with metals using high throughput format and then evaluate the toxicity of resultant e-liquid aerosols in a reconstructed lung cell model. This approach will expand our understanding of potential health impacts associated with added flavor ingredient chemicals and metals to promote more informed regulatory review. This is the first project to the best of our knowledge that evaluates the toxicity of flavor ingredient chemicals and their interaction with metals using these new alternative approaches (NAMs). NAMs are part of the vision of Toxicology in the 21st Century program, a federal agency research collaboration (includes EPA, NIH/NCATS, NIH/NIEHS/NTP, and FDA), which has several goals including shifting toxicology from a reliance on animal models to new and alternative toxicology test methods that are predictive of human toxicity.

The aims of this project are consistent with the FDA’s vision and strategy of reduction, replacement, and refinement of animal use. These aims are also consistent with toxicology research priorities in CTP such as the evaluation of toxicity due to the use of ENDS that contain complex flavor mixtures and how product design characteristics impact user risks due to additional or increased constituent exposures and toxicities including those due to metal flavor complexes. This project investigates research priorities identified in the Division of Non-Clinical Science (DNCS) such as the potential toxicological implications of metals in e-liquids and aerosols, and the different toxicity that results from flavor ingredient chemicals when combined with metals.

3.2 Objectives

The FDA will provide a list of sixty (60) individual flavor ingredient chemicals with potential to interact with the selected metals (Cr, Cd, and Ni). The Contractor shall use all or a subset of individual flavor ingredients according to the requirements of each experiment that are found specifically stated in this section and re-stated in sections 1.3 and 1.4. The subset of individual flavor ingredients shall be determined with appropriate justification by the Contractor in conjunction with the FDA and shall originate from the initial list of 60 individual flavor ingredients provided by the FDA. The test shall be performed on individual flavor ingredients alone, metals alone, or in combination with metals as described in this section and sections 1.3 and 1.4.

To identify the cytotoxic, inflammatory, and genotoxic potential, the Contractor shall use miniaturized Ames test, quantitative high throughput cytotoxicity and cytokine released screening assays (qHTS) to test 60 individual flavors alone, metals alone (Cr, Cd and Ni), and individual flavor ingredient chemicals in the presence of metals in a range of doses in liquid phase. The cytotoxicity assay uses the CellTiter-Blue®, or a similar variant of the neutral red uptake in qHTS format in the normal human bronchial epithelial cell line 16HBE14o-. The level of marker of inflammation cytokine IL-6 released to the supernatant of qHTS cytotoxicity assays shall be measured. In addition, the Contractor shall test a subset of 30 selected, in conjunction with the FDA, flavors alone, metals alone, and individual flavor chemicals in the presence of metals in range of doses in liquid phase with the in vitro Mouse Lymphoma Assay (MLA) (OECD TG 490), and in vitro Mammalian Cell Micronucleus Test (OECD TG 487). The genotoxicity assays (MLA and Mammalian Cell Micronucleus Test) shall follow recommendations found in the OECD guidelines.

For testing a subset of ten, selected in conjunction with the FDA, flavor ingredients alone, metals (Cr, Cd, and Ni) alone and ten, flavor-metal complexes, the Contractor shall obtain physiologically relevant data using commercially available reconstructed normal primary human lung tissues in air-liquid interface that are exposed to aerosolized flavor ingredient chemicals alone and flavor-metal mixtures and report cell viability, oxidative stress, inflammatory mediators, lung barrier function, and DNA damage using the next generation sequencing.

The Contractor shall determine the IC50 (inhibitory concentration 50%) from dose response curves, parameters associated to synergy in assays that test combination of flavor ingredient chemicals, and for genotoxic and mutagenic assays, the no-observed genotoxic level (NOGELs), and other related parameters, for example, Benchmark Dose (BMD). The Contractor shall provide statistical analysis of the results including variance analysis.

3.3 Scope

The FDA will produce a library of 60 flavor (see Attachment 1, Appendix 1) ingredient chemicals based on the predicted propensity of flavor ingredient chemicals to interact with metals and on the toxicity information available for each flavor ingredient. The Contractor shall investigate the toxicity of 60 flavor ingredient chemicals alone and combined with metal using miniaturized Ames assay and cytotoxicity and markers of inflammation using qHTS. The Contractor shall perform cytotoxicity assays and measurements of cytokine levels in the supernatant to determine appropriate sublethal concentrations of flavor ingredient chemicals and metals prior to use in subsequent mutagenic and genotoxic assays, which consist of assays in cell lines and in bacteria. The Micronucleus and MLA assays following OECD guideline shall be used to further test the mutagenicity of 30 selected flavors, metals (Cr, Cd, and Ni) and flavor metal complexes. The Contractor shall determine based on measurables of the mutagenic and genotoxic experiments a list of 10 flavor ingredient chemicals, 3 metals (Cr, Cd, and Ni) and 10 flavor-metal complexes and their concentrations to be tested in the reconstructed normal human lung tissue model to measure the effects on metabolism, membrane integrity, cell viability, inflammation, and DNA damage using next generation sequencing. The Contractor shall perform all data analyses, provide FDA all the raw data, and write study reports. All results and source documents shall be submitted to the Contracting Officer’s Representative (COR). The Contractor may subcontract to a research facility to complete any parts of the work as it considers necessary.

3.4 Tasks

The FDA COR will approve all tasks and deliverables. The Contractor shall incorporate the FDA’s feedback into its final versions of all deliverables. The Contractor shall deliver all documents to the COR electronically, not in hard copy form, with the document title, contract, and task numbers identified in the subject line of the email submission. Deliverables shall be in MS Word, MS Excel, .PDF, and/or SAS transport (.xpt) format. All submitted documents shall be verified virus-free. The Contractor shall perform the following tasks and provide the following deliverables (as defined below). These requirements are to be completed under firm-fixed price.

Task 1 – Kickoff Meeting

The objective of this task is to ensure that FDA and the Contractor are in alignment with the objective, scope, tasks, budget, and timeline of the project. The Contractor shall conduct a kickoff meeting with the COR and the FDA scientific leads ten calendar days after issuance of the contract. Within two business days after the kickoff meeting, the Contractor shall provide, as a deliverable, comprehensive meeting minutes, including start and end time of the meeting; names and roles of the attendees; a summary of issues and discussions; action items; and due dates.

Task 2 – Biweekly Meetings

The objective of this task is to establish status updates of the project. The Contractor shall conduct a bi-weekly web meeting, using either Zoom or Microsoft Teams, at least every ten business days after the kick-off meeting. During these bi-weekly meeting, the Contractor shall update FDA on what has been accomplished, what is due to be completed, and what are any obstacles (i.e., any deviations from the planned study protocols and any other issues that develop during the performance of the contract) that were experienced or are anticipated. Additionally, the Contractor shall provide an agenda and record the meeting minutes and send to FDA for review. FDA may provide feedback, comments, or questions during the meeting. Within two business day from the biweekly meeting, the deliverables of this task are: (1) the Contractor shall provide comprehensive meeting minutes, including start and end time of the meeting; names and roles of the attendees; a summary of issues and discussions; action items; and due dates; (2) any data shared during the meeting, whether verbal or PowerPoint presentation, must be turned over to the FDA during the same time as the meeting minutes. FDA will review meeting minutes and send to the Contractor for any comment. The Contractor shall be responsible to address any comments before finalizing the minutes.

FDA or the Contractor may request additional meetings should issues arise during the project. Both FDA and the Contractor must be in agreement on the need for additional meetings.

Task 3 – Monthly Progress Reports

The Contractor shall submit monthly progress reports on the 10th day of each month to the COR detailing study progress, challenges (and solutions to those challenges) and other study details. Progress reports shall indicate which deliverables or percentages of deliverables were completed during the month and overall progress of the contract.

Task 4 – Protocol Development and Study Implementation

The objective of this task is to establish and review the detailed experimental protocols for this project. The Contractor shall write and submit detailed protocols for each experiment within 30 calendar days of the contract award. Each protocol must include the following: Title page; Table of Contents; Purpose statement; Scope of the experiment; Experimental groups; Materials and reagents with vendor information; Step-by-step methodology; Location of experiments; and Version history including date and time of the protocol. The delivery of this task is in the form of Microsoft Word or PDF documents to FDA COR. These protocols will be reviewed by an expert external committee who will provide feedback and comments which need to be ratified by FDA before the start of the experiments. The external protocol review may take upwards of three-to-four months to finalize.

The Contractor shall follow all federal, state, and local environmental, health, sanitation, and emissions regulations. In addition, the Contractor shall follow all applicable Environmental Protection Agency (EPA) and Occupational Safety & Health Administration (OSHA) regulations. Disposal of laboratory waste must follow the above environmental, health and safety regulations.

Good Laboratory Practices In accordance with the Code of Federal Regulations (CFR) Title 21, Part 58, Good Laboratory Practice (GLP) for Nonclinical Laboratory Studies, the Contractor shall practice Good Laboratory Practices for all applicable work for this contract. FDA understands that GLP-compliance is not always practical and expects that the Contractor use its best, reasonable judgement in determining when GLP-compliance is or is not practical and appropriate. For instances where GLP-compliance is determined not to be practical and appropriate, the Contractor shall use laboratory practices that are near GLP compliance and that can be considered to be GLP-like.

Assays and experiments used to evaluate, mutagenicity and genotoxicity shall conform to OECD, ISO, and other standardized testing guidelines when available. If a standardized testing guideline is unavailable, the Contractor shall provide sufficient justification for use of non-standardized testing guidelines, or any guidelines not specifically mentioned in this proposal. The Contractor shall prepare an appropriate protocol for the relevant procedure in consultation with FDA. For protocols proposing to use guidelines or procedures outside of the national or international standardized testing guidelines, the Contractor shall provide the relevant Standard Operating Procedures (SOPs) and method validation, as appropriate, to the COR. Additionally, the contractor should report any deviations from their established guidelines.

Task 5 – Determination of synergy of flavor chemicals and metals using qHTS

The Contractor shall conduct cell viability assay in qHTS format of the library of 60 flavor ingredient chemicals alone, metals (Cr, Cd, and Ni) alone, and flavor-metal covering each possible combination. The contractor shall demonstrate that the method is robust and validated. The contractor may use the fluorescent method CellTiter-Blue in a high throughput format such as 1536-well or 384-well plate format and the cell line 16HBE14o- seeded at approximate 800 cells per well. The viability of cells shall be measured 24- 48 hours after exposure to test articles, but before cell division, and measurements of the fluorescence (if using CellTiter-blue) made after incubation time of 6 hours with test reactants prepared as described in the manual[footnoteRef:2]. [2: https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/celltiter-blue-cell-viability-assay-protocol.pdf]

The Contractor shall evaluate the supernatant after the cell viability assay for released marker of inflammation cytokine IL-6 levels. The contractor shall demonstrate that the method of choice is robust and validated. The contractor may use the time-resolved fluorescence resonance energy transfer (TR-FRET) assays (PerkinElmer, TRF1223M) according to the manufacturer’s protocol and with fluorophores compatible with CellTiter-Blue.1

The Contractor shall test the viability assay and inflammation cytokine (IL-6 cytokine) measurement in samples by triplicate, with flavor ingredient chemicals in the concentration range of 0.1 – 1000 µM, whereas the concentration range for metals Cr, Cd, and Ni shall be 0.1 – 500 µM, whereby doses are spread out in 10, log base doses, for example, 0, -1, -2, -3, -4, -5, -6, -7, -8, -9 to obtain 10 by 10 assay matrixes per metal-flavor combination.

The Contractor, shall implement appropriate background detection and subtraction techniques to correct signal readout as applicable to methods related to emission, absorption, or fluorescence spectrometry, as required. For example, with regard to autofluorescence detection, the Contractors shall do counter-screens to detect intrinsic fluorescence or wavelength shifts in fluorescence of the min. and max. concentration of chemicals and metals, alone, in combination, and with assay reactants, to avoid assay interference with biological activity readings.5

The Contractor shall use an adequate number of controls per plate in qHTS assays, such as the first and last rows and columns as control wells alternating positive and negative controls or as required by the manufacture’s protocol. In addition to those controls in the protocol, the Contractor shall use ethyl maltol and nicotine in the cell viability/cytokine assay and benzo[a]pyrene in the genotoxic assays, whereas the negative control is vehicle alone. The test articles shall be added directly to plates by some type of automatic liquid handling specified by the Contractor and taken to a final concentration using the vehicle. The Contractor shall use negative control plates at the beginning and end of all the runs to account for any plate position related variation.

The Contractor shall prepare high concentration stocks of metal that is adequate for qHTS format, for example, dilute at 5-10 mM in 1% nitric acid. The contractors shall verify metal concentration by Inducted Coupled Plasma Mass Spectrometry (ICP-MS). The Contractor shall prepare high concentration stocks (1 mM or more) of flavor chemicals in a solvent compatible with the experiment, for example dimethyl sulfoxide (DMSO) should be avoided because it is known to interact with metals. Then the metal concentration scaled down to desired concentration by dilution of metal stocks in deionized water adjusted to pH 7 and flavor ingredient chemicals in water, with 1% of organic solvent. The Contractor shall evaluate and take preventive measures to avoid precipitation of semi-soluble as well as evaporation of semi-volatile compounds.

The contractor shall perform miniaturized in vitro bacterial reverse gene mutation test assay (Ames), such as the 24-well plate format based on OECD TG 471 for detection of genotoxicity of the library of 60 flavor ingredient chemicals alone, metals alone (Cr, Cd, and Ni) and in combination with metals covering each possible combination, utilizing adequate controls and doses based on the results from qHTS assays. In addition, the Contractor shall perform two in vitro mammalian cell assays with concordance to the OECD-compliant assay recommendations for testing of 30 chosen flavor ingredient chemicals alone, metals alone, and metal-flavor mixtures.[footnoteRef:3] For the in vitro mammalian assays, the Contractors shall use the mutation detection Mouse Lymphoma Assay (MLA)(OECD TG 490), and the in vitro Mammalian Cell Micronucleus Test (OECD TG 487). [3: https://www.oecd.org/env/ehs/testing/Draft%20Detailed%20Review%20Paper%20miniaturised%20versions%20%20bacterial%20reverse%20gene%20mutation%20test.pdf]

For all the methods, the Contractor shall demonstrate proficiency by the laboratory in the same or similar technical procedures, for example, test miniaturization steps and response readout, also indicating previously turnaround time achieved in similar projects.

The Contractor shall perform the genotoxic assays using sublethal doses based on the viability assays such that the higher dose (Hdose) is 0.67SD of IC50 or EC50, or of the highest sublethal level observed in non-optimized dose-response curves. The doses are spaced equally on a log scale where the lowest dose (Ldose) is 0.14*IC50 or EC50, and the step calculated as (Hdose/Ldose)1/n, where n is the number of doses. To calculate doses: dose1=Ldose, dose2=Ldose*step, dose3 = dose2*step, dosen=Hdose. The metabolic activator, when required is the S9 microsomal fraction of the rat liver extract.

In the miniaturized Ames test, the Contractor shall use the 24-well plates format or similar. The Contractor shall expose the test article to five S. typhimurium strains in five doses, sorted in 6 by 6 assay matrix, after 48-72 hours of incubation with test chemicals and in the presence and absence of metabolic activator, based on miniaturized Ames OECD guideline.2 For deviations to the protocol, the Contractor shall include justification as for the appropriateness of the deviation.

The Contractor shall perform the mammalian genotoxicity test with cells and incubation time according to protocols based on OECD guidelines. The Contractor shall test three concentrations of test chemical and metals assorted in 3 by 3 assay matrix.

The Contractor shall use Minimal Essential Medium (MEM) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1 mM sodium pyruvate, and 10% fetal bovine serum to perform the tests. To control for exogenous metal contamination, the Contractor shall determine the concentration of elemental metals Ni, Cr, and Cd in every culture media required in all experiments using ICP-MS and if the concentration range is found to interfere with the concentration range of the experiments, proceed to strip metals from the culture media using ion exchange columns and reconstitution with metals or obtaining customized media stripped of Ni, Cr, and Cd from a manufacturer.6,7

The Contractor may subcontract to a research facility to complete any parts of the qHTS or genotoxicity assays as it considers necessary.

The contractor shall work with FDA to choose:

· In total, thirty (30) of flavor ingredient chemicals, metals (Cr, Cd, and Ni), and metal flavor mixtures to test in the Mouse Lymphoma Assay (MLA)(OECD TG 490), and the in vitro Mammalian Cell Micronucleus Test

· Ten (10) flavor ingredient chemicals, three (3) metals (Cr, Cd, and Ni) and ten (10) metal flavor mixtures with mutagenic effects based on statistical analysis of the results to be tested in the reconstructed cell tissues as described in the Task 6 at a concentration above the NOAEL.

A higher priority shall be assigned to flavor ingredient chemicals or chemical metal complex that significantly induce IL-6 expression, or with genotoxic effects.

Task 6: ALI exposure of in vitro 3D human upper airway epithelium

The contractor shall purchase and maintain 3D normal human upper airway epithelium (for example MUCILAIR™) in 24-well format Transwell® cell culture inserts according to the manufacturer instructions. One day (24h) before exposure, basal medium shall be changed, collected and stored at −20°C for further testing and the transepithelial electrical resistance (TEER) shall be tested. The aerosol of flavor chemical ingredient or flavor-metal complex in 70% propylene glycol (PG)/30% vegetable glycerin (VG) shall be generated, without heating, in compliance to CORESTA recommended method (CRM) No. 81 protocol, with puff duration of 3 second, puff volume of 55 ml, one puff every 30 second, 200 puff in total and with 24h rest period. The exposure protocol as defined in CORESTA and the duration of exposure shall be modified according to a preliminary dose range study where appropriate. The aerosol shall be use undiluted and at dilution rates of 0.25 and 0.5 l/min with ambient air. The Air liquid interface (ALI) exposure system shall be used to expose 3D human upper airway epithelium by triplicate to freshly generated diluted or undiluted aerosol mentioned above three times a week for 21 days. The Contractor shall demonstrate their capability to expose 3D cells and generate aerosol with complete information about available instruments and previous work done with similar cell cultures.

The following endpoints shall be measured to assess toxicity of exposure to the 3D human upper airway epithelium. Any deviation shall be accompanied by scientific rationales and validation of alternative methods by the Contractor.

· Barrier integrity: shall be examined using TEER every day post-exposure and permeability assay (identify and quantify levels of active efflux).

· Cell viability: shall be examined using trypan blue assay at 7-, 14-, and 21- days post exposure.

· Oxidative stress: shall be examined using oxygen radical antioxidant capacity (ORAC) assay. Basal medium shall be collected at 24h, 7-, 14- and 21-days post exposure and stored at −20°C for testing.

· Inflammatory mediators: Seven cytokines (IL-1β, IL-6, IL-10, CXCL1, CXCL2, CXCL5 and CXCL10) shall be measured using commercially available enzyme-linked immunosorbent assay kits. Basal medium shall be collected at 24h, 7-, 14- and 21-days post exposure and stored at −20°C for testing.

· Mutagenesis at the end of the exposure period (21 days):

· The error‐corrected next‐generation sequencing method Duplex Sequencing shall be used to detect mutations, using a human panel, which is used to sensitively detect DNA damage after aerosol exposure.3

· Comet assay

Task 7 - Execute the Data Analysis Plan

The Contractor shall perform the data analysis indicated in the COR approved Data Analysis Plan following steps described in Task 7 below, and Task 5 and 6 above. The Data Analysis Plan shall include FDA review of all draft deliverables and as needed, subsequent contractor revision, and delivery of final reports, as specified in Task 9, 10, 11.

For the qHTS format assays, the Contractor shall verify the quality of each qHTS assays by deconvolution of the Z and Z’- Factors from the sample and control effects, in assay and control plates, defining the mean and standard deviation of sample and controls, where a Z or Z’-Factor <0.5 is considered a low quality assay.8

In addition, for qHTS assays, the Contractor shall verify the reproducibility of the biological effect by the analysis of each effect variance obtained by calculating the standard error of the mean (SEM) considering triplicate measurements.

The Contractor shall identify any positional bias in plates by normalization of all qHTS data to B scores that uses a function refractive to outliers (Tukey’s two-way median polish) to estimate column and row effects (edge effects) in plates, thus reducing the number of false negatives.9 The Contractor shall identify chemical flavors or metals, combined or alone, as hits when the effect produced is three times the standard deviation of the mean (B-score > 3SD). The Contractor shall identify plates with number of hits that are larger than those expected to occur by chance, for example, using Pareto plots.10

The Contractor shall use qHTS data to plot a dose-response curve from cell viability and cytokine levels assays to calculate the IC50 defined as the concentration required to decrease the cell population by 50%, or the effective dose 50% (EC50) for cytokine measurements, using a four-points Hill function fit to the dose-response curve that includes the max and min response, the slope of the dose-response curve, and the IC50 or EC50, including values of coefficient of determination R2 for the assessment of goodness of fit.

The Contractor shall investigate discrepancies of the min/max response with respect to the positive/negative controls in the qHTS assays and report %CV >10% from min/max and control plates, and identify outliers in dose-response curves by an statistical method.11

The Contractor shall evaluate synergy of flavor chemicals and metals in the assays and deconvolve parameters of synergy. For example, obtain three types of synergy: efficacy (β), potency (α), and cooperativity (γ), by fitting qHTS data to a 2D Hill equation. Several software are available for free, such as MuSyC model. The Contractors shall verify the concordance of this results to other models such as Explicit Mean Equation as defined by Lederer et. al.12, and the models Bliss and Loewe. The data analysis tools previously developed are available online at music.lolab.xyz or the SynergyFinder software.13,14

For the binary interpretation of the results from the mutagenicity and genotoxic assays, the Contractor shall define the result of an assay as positive or negative result. The Contractor shall follow the corresponding OECD guidelines for the analysis, interpretation, and reporting of the results obtained from the miniaturized bacterial reverse gene mutation test assay (Ames) (OECD TG 471), the Mouse Lymphoma Assay (MLA)(OECD TG 490), and the in vitro Mammalian Cell Micronucleus Test (OECD TG 487). The Contractor shall provide scientific justification, citing previously published references, when deviation from the guideline for interpretation of the results is consider necessary. The Contractor shall use “expert judgement” and “biological relevance” when necessary for interpretation of not clearly positive or not clearly negative results, according to the guidelines, and in such cases provide scientific justification citing previously published references as supportive information. The Contractor shall consider as part of “expert judgment” to repeat assays using a different set of experimental conditions.

For the semi-quantitively analysis of the results the Contractor shall analyze the dose-response plot generated in the mutagenicity, genotoxicity, and cytotoxicity assays to identify a no-observed adverse effect level (NOAEL) or the lowest experimental point, and its linear extrapolation to zero response and corresponding concentration. The Contractor shall provide scientific rational in those instances when a semi-quantitative analysis of the data is not obtained. For this analyses the Contractor shall represent the dose-response as number of revertant colonies per mass of test material for the Ames test, the mutant frequency per mass of test material for the MLA, and frequency of micronucleated cells per mass of test material for the Micronucleus test or provide scientific justification for an alternative if required. The data table shall contain descriptive statistics and plot shall contain error bars in each datapoint. The dose-response plot at doses below NOAEL that deviate from linearity, shall be fit to bilinear models of regression e.g., hockey stick model, and the no-observed genotoxic level (NOGELs) and the next highest dose tested or lowest observed genotoxic effect level (LOGEL) identified.15-17 Then, the Contractors shall evaluate the non-monotonic dose response curves to estimate two points of departure (PoD), such as threshold effect level (Td) and benchmark dose (BMD), using previously published statistical and mathematical methods.18 The statistically determined Td is defined as a dose below which there is a non-significative different effect and above which there is a significative effect when compared to background. After obtaining Td, several other parameters are estimated for good fit, such as slope above Td, y-intercept, and 95% confidence intervals. Then, using a published mathematical model the BMD can be derived which is a dose causing an effect of 10% above the background level or benchmark response (BMR), and the lower limit of the one-sided, 95% confidence interval for the BMD (BMDL10).

For the mutagenicity and genotoxicity assays the Contractor shall verify the reproducibility of the biological effect by the analysis of each effect variance obtained by calculating the standard error of the mean (SEM) considering triplicate measurements. For all assays the Contractor shall provide appropriate descriptive statistics.

The Contractor shall perform descriptive statistics on data from the 3D cell experiments, presented in tables and plotted with error bars. The Contractor shall use representation of data using the most common units in the field to allow for comparation with previously published results. The Contractor shall follow the data analysis recommended by the manufacturer of the panel used for Duplex sequencing and provide critical mutant frequency, mutant count, per locus or cell, and follow previously published graphical representation of data. [footnoteRef:4] [4: Valentine III C, et al. Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing. PNAS (2020) 117(52)33414-33425 https://www.pnas.org/doi/10.1073/pnas.2013724117]

Task 8: Outline of the Final Report

The Contractor shall submit an Outline of the final Study Report to FDA incorporating all suggestions and edits from previous bi-weekly meetings received from the COR and use it to prepare a Draft Final Report. All the data in the final Study Report shall be properly audited. A Quality Assurance Statement shall be issued by the contractor and included in the Final Report. The Final Report shall be supplemented with electronic copies of the raw data associated with performance of this contract. The raw data shall be in MS Word, MS Excel, .PDF, and/or SAS transport (.xpt) format. All submitted documents shall be verified virus-free. FDA will review the Final Report prior to its acceptance as the final deliverable and conclusion of the contract.

Task 9: Draft Final Report

The Contractor shall submit a draft of the final report within 11 months of contract award. The Contractor shall prepare a detailed final report including: 1) an executive summary; 2) methods, including how the data was collected and analyzed; 3) results of the analysis; 4) discussion, including problems encountered during the study and their solutions; and 5) conclusions; and 6) related appendices. As a part of the final report, the Contractor shall submit analytical procedures (including details regarding extraction procedures and/or any other procedures used during sample preparation), validation reports, system suitability, and any other pertinent information for measuring the analytes indicated in Task 5. The Contractor shall incorporate any revisions as needed based on comments received from FDA.

Task 10: Final Report

The Contractor shall submit all raw data and a Final Report within 12 months of contract award that presents the objectives, methods, results of the analyses, and a summary of the findings and conclusions of the project. The Final Report shall include the required elements outlined for the draft report (Tasks 10 and 11) and shall incorporate any revisions based on comments received from the FDA. Contractor will be responsible to address FDA’s comments before finalizing any report.

The contractor shall include the following in the draft and final report:

1. Specific protocols, methods, materials, and equipment used for each experiment, including test material, doses, cell line and engineered-tissue model, and supplier information (including city, state, and country for all animals, test material, equipment, and materials), and software information shall be provided to FDA.

1. Data analyses shall include appropriate statistical analyses as outline in Task section.

1. Interpretation and summary of results. A brief paragraph summarizing the pertinent conclusions of the study shall be provided.

1. All individual data needed for conclusions shall be presented in coherent tables. A copy of the raw data shall be provided separately in Attachment 1, appendix 1.

1. Documentation shall be provided to demonstrate the sensitivity, linearity, specificity, and reproducibility of the analytical method, relevant test guidelines (i.e., OECD guidelines, etc.) and procedural documents (i.e., the study protocol). For protocols proposing to use guidelines or procedures outside of the national or international standardized testing guidelines, the Contractor shall provide the relevant Standard Operating Procedures (SOPs) and method validation, as appropriate to the COR. If the FDA accepts an alternative method proposed by the contractor, then the alternative method will only be used to conduct this part of the study.

1. FDA will retain ownership of the data produced during performance of this contract and retains sole responsibility to draft, prepare, review, and submit any subsequent manuscripts using this data for publication in any format.

The Contractor shall provide the raw data and the Final Report to the COR in the following manner:

1. Microsoft Word electronic versions of the Final Report and all text-based appendices.

2. A print-ready PDF electronic version of the Final Report and all appendices.

3. Files of all raw data in their native file format (Excel).

Task 11: Final Briefing

Within 12 months of the award, the Contractor shall conduct a final briefing to FDA personnel via web conference. This briefing shall consist of a 30- to 40-minute presentation of the project and shall include a background on the problem, descriptions of methods used, the results of the tasks, and a brief discussion of the results and problems encountered during the study. The Contractor shall be prepared with appropriate briefing materials and shall send their presentation materials to the COR for review within two days prior to the presentation date.

3.5 Deliverables

The Contractor shall complete and deliver the following deliverables according to the following schedule:

Milestones Projected Completion Date

(The FDA recommends use of single lot for all flavor ingredient chemicals and metals)

0.5 month from completion of the external review

Within 10 business days following the completion of the study

References

SECTION 4 CONTRACT REQUIREMENTS

4.1 CONFLICTS OF INTEREST

The purpose of this section is to ensure that the contractor and its subcontractors:

(1) Are not biased because of their financial, contractual, organizational, or other interests which relate to the work under this contract, and

(2) Do not obtain any unfair competitive advantage over other parties by virtue of their performance of this contract.

(b) Scope. The language of section VII applies to performance or participation by the contractor, its parents, affiliates, divisions and subsidiaries, and successors in interest (hereinafter collectively referred to as “contractor”) in the performance of this contract as a prime contractor, subcontractor, co- sponsor, joint venture, consultant, or in any similar capacity.

(c) Definition of Conflict of Interest Conflict of interest means that because of other activities or relationships with other persons or organizations, a person is unable or potentially unable to render impartial assistance or advice to the Government, that the person's objectivity in performing the contract is or might be otherwise impaired, or that the person has or might acquire an unfair competitive advantage (see FAR 9.501).

(d) Contractor’s Conflict of Interest Responsibilities The individual contractor must be free of interest prior to performing under this contract. Any time prior to or during the performance of the contract the individual contractor believes that a potential or actual conflict exists, the individual should notify the contract officer and project officer. The contract officer will determine whether or not a conflict of interest exists and how to resolve or mitigate it. The contractor should not commence or continue working on the contract until directed by the contract officer.

(e) Warrant and Disclosure. The warrant and disclosure requirements apply to both the contractor and all subcontractors. The contractor warrants that, to the best of the contractor's knowledge and belief, there are no relevant facts or circumstances which would give rise to an organizational conflict of interest, as defined in FAR Subpart 9.5, and that the contractor has disclosed all relevant information regarding any actual or potential conflict. The contractor agrees it shall make an immediate and full disclosure, in writing, to the Contracting Officer of any potential or actual organizational conflict of interest or the existence of any facts that may cause a reasonably prudent person to question the contractor's impartiality because of the appearance or existence of bias or an unfair competitive advantage. Such disclosure shall include a description of the actions the contractor has taken or proposes to take in order to avoid, neutralize, or mitigate any resulting conflict of interest.

Prior to award, contractors that disclose potential or actual conflicts of interest shall submit a mitigation of conflicts of interest plan along with their technical and business proposals.

(f) Remedies. The Contracting Officer may terminate this contract for convenience, in whole or in part, if the Contracting Officer deems such termination necessary to avoid, neutralize or mitigate an actual or apparent organizational conflict of interest. If the contractor fails to disclose facts pertaining to the existence of a potential or actual organizational conflict of interest or misrepresents relevant information to the Contracting Officer, the Government may terminate the contract for default, suspend or debar the contractor from Government contracting, or pursue such other remedies as may be permitted by law or this contract.

(g) Subcontracts. The contractor shall include a clause substantially similar to this clause, including paragraphs (f) and (g), in any subcontract or consultant agreement.

(h) Prime Contractor Responsibilities– The contractor shall identify and avoid, neutralize, or mitigate any subcontractor organizational conflict prior to award of the contract to the satisfaction of the Contracting Officer. If the subcontractor's organizational conflict cannot be avoided, neutralized, or mitigated, the contractor must obtain the written approval of the Contracting Officer prior to entering into the subcontract. If the contractor becomes aware of a subcontractor's potential or actual organizational conflict of interest after contract award, the contractor agrees that the Contractor may be required to eliminate the subcontractor from its team, at the contractor's own risk. The contractor shall obtain from its subcontractors or consultants the disclosure required in FAR Part 9.507

(i) Waiver. The contractor may seek a waiver from the Head of the Contracting Activity by submitting such waiver request to the Contracting Officer, including a full written description of the requested waiver and the reasons in support thereof.

4.2 Government-Furnished Property, Material, Equipment, or Information (GFP, GFM, GFE, or GFI) None. GFP will not be furnished to the Contractor.

4.3 Security

There are no unique security requirements associated with performance of this contract.

4.4 Travel

There are no travel requirements associated with…