Final solicitation_Comprehensive toxicity analysis of individual ENDS .pdf

SEE ADDENDUMIS CHECKED

CODE 18a. PAYMENT WILL BE MADE BY

CODE

FACILITYCODE

17b. CHECK IF REMITTANCE IS DIFFERENT AND PUT SUCH ADDRESS IN OFFER

OFFEROR

WO75 CODE 16. ADMINISTERED BYCODE

X

X

SIZE STANDARD:

% FOR:SET ASIDE:UNRESTRICTED ORDAP

RFPIFB

10. THIS ACQUISITION ISCODE

RFQ

14. METHOD OF SOLICITATION

13b. RATING

NAICS:

SMALL BUSINESS

08/04/2023 1300 ET

07/20/2023

LISA JACKSON

(No collect calls)

INFORMATION CALL:

FOR SOLICITATION 8. OFFER DUE DATE/LOCAL TIMEb. TELEPHONE NUMBER a. NAME

4. ORDER NUMBER3. AWARD/ 6. SOLICITATION

75F40123R00115

5. SOLICITATION NUMBER

SOLICITATION/CONTRACT/ORDER FOR COMMERCIAL ITEMS 1. REQUISITION NUMBER PAGE OF

1 2 OFFEROR TO COMPLETE BLOCKS 12, 17, 23, 24, & 30

TELEPHONE NO.

17a. CONTRACTOR/

Silver Spring MD 20993 Building 75 10903 New Hampshire Avenue The US Food and Drug Administration

WO75

15. DELIVER TO

Beltsville MD 20705 4041 Powder Mill Road

ATTN:

9. ISSUED BY

7.

2. CONTRACT NO.

EFFECTIVE DATE

18b. SUBMIT INVOICES TO ADDRESS SHOWN IN BLOCK 18a UNLESS BLOCK BELOW

ISSUE DATE

DELIVERY FOR FOB DESTINA-

TION UNLESS BLOCK IS

MARKED

11.

SEE SCHEDULEX

12. DISCOUNT TERMS

THIS CONTRACT IS A

RATED ORDER UNDER

DPAS (15 CFR 700)

13a.

SERVICE-DISABLED

VETERAN-OWNED

SMALL BUSINESS

HUBZONE SMALL

BUSINESS

8(A)

DHHS/FDA/OAGS/DAP

WOMEN-OWNED SMALL BUSINESS

(WOSB) ELIGIBLE UNDER THE WOMEN-OWNED

SMALL BUSINESS PROGRAM

EDWOSB

24.

AMOUNT

23.

UNIT PRICE

22.

UNIT

21.

QUANTITY

20.

SCHEDULE OF SUPPLIES/SERVICES

19.

ITEM NO.

Delivery: 09/30/2023 Period of Performance: 09/30/2023 to 09/29/2025

1 Comprehensive toxicity analysis of individual ENDS

(Use Reverse and/or Attach Additional Sheets as Necessary)

HEREIN, IS ACCEPTED AS TO ITEMS:

DATED

ERIKA A. EAM

. YOUR OFFER ON SOLICITATION (BLOCK 5),

INCLUDING ANY ADDITIONS OR CHANGES WHICH ARE SET FORTH

COPIES TO ISSUING OFFICE. CONTRACTOR AGREES TO FURNISH AND DELIVER

ARE

ARE

31c. DATE SIGNED

27b. CONTRACT/PURCHASE ORDER INCORPORATES BY REFERENCE FAR 52.212-4. FAR 52.212-5 IS ATTACHED. ADDENDA

31a. UNITED STATES OF AMERICA (SIGNATURE OF CONTRACTING OFFICER)

30c. DATE SIGNED 31b. NAME OF CONTRACTING OFFICER (Type or print)

ALL ITEMS SET FORTH OR OTHERWISE IDENTIFIED ABOVE AND ON ANY ADDITIONAL

SHEETS SUBJECT TO THE TERMS AND CONDITIONS SPECIFIED.

27a. SOLICITATION INCORPORATES BY REFERENCE FAR 52.212-1, 52.212-4. FAR 52.212-3 AND 52.212-5 ARE ATTACHED. ADDENDA

26. TOTAL AWARD AMOUNT (For Govt. Use Only)

OFFER

STANDARD FORM 1449 (REV. 2/2012)

Prescribed by GSA - FAR (48 CFR) 53.212

ARE NOT ATTACHED.

ARE NOT ATTACHED.

AUTHORIZED FOR LOCAL REPRODUCTION

PREVIOUS EDITION IS NOT USABLE

30b. NAME AND TITLE OF SIGNER (Type or print)

30a. SIGNATURE OF OFFEROR/CONTRACTOR

28. CONTRACTOR IS REQUIRED TO SIGN THIS DOCUMENT AND RETURN

25. ACCOUNTING AND APPROPRIATION DATA

29. AWARD OF CONTRACT:

REF.

32e. MAILING ADDRESS OF AUTHORIZED GOVERNMENT REPRESENTATIVE

32c. DATE 32b. SIGNATURE OF AUTHORIZED GOVERNMENT REPRESENTATIVE

ACCEPTED, AND CONFORMS TO THE CONTRACT, EXCEPT AS NOTED:

32a. QUANTITY IN COLUMN 21 HAS BEEN

RECEIVED INSPECTED

40. PAID BY39. S/R VOUCHER NUMBER38. S/R ACCOUNT NUMBER

37. CHECK NUMBER

FINALPARTIAL

36. PAYMENT

FINALPARTIAL

35. AMOUNT VERIFIED

CORRECT FOR

34. VOUCHER NUMBER33. SHIP NUMBER

COMPLETE

32g. E-MAIL OF AUTHORIZED GOVERNMENT REPRESENTATIVE

42d. TOTAL CONTAINERS42c. DATE REC'D (YY/MM/DD)

42b. RECEIVED AT (Location)

42a. RECEIVED BY (Print)

41c. DATE41b. SIGNATURE AND TITLE OF CERTIFYING OFFICER

41a. I CERTIFY THIS ACCOUNT IS CORRECT AND PROPER FOR PAYMENT

STANDARD FORM 1449 (REV. 2/2012) BACK

24.

AMOUNT

23.

UNIT PRICE

22.

UNIT

21.

QUANTITY

20.

SCHEDULE OF SUPPLIES/SERVICES

19.

ITEM NO.

32f. TELEPHONE NUMBER OF AUTHORIZED GOVERNMENT REPRESENTATIVE

32d. PRINTED NAME AND TITLE OF AUTHORIZED GOVERNMENT REPRESENTATIVE

2 2 of

FDA RFP No.: 75F40123R00115

Food and Drug Administration (FDA)

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

2 | P a g e

Section B - Continuation of Standard Form (SF) 1449 This is a combined synopsis/solicitation for commercial items prepared in accordance with the format in Subpart 12.6, as supplemented with additional information from FAR Part 15. This solicitation is being issued as a Request for Proposal (RFP). The solicitation document and incorporated provisions and clauses are those in effect through Federal Acquisition Circular (FAC) 2023-04 effective June 2, 2023. The North American Industrial Classification System (NAICS) code for this procurement is 541690, Other Scientific and Technical Consulting Services.

B.1 GENERAL

The contractor will provide services associated with research requirements outlined in this SOW to research 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.

(a)

(b) The contract term is a total of two (2) years from the date of award; and

(c) The contract will be for Commercial Services, Firm-fixed Price contract.

B.2 PRICE SCHEDULE

All cost proposed prices for each task must include labor prices (labor rates proposed must be fully loaded rates, labor categories, and all Other Direct Cost (ODC’s).

Task Number

Description Price

1 Kickoff Meeting $ 2 Bi-Weekly Meetings $ 3 Monthly Progress Reports $ 4 Protocol Development $ 5 Determination of synergy

5.1 Acquire reactants

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

5.2 Cell viability and cytokine levels $

5.3 Ames Assay $

5.4 Comet Assay $

5.5 Micronucleus Assay $

5.6 Acquire biologicals for 3D model $

6 3D model exposure $ 7 Study Implementation $ 8 Execute the Data Analysis Plan $ 9 Outline Final Report

10 Draft Final Report $ 11 Final Report $ 12 Final Briefing $

Total Price $

3 | P a g e

B.3 STATEMENT OF WORK

B.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.

B.3.2 Objectives The FDA will provide a final list of 125 individual flavor ingredient chemicals selected from a primary chemical list for testing included in Appendix 1. The final list of chemicals will be selected based on potential interaction with the selected metals (Cr, Cd, and Ni) and we anticipate that the chemical complexity in this final library will be similar to the preliminary list (Appendix 1) and will have no impact on the performance of the experiments.

The Contractor shall use in vitro quantitative high throughput screening (qHTS) to identify the cytotoxic and genotoxic potential of individual flavor ingredient chemicals alone, metals alone, and individual flavor ingredient chemicals in the presence of metals in a range of doses using CellTiter- Blue®, a variant of the neutral red uptake, in the human bronchial epithelial cell line 16HBE14o- and

4 | P a g e their levels of the inflammatory cytokine IL-6 released to the supernatant, in vitro micronucleus assay using human lymphoblast TK6 cell line, the comet assay in TK6 cell line, and the bacterial reverse gene mutation test (Ames).

The Contractor shall obtain physiologically relevant data using commercially available reconstructed human lung tissues that are exposed to aerosolized flavor ingredient chemicals alone and flavor-metal complexes and report cell viability, oxidative stress, inflammatory mediators, lung barrier function, and DNA damage.

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.

B.3.3 Scope The Contractor shall investigate the toxicity of 125 flavor ingredient chemicals alone and combined with metal using high throughput assays. The FDA will produce a final library of 125 flavor ingredient chemicals based on the predicted propensity of flavor ingredient chemicals to interact with metals. 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 to use in subsequent mutagenic and genotoxic assays, which consist of assays in cells lines and in bacteria. The Contractor shall determine based on measurables of the mutagenic and genotoxic experiments a list of 20 flavor ingredient chemicals and 20 flavor-metal combinations and their concentrations to be tested in the reconstructed human lung tissue model to measure the effects on metabolism, membrane integrity, cell viability, inflammation, and DNA damage. The Contractor shall perform all data analyses, provide FDA all of the raw data, and write study reports. All results and source documents shall be submitted to the Contracting Officer’s Representative (COR.)

B.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).

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 ten calendar days after issuance of the task order. 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 starting

5 | P a g e after the completion of the external review and before the start of experiments. 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 biweekly meeting, the deliverable 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 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 needs 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.

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 125 flavor ingredient chemicals and metals (Cr, Cd, and Ni) covering each possible combination using CellTiter-Blue in a high throughput 1536-well (or 384-well) plate format in 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 made after incubation time of 6 hours with test reactants prepared as described in the manual1.

The Contractor shall evaluate the supernatant after the cell viability assay for inflammatory cytokine IL-

1 https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/101/celltiter-blue-cell-viability-assay-protocol.pdf

6 | P a g e

6 levels using time-resolved fluorescence resonance energy transfer (TR-FRET) assays (PerkinEler, TRF1223M) according to the manufacturer’s protocol and with fluorophores compatible with CellTiter- Blue.1

The Contractor shall prepare high concentration stocks of metal at 5-10 mM in 1% nitric acid and this concentration verified by Inducted Coupled Plasma Mass Spectrometry (ICP-MS) and 1mM flavor chemical stock in dimethyl sulfoxide (DMSO), 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% DMSO.

The Contractor shall perform qHTS for detection of mutagenic and genotoxic endpoints of the library of flavor ingredient chemicals and metals covering each possible combination using two mammalian cell assays and the bacterial reverse gene mutation test (miniaturized Ames test, scale is 24-well plates). The Contractors shall use two mammalian high throughput assays such as DNA damage detection assay (CometChip, scale is 96-well plates) that is based on the comet assay and the in vitro micronucleus assay using TK6 cells (the scale is 96-well plates).

The Contractor shall test the viability assay and 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 8, log base doses, for example, 0, -1, -2, -3, -4, -5, -6, -7, -8, -9 to obtain 8 by 8 assay matrixes per metal-flavor combination.

The Contractor shall perform the mutagenicity and 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 liver extract.

The Contractor shall perform the miniaturized Ames test on the plate incorporation modality, 24-well plates, on five S. typhimurium strains in six doses, by triplicate, sorted in 6 by 6 assay matrix divided in two plates, after 48-72 hours of incubation with test chemicals and in the presence and absence of metabolic activator, as described in OECD guidelines 358 and 471.2

The Contractor shall perform the CometChip assay such as Trevigen in 96-well plates, three doses sorted in 5 by 5 assay matrix after 24 hours of incubation with test chemicals in TK6 cells, following the manufacture’s protocol with modifications as reported in Heflich et al.3,4

The Contractor shall perform the in vitro micronucleus assay in 96-well plates, three doses, following manufacture’s protocols, using TK6 cells in 5 by 5 assay matrix after 6 hours of incubation with test chemicals in the presence and absence of metabolic activator and after 24 hours of incubation with metabolic activator.23

2 https://www.eurofinsdiscoveryservices.com/services/adme-tox/in-vitro-toxicology/genotoxicity/in-vitro-micronucleus-assay 3 https://gentronix.co.uk/services/clastogens-and-aneugens/96-well-mnt-and-fish/

7 | P a g e

The Contractor shall perform autofluorescence detection 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 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. For positive control in the cell viability and cytokine screening the Contractor shall use ethyl maltol and nicotine in the cell viability/cytokine assay and benzo[a]pyrene in the mutagenic and genotoxic assays, whereas the negative control is 1% DMSO vehicle. The test flavor ingredient chemicals and metals will be added directly to plates by automatic liquid handling with a final concentration of 1% DMSO. 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 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 test. 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.6,7

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

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, report %CV >10% from min/max and control plates, and identify outliers in dose-response curves by an statistical method.11

8 | P a g e

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

The Contractor shall analyze the dose-response plot generated in the mutagenicity and genotoxicity 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 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).

The contractor shall work with the FDA to choose 20 flavor ingredient chemicals and 20 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 next task at a concentration above the NOGEL. A higher priority shall be assigned to flavor ingredient chemicals or chemical metal complex that significantly induce IL-6 expression, or with mutagenic or genotoxic effects.

Task 6: ALI exposure of in vitro 3D human upper airway epithelium The contractor shall purchase and maintain 3D human upper airway epithelium (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 in compliance to CORESTA recommended method (CRM) No. 81 protocol, with puff duration of 3S, puff volume of 55ml, one puff every 30 second, 200 puff in total and with 24h rest. 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 following endpoint shall be measured to assess toxicity of exposure to the 3D human upper airway epithelium:

• Barrier integrity: shall be examined using TEER (at 24h, 7, 14 and 21days post-exposure) and permeability assay (identify and quantify levels of active efflux).

• Cell viability: shall be examined use trypan blue assay at 21 days post exposure.

9 | P a g e

• Oxidative stress: shall be examined use 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: The error‐corrected next‐generation sequencing method Duplex Sequencing shall be used to detect mutations, which is used to sensitively detect DNA damage after aerosol exposure.3

Task 7: Study Implementation 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.

7.1 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 cytotoxicity 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 8: Execute the Data Analysis Plan The Contractor shall perform the data analysis indicated in the COR approved Data Analysis Plan. The Data Analysis Plan shall include FDA review of all draft deliverables and as needed, subsequent contractor revision, and delivery of final reports.

Task 9: 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

10 | P a g e 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 10: 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 11: 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 9 and 10) 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.

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

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

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

5. 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.

6. 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.

11 | P a g e

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 12: 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.

Deliverables

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

Task Number

Work Milestones

Projected Completion Date

1 Kickoff Meeting Within 2 weeks from date of award

2 Bi-Weekly Meetings Every two weeks starting following the completion of the external review and before start of experiments

3 Monthly Progress Reports 10th day of every month 4 Protocol Development Submit protocols for external review within 30 calendar days of award.

5 Determination of synergy

5.1 Acquire reactants

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

0.5 month from completion of the external review

5.2 Cell viability and cytokine

levels

0.5 month from acquiring reactants and 1 month thereafter for completion

5.3 Ames Assay 0.5 month from completion of

cytokine level assay and 1 month thereafter for completion

5.4 Comet Assay 0.5 month from completion of

Ames assay and 1 month thereafter for completion

12 | P a g e

5.5 Micronucleus Assay 0.5 month from completion of

Comet Assay and 1 month thereafter for completion

5.6 Acquire biologicals for 3D

model

Within 1 month of completion of Micronucleus Assay, and 1 week before starting ALI exposure

6 3D model exposure 1 week from acquisition of biologicals and 2 months thereafter for completion

8 Execute the Data Analysis Plan 0.5 month from completion of each experiment

9 Outline Final Report

Within 10 business days following the completion of the study

10 Draft Final Report 3 weeks from Outline Final Report and within 11 months of award

11 Final Report Within 20 business days following receipt of final FDA comments in the prior biweekly meeting and within 12 months of award

12 Final Briefing Within 12 months from award and no more than 0.5 month from completion of Final Report

References

1. Carlstron J, Hurt S, Bosse R. High Throughput Quantitation of Cytokine Biomarkers using LANCE Ultra TR-FRET Assays. PerkinElmer, Inc. Accessed December 10, 2022.

https://resources.perkinelmer.com/lab-solutions/resources/docs/app_lance_ultra_inflammation_biomarkers.pdf

2. Escobar PA, Kemper RA, Tarca J, et al. Bacterial mutagenicity screening in the pharmaceutical industry. Mutat Res. Apr-Jun 2013;752(2):99-118. doi:10.1016/j.mrrev.2012.12.002

3. Wang Y, Mittelstaedt RA, Wynne R, et al. Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing. Environ Mol Mutagen. Jun 2021;62(5):306-318. doi:10.1002/em.22444

4. Sykora P, Witt KL, Revanna P, et al. Next generation high throughput DNA damage detection platform for genotoxic compound screening. Scientific Reports. 2018/02/09 2018;8(1):2771.

doi:10.1038/s41598-018-20995-w

5. Simeonov A, Jadhav A, Thomas CJ, et al. Fluorescence Spectroscopic Profiling of Compound Libraries. Journal of Medicinal Chemistry. 2008/04/01 2008;51(8):2363-2371. doi:10.1021/jm701301m

6. Kocaoba S. Determination of some heavy metals from aqueous solutions using modified Amberlite XAD-4 resin by selective solid-phase extraction. Journal of Analytical Science and Technology. 2022/05/16 2022;13(1):15. doi:10.1186/s40543-022-00324-7 https://resources.perkinelmer.com/lab-solutions/resources/docs/app_lance_ultra_inflammation_biomarkers.pdf https://resources.perkinelmer.com/lab-solutions/resources/docs/app_lance_ultra_inflammation_biomarkers.pdf

13 | P a g e

7. Rayner MH, Suzuki KT. A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum. Biometals. Jul 1995;8(3):188-92. doi:10.1007/bf00143374

8. Zhang J-H, Chung TDY, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. SLAS Discovery. 1999/04/01/ 1999;4(2):67-73.

doi:https://doi.org/10.1177/108705719900400206

9. Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R. Statistical practice in high-throughput screening data analysis. Nature Biotechnology. 2006/02/01 2006;24(2):167-175. doi:10.1038/nbt1186

10. Brideau C, Gunter B, Pikounis B, Liaw A. Improved Statistical Methods for Hit Selection in High-Throughput Screening. Journal of Biomolecular Screening. 2003;8(6):634-647.

doi:10.1177/1087057103258285

11. Murray D, Wigglesworth M, Bittker JA, Ross NT.

12. Lederer S, Dijkstra TMH, Heskes T. Additive Dose Response Models: Explicit Formulation and the Loewe Additivity Consistency Condition. Original Research. Frontiers in Pharmacology. 2018- February-06 2018;9doi:10.3389/fphar.2018.00031

13. Wooten DJ, Meyer CT, Lubbock ALR, Quaranta V, Lopez CF. MuSyC is a consensus framework that unifies multi-drug synergy metrics for combinatorial drug discovery. Nature Communications. 2021/07/29 2021;12(1):4607. doi:10.1038/s41467-021-24789-z

14. Zheng S, Wang W, Aldahdooh J, et al. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets. Genomics, Proteomics & Bioinformatics.

2022/06/01/ 2022;20(3):587-596. doi:https://doi.org/10.1016/j.gpb.2022.01.004

15. Piersma AH, Hernandez LG, van Benthem J, et al. Reproductive toxicants have a threshold of adversity. Critical Reviews in Toxicology. 2011/07/01 2011;41(6):545-554.

doi:10.3109/10408444.2011.554794

16. Cao X, Mittelstaedt RA, Pearce MG, et al. Quantitative dose–response analysis of ethyl methanesulfonate genotoxicity in adult gpt-delta transgenic mice. Environmental and Molecular Mutagenesis. 2014;55(5):385-399. doi:https://doi.org/10.1002/em.21854

17. Gollapudi BB, Johnson GE, Hernandez LG, et al. Quantitative approaches for assessing dose– response relationships in genetic toxicology studies. Environmental and Molecular Mutagenesis.

2013;54(1):8-18. doi:https://doi.org/10.1002/em.21727

18. Johnson GE, Soeteman-Hernández LG, Gollapudi BB, et al. Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment.

Environmental and Molecular Mutagenesis. 2014;55(8):609-623. doi:https://doi.org/10.1002/em.21870

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.

https://doi.org/10.1177/108705719900400206 https://doi.org/10.1016/j.gpb.2022.01.004 https://doi.org/10.1002/em.21854 https://doi.org/10.1002/em.21727 https://doi.org/10.1002/em.21870

14 | P a g e

(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

15 | P a g e 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.

GOVERNMENT FURNISHED PROPERTY

Government Furnished Property (GFP) will not be furnished for this requirement

SECURITY

During the course of performance FDA will provide the Contractor with data that is strictly confidential and shall not be disclosed to other than authorized individuals and organizations.

Contractor personnel must sign the provided FDA Form 3398 - Commitment to Protect Non- Public Information within five days of receiving the form and prior to commencing any work involving information under issued tasks. The Form will specify all information designated as non-public.

Where task orders require Contractor access to FDA systems using FDA provided laptops or access to FDA Document Control Centers and Mailrooms, all Contractor personnel utilizing those systems shall complete and sign the equip85, eArrive, and HHS 745 forms in order to be badged and gain systems access. The Contractor shall ensure the completion of these forms.

Badging may take 21- 31 business days and the Contractor shall reflect that in the timeline for a task order.

The Contractor shall ensure compliance with IT security requirements, including the following:

DHHS Information Security and Privacy Program http://www.hhs.gov/ocio/securityprivacy/index.html

Federal Information Security Management Act (FISMA) http://www.whitehouse.gov/sites/default/files/omb/memoranda/m03- 19.pdf National Institute of Standards and Technology (NIST) SP800-53 http://www.hhs.gov/ocio/securityprivacy/index.html http://www.whitehouse.gov/sites/default/files/omb/memoranda/m03-19.pdf http://www.whitehouse.gov/sites/default/files/omb/memoranda/m03-19.pdf

16 | P a g e http://csrc.nist.gov/publications/nistpubs/800-53-Rev3/sp800-53-rev3-final_updated- errata_05- 01-2010.pdf

FDA Security Authorization process, which closely resembles NIST SP800-37 http://csrc.nist.gov/publications/nistpubs/800-37-rev1/sp800-37-rev1-final.pdf

Format and Media: The Contractor shall furnish deliverables in a manner consistent with the FISMA and NIST security requirements of the information provided. For example, the Contractor shall not send Sensitive Personal Identifying Information via e-mail across the internet.

PLACE OF PERFORMANCE

All services will be performed at the Contractor’s facilities.

PERIOD OF PERFORMANCE

The period of performance (PoP) for the contract will be two (2) years from the date of award.

[End of Section B]

SECTION C - CONTRACT CLAUSES

C.1 52.212-4 CONTRACT TERMS AND CONDITIONS—COMMERCIAL PRODUCTS

AND COMMERCIAL SERVICES. (DEC 2022). This clause is incorporated by reference (refer to SF1449 block 27a)

ADDENDUM TO FAR 52.212-4:

C.2 ADDITIONAL CLAUSES INCORPORATED BY REFERENCE

This contract incorporates one or more FAR and HHSAR clauses by reference, with the same force and effect as if they were given in full text. Upon request, the Contracting Officer will make their full text available. Also, the full text of a clause may be accessed electronically at this address:

https://www.acquisition.gov/browse/index/far and http://www.hhs.gov/grants/contracts/contract-policies-regulations/hhsar/index.html http://csrc.nist.gov/publications/nistpubs/800-53-Rev3/sp800-53-rev3-final_updated-errata_05-01-2010.pdf http://csrc.nist.gov/publications/nistpubs/800-53-Rev3/sp800-53-rev3-final_updated-errata_05-01-2010.pdf http://csrc.nist.gov/publications/nistpubs/800-53-Rev3/sp800-53-rev3-final_updated-errata_05-01-2010.pdf http://csrc.nist.gov/publications/nistpubs/800-37-rev1/sp800-37-rev1-final.pdf https://www.acquisition.gov/browse/index/far http://www.hhs.gov/grants/contracts/contract-policies-regulations/hhsar/index.html

17 | P a g e

Reg Clause Date Clause Title FAR 52.203-16 Jun 2020 Preventing Personal Conflicts of Interest FAR 52.203-17 Jun 2020 Contractor Employee Whistleblower Rights and

Requirement To Inform Employees of Whistleblower Rights

FAR 52.204-4 May 2011 Printed or Copied Double-Sided on Postconsumer Fiber Content Paper

FAR 52.204-12 Oct 2016 Unique Entity Identifier Maintenance

FAR 52.204-13 Oct 2018 System for Award Management and Maintenance FAR 52.204-18 Aug 2020 Commercial and Government Entity Code Maintenance FAR 52.204-19 Dec 2014 Incorporation by…