The Science of Substance | 1. FY 2020 in Review

FISCAL YEAR FISCAL 2020 ANNUAL REPORT FOR REPORT FOR ANNUAL The Science of Substance 2020 Contents

1.0 FY 2020 in Review 1 4.0 Science, Innovation, and Methods

1.1 Letter from the Director 2 Development 29

1.2 Timeline 4 4.1 Development of New Approach Methodologies 30

1.3 NTP Public Health Impact 5 NICEATM Activities 31 4.2 Tox21 40 2.0 About NTP 9 4.3 Validation and Adoption of NAMs 42

2.1 Organization and Structure 10 ICCVAM Test Method Evaluation Activities 43

2.2 Funding 12 ICCVAM International Validation Activities 44 Progress toward Strategic Roadmap Goals 46 3.0 Publishing, Collaboration, and Science Measuring Progress in Adopting Alternatives and Reducing Animal Use 47 Communications 1 5

3.1 Completed NTP Reports and Publications 16 5.0 Testing and Assessment 49 3.2 Advisory Groups 20 5.1 NTP at NIEHS/DNTP 52 3.3 Scientific Panels 21 5.1.1 Chemical Screening 52 3.4 Workshops and Meetings 22 5.1.2. Chemical Testing 55 NICEATM Meetings and Workshops 22 5.1.3 Noncancer Research 55 ICCVAM Meetings 24 5.1.4 Cancer Health Effects Research 56 3.5 Collaboration and Scientific Leadership 25 5.2 NTP at FDA/NCTR 60 FDA/NCTR 25 5.3 NTP at NIOSH 62 CDC/NIOSH 25 NIH/NCATS/DPI 26 DOE/ORNL 26 EPA/NCCT 26 EPA/NCEA 26 NIST 27

3.6 Training Opportunities 27 1.0 FY 2020 in Review The Science of Substance | 1. FY 2020 in Review 3

Publication of 260 peer-reviewed research articles and reports on articles and reports on 260 peer-reviewed research Publication of , 2-hydroxy- concern, including perfluorooctanoic substances of and traffic-related (used in ), 4-methoxybenzophenone air pollution. for neonicotinoid pesticides, Parkinson’s Release of scoping reviews disease, and progestogens. for potential listing in the 15th Report on Reviews of eight substances mandated, science-based, public Carcinogens, a congressionally prepares for the Health and Human health document that NTP Services Secretary.

f f f The National Toxicology Program (NTP) annual report for fiscal year (FY) year (FY) fiscal for report annual (NTP) Program Toxicology National The meanings carries two . This title of Substance titled The Science 2020 is we the substances First, it represents we perform. the vital work about public to inform necessary scientific knowledge provide the study to the it reflects the substantive way decision makers. Second, health policy and health concerns touches on emerging public science we conduct every day. improves lives including: spotlights our many accomplishments, This annual report and reviewing literature, NTP develops In addition to evaluating substances methods to stay on the cutting edge of new technologies and research 2020, this work included several activities to toxicological science. In FY testing, such as a workshop to discuss advance alternatives to animal a series of webinars on the application and acute systemic toxicity testing, and development of human-based in benefits of recombinant antibodies, approach methodologies. vitro systems and computational In FY 2021, we will continue NTP’s critical role in the generation, that interpretation, and communication of toxicological information public health. strengthens science and informs decisions that protect is key. We should Communication and collaboration among stakeholders establish common advocate to ensure that toxicologists around the world research information. approaches for collecting, sharing, and archiving their stature as a My goal is to enhance our program’s effectiveness and our role as a valuable global leader in toxicology research and to develop resource that informs decisions for a healthier tomorrow. Ph.D. Woychik, Rick Director, National Toxicology Program National Institute of Environmental Health Sciences Rick Woychik, Ph.D., Rick Woychik, Ph.D., was appointed Director of the National Institute of Environmental Health Sciences and the National Toxicology Program in June 2020

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1 Letter from the Director from the Letter The Science of Substance | 1. FY 2020 in Review Review in 2020 FY 1. | Substance of Science The 2 The Science of Substance | 1. FY 2020 in Review 5

Report on Carcinogens informs the TRI. 14th identifies 1-BP as “reasonably anticipated to be a human to be a human 14th Report on Carcinogens identifies 1-BP as “reasonably anticipated 14th Report on Carcinogens informed the rule. .3 dust-lead clearance levels for floors and window sills after completing an abatement activity.dust-lead clearance levels for floors lead exposure. EPA relied on NTP research on the health effects of low-level EPA is soliciting information from the public (84 FR 66369) as it considers proposing a future to reporting under Section rule on adding certain PFAS to the list of toxic chemicals subject Act and Section 6607 of the 313 of the Emergency Planning and Community Right-to-Know Pollution Prevention Act. EPA is collaborating with NTP to study the toxicology of PFAS. the Toxics Release EPA is proposing corrections to existing regulatory language for Inventory (TRI) Program (84 FR 65739), including updated identifiers, formulas, and names for certain TRI-listed chemicals. and do not alter existing These proposed corrections maintain previous regulatory actions reporting requirements or impact compliance burdens or costs. The The Environmental Protection Agency (EPA) issued a final rule (86 FR 983) lowering theThe Environmental Protection Agency known as 1-BP) to the ) to add n-propyl bromide (also EPA is granting petitions (85 FR 36851 in the Clean Air Act. list of hazardous air pollutants contained The of carcinogenicity from studies in experimental animals. carcinogen” based on evidence Safety Administration, on behalf of the U.S. EPA and the National Highway Traffic final rules (85 FR 24174) to amend and establish Department of Transportation, issued passenger cars dioxide and fuel economy standards for model year 2021–2026 and light trucks. The 1

ƒ Substances and Polyfluoroalkyl Per- ƒ ƒ Program Release Inventory Toxics ƒ ƒ ƒ Federal Regulatory Updates Dust-lead Clearance Levels ƒ Bromide n-Propyl ƒ ƒ Fuel Emissions Vehicle ƒ ƒ NTP contributes study data and recommendations used by recommendations study data and NTP contributes to agencies research and regulatory health state and federal passed following regulations public health. The help protect by NTP research. in FY 2020 were informed NTP Public Health Impact Health NTP Public presented an overview of webinar presented an overview of September 2–3, 2020 September 2–3, AdvisoryScientific Committee on Methods Meeting Toxicological Alternative During the meeting, the committee heard presentations on synthetic antibody technology, computational toxicology tools, and approaches to curate and characterize data for alternative methods use. April 6 NIEHS Science Symposium: Honoring Linda Birnbaum FormerDr. NTP Director The National Institute of Environmental Health Sciences (NIEHS) hosted the 2020 Birnbaum Research Symposium to honor the contributions of former NIEHS and NTP Director Dr. Linda Birnbaum, who retired in October 2019. October 30–31 October Gaps:Mind the Prioritizing Activities to Meet Regulatory Acute Needs for Systemic Toxicity , organized by NTP During the workshop for the Evaluation Interagency Center Methods of Alternative Toxicological the Physicians Committee (NICEATM) and attendees for Responsible Medicine, for assessing acute discussed the potential without animal testing. systemic toxicity January 21 of Practice Communities ICCVAM Use Affinity of Animal-free Webinar: Reagents The monoclonal and polyclonal antibodies to and examined animal-free technologies produce affinity reagents. 2019 2019 2020 2020 2020 2020 2020

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May 21 Public Forum ICCVAM During the forum, the International Coordinating Committee on the Validation of Alternative Methods (ICCVAM) members presented on their activities to advance new approaches to testing chemicals and medical products and reducing the amount of testing required. February21 of Scientific CounselorsNTP Board Meeting During the meeting, members discussed recent activities related to exposures (per- and polyfluoroalkyl substances [PFAS], nanoplastics, and microplastics), disease (hypertensive disorders of pregnancy), and new hazard-assessment approaches. Peer-review Meeting: Draft NTP NTP Meeting: Draft Peer-review Toxicology Reports on the Technical Studies of and Carcinogenesis 2-Hydroxy-4-methoxybenzophenone and Acid Perfluorooctanoic During the meeting, panelists discussed studiesdraft NTP technical reports on the (TRof 2-hydroxy-4-methoxybenzophenone (TR 598). 597) and perfluorooctanoic acid December 12 December 12 Timeline The Science of Substance | 1. FY 2020 in Review Review in 2020 FY 1. | Substance of Science The 4 The Science of Substance | 1. FY 2020 in Review 7 9-THC) to the list 9-tetrahydrocannabinol (Δ 9-tetrahydrocannabinol 9-THC cannabis (marijuana) smoke and Δ added cannabis (marijuana) that exposures to bisphenol A (BPA) from certain polycarbonate certain polycarbonate A (BPA) from to bisphenol that exposures OEHHA determined to needed dose concentration allowable exceed the maximum products did not eyewear under Proposition 65. require a warning possible health effects on the cited NTP research on BPA, which has The California issuance children. gland of fetuses, infants, and brain and prostate OEHHA 65. toxicity for purposes of Proposition by the state to cause reproductive of chemicals known addition. helped inform the and carcinogenic rodent studies NTP toxicological

California Updates Regulatory A Bisphenol ƒ ƒ and Δ Cannabis Smoke ƒ ƒ

α,α,α-

Air Toxics adopted new the 14th Report on Carcinogens. found sufficient evidence that blood lead concentrations are associated are associated evidence that blood lead concentrations research found sufficient

. trifluorotoluene for use in the Air Toxics Hot Spots Program Proposition 65 No Significant Risk Level of 23 micrograms OEHHA also proposed adopting a per day for p-chloro-α,α,α-trifluorotoluene. data for p-chloro-α,α,α-trifluorotoluene in research NTP summarized carcinogenicity . published in 1992, 2009, and 2018 reference exposure concentrations for the chemical solvent toluene for use in the reference exposure concentrations Hot Spots Program. studies, where necrosis of the NTP published research on the effects of toluene in rodent brain was observed. and slope factor for p-chloro- OEHHA is also adopting a new cancer inhalation unit risk The California Office of Environmental Health Hazard Assessment (OEHHA) has The California Office of Environmental ) to the National Primary Drinking Water Drinking Water National Primary ) to the revisions (84 FR 61684 regulatory EPA is proposing Water Act. Safe Drinking authority of the copper under the for lead and Regulation to require use of lead- changes (85 FR 54235) to existing regulations EPA has also finalized facilities. and residential and nonresidential in public water systems free plumbing materials NTP findings EPA used this research and its effects in adults and children. with adverse health in impacts associated with reductions sources to assess the health as one of two main lead ingestion. its public health goal (PHG) for 1,2-dibromo-3-chloropropane, a soil OEHHA updated its public health goal (PHG) for 1,2-dibromo-3-chloropropane, a effects. fumigant formerly used in agriculture that causes adverse health adverse health effects A PHG is the concentration of a drinking water contaminant at which are not expected to occur from a lifetime of exposure. OEHHA relied on NTP data from inhalation studies in rodents. OEHHA also updated its PHG for four trihalomethanes found in drinking water as a result of disinfection methods: , bromodichloromethane, dibromochloromethane, and bromoform. In its guidance, OEHHA cites numerous NTP studies, including OEHHA proposed adopting Proposition 65 No Significant Risk Levels for , OEHHA proposed adopting Proposition , which are all used in cosmetic treatments dibromoacetic acid, and (such as chemical peels and tattoo removal) and as topical medication. This action was supported by NTP research on trichloroacetic acid and dichloroacetic acid and its review of carcinogenicity studies on dibromoacetic acid.

ƒ ƒ ƒ ƒ California Updates Regulatory Solvents Toluene ƒ Federal Regulatory Updates Federal Water Copper in DrinkingLead and ƒ ƒ ƒ 1,2-Dibromo-3-Chloropropane and Trihalomethanes ƒ ƒ ƒ ƒ ƒ Halogenated Acetic ƒ ƒ The Science of Substance | 1. FY 2020 in Review Review in 2020 FY 1. | Substance of Science The 6 The Science of Substance | 1. FY 2020 in Review 9 .0 2 About NTP

The National Toxicology Program The National Toxicology Program (NTP) provides the scientific basis for programs, activities, and policies that promote health or lead to the prevention of disease. Founded in 1978, NTP plays a critical role in generating, interpreting, and sharing toxicological information about hazardous substances in our environment. The Science of Substance | 1. FY 2020 in Review Review in 2020 FY 1. | Substance of Science The 8 The Science of Substance | 2. About NTP 11

Counselors Scientific Advisory Scientific Advisory Board of Scientific Board of Scientific Toxicological Methods Special Emphasis Panels External and Science Science External and Committee on Alternative Committee on Alternative Oversight and Review Oversight and NTP Director Policy Oversight NTP Executive Committee Assistant Secretary for Health Secretary for Assistant NIH/NIEHS FDA/NCTR CDC/NIOSH CDC/NIOSH NTP Core Agencies NTP is located administratively at NIEHS. Rick Woychik, Ph.D., was appointed at NIEHS. Rick Woychik, Ph.D., was appointed NTP is located administratively June 2020 after serving as acting director since director of NIEHS and NTP in D.V.M., Ph.D., is NTP associate director and October 2019. Brian Berridge, of NTP (DNTP) at NIEHS, which is a focal point scientific director of the Division research practices and for many NTP activities. NIEHS and NTP support best technology to discover how the environment affects embrace developments in in the field of environmental health sciences. people and to maintain leadership of NIOSH. Staff from two NIOSH divisions participateJohn Howard, M.D., is director Research Branch, andin NTP activities. Elizabeth Whelan, Ph.D., chief of the Field NTP activities withinCheryl Estill, Ph.D., Exposure Assessment Team Lead, manage Ph.D., overseesthe Division of Field Studies and Engineering. Donald Beezhold, participation in NTPactivities in the Health Effects Laboratory Division. NIOSH’s and safety under theis consistent with its mandate to protect worker health Mine Safety and Health Act.Occupational Safety and Health Act and the Federal management oversight William Slikker Jr., Ph.D., director of FDA/NCTR, provides a center within FDA, and coordination of NTP activities within the center. As Rear Admiral Denise NCTR also is under the leadership of FDA’s chief scientist, methods Hinton, M.S. Scientists at NCTR provide innovative technologies, that support development, vital scientific training, and technical expertise product centers. NCTR the NTP mission, as well as the regulatory roles of FDA and are critical in conducts an array of studies that reflect the NTP mission roles. supporting FDA product centers and their regulatory

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2 Three agencies form the core of NTP: the National Institute the core of NTP: the National Institute Three agencies form Sciences (NIEHS) of the National of Environmental Health National Institute for Occupational Institutes of Health (NIH); of the Centers for Disease Safety and Health (NIOSH) (CDC); and Food and Control and Prevention primarily through the National Center Administration (FDA), (NCTR). for Toxicological Research Organization and Structure and Organization The Science of Substance | 2. About NTP About 2. | Substance of Science The 10 The Science of Substance | 2. About NTP 13 Contractor Contractor Services Kelly Government Battelle Memorial Institute Midwest Research Institute Research Triangle Laboratories Experimental Pathology of American Holdings Laboratory Corporation Sciome, LLC Interior/Federal Consulting Department of the Center Group/National Business Institute for Department of Energy with Oak Ridge Science and Education Integrated Laboratory Systems Southern Research Institute Battelle Memorial Integrated Laboratory Systems Burleson Research Technologies Instem Experimental Pathology Laboratories Integrated Laboratory Systems PAI/Charles River Laboratories and National Academies of Sciences, Engineering, Medicine Taconic Biosciences Planview CSS-Dynamac Corporation ICF International, Inc. Social and Scientific Systems Vistronix Texas A&M University Battelle Memorial mice 1 Description Description staff and scientific Administrative chemistry services Analytical repository Archives and specimen project Bioinformatics methylation Bioinformatics support Botanicals Safety Consortium Collaborative work with Ramazzini Institute methods Evaluation of alternative toxicological Evaluation of toxicity after early life exposure chemicals Evaluation of the toxicity of selected Genetic toxicity testing support services Immunotoxicity system In-life data collection and management Pathology support Peer review of revised NTP monograph Production of B6C3F Project Portfolio Meeting Software (PPMPro) Quality assessment support/audits and inspections Scientific information management and literature- based evaluations for NTP Statistical support Support for toxicological data Tissue Chip Consortium (TCC) Membership Toxicological and carcinogenic potential of chemicals

FY 2021 ($140.3 million projected) $114 . 6 NIEHS $13.7 NIOSH $12.0 NCTR

FY 2020 $114 .7 NIEHS $12.8 NIOSH $12.4 NCTR ($139.9 million)

FY 2019 NIOSH $12.3 NCTR $113 . 3 NIEHS $11. 3 ($136.9 million)

.2 FY 2018

2 $12.8 NIOSH $11. 8 NCTR $112 .1 NIEHS NTP conducts its research through in-house studies at the NTP conducts its research through in-house laboratories or three core agencies or through contract In FY 2020, interagency agreements with other agencies. CDC and FDA NIEHS funded the contracts listed in the table. some of their may have additional contracts that support voluntary NTP efforts. —NIEHS, FDA, and NIOSH—to support its activities. These and NIOSH—to support —NIEHS, FDA, have been appropriations are specified after annual allocations NTP budget for FY 2020 was $140 million. determined. The total ($136.7 million) Funding Funding agencies from the three core voluntary allocations NTP relies on The Science of Substance | 2. About NTP About 2. | Substance of Science The 12 The Science of Substance | 1. FY 2020 in Review 15 .0 3 Publishing, Publishing, and Science and Science Collaboration, Collaboration,

Communications

Working together with the Working together with the scientific community and reporting on ideas and findings is essential for protecting public health. Each year, the National Toxicology Program (NTP) publishes dozens of reports and hosts scientific panels, workshops, meetings, and trainings to advance the field. The Science of Substance | 1. FY 2020 in Review Review in 2020 FY 1. | Substance of Science The 14 The Science of Substance | 3. Publishing, Collaboration, and Science Communications 17 Published Date May 2020 May 2020 June 2020 June 2020 June 2020 June 2020 July 2020 July 2020 November 2019 June 2020 July 2020 September 2020 September 2020 September 2020 December 2019 Substance or Topic Addressed Substance or Topic 2-Hydroxy-4-methoxybenzophenone 2-Hydroxy-4-methoxybenzophenone Acid Perfluorooctanoic Tris(chloropropyl) Phosphate 4-Methylcyclohexanemethanol Vinpocetine Dimethylaminoethanol Bitartrate Fullerene C60 (1 µm and 50 nm) Abrasive Blasting Agents 1020 Long Multiwalled Carbon Nanotubes Gum Guggul Extract Formulation 4-Methylcyclohexanemethanol Scoping Review of Potential Human Health Effects Associated with Exposures to Neonicotinoid Pesticides Scoping Review of Paraquat Dichloride Exposure and Parkinson’s Disease Scoping Review of Prenatal Exposure to Progestogens and Adverse Health Outcomes Traffic-related Air Pollution and Hypertensive Disorders of Pregnancy 01 02 03 04 87 91 94 99 01 15 16 17 07 597 598 Report Number Report Series Technical Technical Reports Developmental and Reproductive Toxicity Reports Toxicity Reports Immunotoxicity Report Research Reports Monograph All published NTP reports are peer reviewed by experts who are screened for for screened are who by experts reviewed are peer reports NTP All published two technical NTP completed In 2020, their service. of interest before conflicts four toxicity toxicity reports, reproductive and four developmental reports, one monograph, reports, and three research report, one immunotoxicity reports, below. as listed , which document the toxicity reports, which document the Developmental and reproductive test animal development and reproduction potential of agents to affect studies, generally no NTP toxicity reports, which document shorter-term longer than 13 weeks Immunotoxicity reports, which evaluate the potential of environmental system and occupational substances to damage the immune NTP research reports, which provide the results of research studies, rapid under the scope communications, and literature surveys that do not fall of the other report series NTP monographs, which assess the evidence that exposure to a substance the science causes adverse health effects or presents the state of Report on Carcinogens monographs, which are prepared for candidate evaluations and substances selected for review and consist of cancer substance profiles , which document long-term toxicology and toxicology and NTP technical reports, which document long-term for 2 years carcinogenicity studies, generally

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3 f f f f f f f NTP scientists published 260 journal articles and book 260 journal articles and book NTP scientists published Citations for these publications chapters in FY 2020. of . In addition, the findings are provided in Appendix I projects are published in the NTP studies and research reports: following types of NTP and Publications and Completed NTP Reports Reports NTP Completed The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The 16 18

The Science of Substance | 3. Publishing, Collaboration, and Science Communications Supplements Botanical Dietary HIGHLIGHT a rangeofenhancementpurposes. plant extractsthatpeopletakefor made fromplants,plantparts,or dietary supplements,areproducts sometimes calledherbalsorherbal Botanical dietarysupplements, 18% 2018. HerbalGram. 2019;123:62-73. Supplement Sales inUSIncreaseby9.4% J, Morton-ReynoldsC.2019.Herbal 1 Approximately supplements in2018. billion onherbaldietary consumers spent$8.8 dietary supplements.U.S. products, including of U.S.adultsusenatural SmithT,GillespieM,EcklV,Knepper

1 miscarriage or harmfetaldevelopment. to avoidvinpocetinebecause itmaycause on thelabelforwomen of childbearingage vinpocetine productsto includeawarning findings, FDAadvisedcompanies marketing exposed tovinpocetine. Becauseofthese harmful reproductiveeffectsinanimals published areportonitsstudythatfound function andweightloss.InJune2020,NTP for usessuchasimprovementofbrain specific plants.Thesubstanceismarketed from eithertheseedsorleavesof vinca alkaloidvincamine,whichisextracted Vinpocetine isasyntheticderivativeofthe Vinpocetine Supplements onDietary FY 2020Reports health careproviders,governmentregulators,andconsumers. effects. NTPstudiesofferscientificdatathatcaninformdecisionsby about labeling,productquality,healthbenefits,andriskofharmful Public concernaboutbotanicaldietarysupplementsincludesquestions identify anyharmfromshort-andlong-termexposuretothesesubstances. requests fromthepublicandotherfederalagencies.Thestudieshelp NTP teststhesafetyofsupplementsandtheiringredientsinresponseto adulterated ormisbrandedproductsonlyaftertheyreachthemarket. them availabletothepublic.FDAisresponsibleforactingagainst the safetyoftheirproductsandaccuratelylabelingthembeforemaking of dietarysupplementsandingredientsareresponsibleforevaluating foods anddrugproducts.Underthelaw,manufacturersdistributors under adifferentsetofregulationsthanthosecoveringconventional The FoodandDrugAdministration(FDA)regulatesdietarysupplements interact withover-the-counterandprescriptiondrugs. may containactiveingredientswithstrongbiologicaleffectsthatcould in thebody,howitisprepared,anddosetaken.Thesesupplements depends onmanyfactors,suchasitschemicalstructure,howitworks Not allproductsfromplantsourcesaresafe.Thesafetyofabotanical Supplements? Why DoesNTPStudyDietary related tothecompound. effects thatmayor notbe marginal ordiscordantdevelopmental in rats,whichdemonstrated only on itsprenataldevelopment study In June2020,NTPpublishedareport such asspraypaintingandlacquering. occupational andindustrialroutes exposure mayalsooccurthrough general cognitivefunction.Human marketed toimprovememoryand dimethylaminoethanol bitartrateare Dietary supplementscontaining Dimethylaminoethanol bitartrate

19 The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The The Science of Substance | 3. Publishing, Collaboration, and Science Communications 21

Actions Peer-Review Report Peer Review Information Actions Peer-Review Report

NTP assessed the NTP assessed the contribution of combined gestational and lactational exposure to the chronic toxicity and carcinogenicity of perfluorooctanoic acid (PFOA). Uses of Study Substance NTP assessed adverse effects in rodents of the UV filter often added to sunscreens. TR-598 Report Number TR-597 .3 3 Report Peer Review of Draft NTP Technical Report on the Toxicology and Carcinogenesis Studies of 2-Hydroxy-4- methoxybenzophenone Peer Review of Draft NTP Technical Report on the Toxicology and Carcinogenesis Studies of Perfluorooctanoic Acid Scientific Panels Scientific panels, referred to as special emphasis ad hoc scientific panels, NTP convenes issues, review and advice on targeted scientific peer to provide independent test new and revised toxicological of public health concern, such as agents public the opportunity for others. All panel reviews provide methods, and ensure that NTP receives transparent, unbiased, comment. These panels help for use in making credible decisions about and scientifically rigorous input research, and testing priorities, and in evaluating human health hazards, setting test methods for toxicity screening. the results of long-term NTP toxicology and NTP technical reports provide usually involve exposing laboratory animals carcinogenicity studies that for a period of two years. For each report, (rats and mice) to a substance and assessing the methods, results, and the panel is charged with reviewing potential, including carcinogenic activity, conclusions about the toxicologic of selected substances. listed below underwent peer review in FY 2020. The draft technical reports past reports’ peer-review meetings is available Additional information about web page. on the NTP Technical Reports

.2 Past BSC Meetings page on the NTP website.

3 Advisory Groups Advisory and promote the development advance toxicological research NTP strives to from external as such, relies on advice approaches and, and use of innovative guide its science. advisory groups to help federally chartered of NTP’s provides advice on the merits Scientific Counselors (BSC) The Board of programs. At its meeting on February 21, 2020, intramural and collaborative systems, such as organs-on-a-chip or the BSC heard how microphysiological an opportunity to get closer to the complexity of in 3D organ constructs, offer reinvent how toxicology is done. The BSC was briefed vivo systems to potentially research on human exposure to nanoplastics and on recent developments in by national and international regulatory microplastics, including activities They learned about a recently completed agencies to address this problem. literature that confirmed an association of systematic review of the published to hypertensive disorders of pregnancy and about traffic-related air pollution PFAS. Materials from the meeting are available on NTP’s portfolio of studies on the on Alternative Toxicological Methods The Scientific Advisory Committee Coordinating Committee on the Validation (SACATM) advises the Interagency the NTP Interagency Center for the Evaluation of Alternative Methods (ICCVAM), Methods (NICEATM), and the NIEHS and NTP director of Alternative Toxicological activities related about statutorily mandated duties of ICCVAM and NICEATM scientifically valid to promoting the regulatory acceptance of new or revised and the environment toxicological tests that protect human and animal health ensuring human safety while reducing, refining, or replacing animal tests and annual meeting and product effectiveness. The opening session of SACATM’s and future of ICCVAM on September 2–3, 2020, focused on the past, present, partner activities on its 20th anniversary. Other sessions summarized international and characterization and focused on nonanimal-derived antibodies, curation praised advances in of data, and computational resources. SACATM members for chemical safety testing. use of computational methods to reduce animal use be developed to further They suggested resources and approaches that could they could be applied. advance these tools and identified testing areas where page Information about the meeting is available on the Past SACATM Meetings on the NTP website. The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The 20 The Science of Substance | 3. Publishing, Collaboration, and Science Communications 23

Defining how to calculate the LD50 (median lethal dose) of a chemical a chemical dose) of (median lethal the LD50 how to calculate Defining product mixture/formulated optimization or (or assay) development which model gaps for Identifying described the In the first webinar, presented July 23, 2020, researchers of therapeutics for role of nonanimal-derived antibodies in development COVID-19 and other diseases. João Barroso of In the second webinar, presented September 16, 2020, a 2018 EURL ECVAM EURL ECVAM shared a summary of the findings from the validity and Scientific Advisory Committee meeting that reviewed affinity reagents and benefits of using animal-free technology to produce the subsequent EURL ECVAM recommendation. is needed (or assay) outputs regulatory needs for model Understanding that would be useful types of mechanistic information Pinpointing the intelligence in model development feasibility of using artificial Establishing the

f f f f f f f The specific areas discussed included: discussed areas specific The Series Antibody Webinar are used in a range of research, diagnostic,Affinity reagents such as antibodies Because traditional practices for producing suchand regulatory applications. of laboratory animals, even when they are appliedreagents require immunization their use is inconsistent with the spirit of replacing,to nonanimal test methods, use. Use of animal-based affinity reagents alsoreducing, or refining animal methods that use them, which contributes to increasedintroduces variability to the and use of nonanimal-derived antibodies. interest in promoting the acceptance Ethical Treatment of Animals International NICEATM, the People for the and the European Union Reference Laboratory Science Consortium (PETA-ISC), a series of for Alternatives to Animal Testing (EURL ECVAM) co-organized and benefits of free webinars in which experts addressed the application recombinant antibodies. about upcoming The webinar series will continue into FY 2021. Information on the PETA-ISC website. webinars and recordings of past webinars are available Group discussions focused on issues specific to particular classes of chemicals, chemicals, to particular classes of focused on issues specific Group discussions next of activities, and recommending acute toxicity, prioritization mechanisms of an analysis of variability in the in vivo test to steps. Follow-up actions included predictions, a comprehensive assessment of an establish confidence in toxicity mixtures toxicity, and exploration of the addition additivity equation for predicting information to complement in silico predictions. of biological and mechanistic

highlighted these commonly raised issues and provided raised issues and provided This symposium highlighted these commonly .4

3 Workshop: Mind the Gaps: Prioritizing Activities to Meet Regulatory Mind the Gaps: Prioritizing Activities to Meet Regulatory Workshop: Needs for Acute Systemic Lethality performed type of safety Acute systemic toxicity tests are the most commonly considered test worldwide. Participants in this October 30–31, 2019, workshop with chemicals and approaches available to assess acute lethality associated strategies to chemical mixtures, with the purpose of designing comprehensive was organized by predict toxicity while avoiding animal tests. The workshop Medicine. NICEATM and the Physicians Committee for Responsible Symposium Webinar: Use of the Kinetically Derived Maximum Dose in Maximum Dose in of the Kinetically Derived Use Webinar: Symposium Testing Toxicity dose (KMD) refers to the dose at which a The kinetically derived maximum or linear pharmacokinetics is observed. departure from dose proportionality a KMD may not be relevant to human health Toxicity findings at doses above concentrations are orders of magnitude lower. risks when potential exposure in pharmaceutical development, consideration In contrast to its routine use interpretation of animal toxicity studies for of the KMD in the design or relatively rare. Interest is growing in using the KMD to environmental chemicals is data or to set top dose in chronic toxicity studies interpret animal dose-response technical and scientific issues hinder its proper of these chemicals, but many use. needed to develop more consistent, transparent the background information KMD application in risk assessment. approaches to support broader by NTP Interagency Center for the Evaluation of The symposium, organized (NICEATM), the U.S. Environmental Protection Alternative Toxicological Methods and Environmental Agency (EPA) Office of Pesticide Programs, and the Health 30, 2020, and Sciences Institute, was presented virtually on September symposium reviewed attended by more than 400 viewers. Presentations in this commonly raised the application of KMD in toxicity testing. They summarized as an approach to select technical and scientific issues related to the use of KMD study results. doses in toxicology testing studies or to interpret dose-response Workshops and Meetings and Workshops Workshops Meetings and NICEATM The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The 22 The Science of Substance | 3. Publishing, Collaboration, and Science Communications 25 National Institute for Occupational Safety and National Institute for Occupational Safety and .5 3 CDC/NIOSH NIEHS provides support to the through Health (NIOSH) of the Centers for Disease Control and Prevention (CDC) under these agreements multiple interagency agreements. In FY 2020, research other two-dimensional evaluated occupational exposure to graphene and polyfluoroalkyl substances nanomaterials, molds and mycotoxins, and per- and journal articles (PFAS). This research is included among the 76 peer-reviewed NTP (see Appendix I). More published in FY 2020 by NIOSH scientists in support of be found in Section 5.3. information about NTP research at NIOSH in FY 2020 can Collaboration and Collaboration Leadership Scientific agencies with other federal NTP collaborations NIEHS supports agreements. FY 2020 NTP activities madethrough interagency are outlined here. under these agreements FDA/NCTR NIEHS provided financial resources to the Under an interagency agreement, studies on issues of mutual interest to NIEHS and FDA to conduct toxicology Research (NCTR). The interagency FDA at the National Center for Toxicological on endocrine-active agents, dietary supplements, agreement supports studies therapeutics and medicines, cosmetic food additives and contaminants, materials and on the development of novel ingredients, and nanoscale This research supports an increased understanding of toxicological approaches. and dose-response relationships of the the pharmacokinetics, mode-of-action, to risk assessment models. This research is included substances and refinements journal articles published in FY 2020 by NCTR among the 60 peer-reviewed scientists in support of NTP (included in Appendix I). Further information about 5.2. NTP research at NCTR in FY 2020 can be found in Section

are available on the NTP website. public forum on May 21, 2020. Usually an in- ICCVAM held its seventh annual forum was presented as a webinar that was person event, the 2020 public viewers. Representatives of ICCVAM member attended by more than 400 advance new approaches to safety testing agencies described work to products and to reduce the amount of testing of chemicals and medical forum commended the agencies’ progress and required. Commenters at the agencies taking leadership roles in reducing noted the importance of federal to government agencies, viewers represented animal testing. In addition companies, animal welfare organizations, pharmaceutical and chemical test method developers. Presentations from the academic researchers, and on the NTP website. public forum are available ICCVAM Meetings ICCVAM Coordinating the Interagency held by 10 teleconferences supported NICEATM in FY 2020. (ICCVAM) Methods of Alternative on the Validation Committee public meetings. three ICCVAM-sponsored support for also provided NICEATM on January of Practice webinar was held ICCVAM Communities The sixth annual and limitations of nonanimal- webinar reviewed the usefulness 21, 2020. The reagents. to replace animal-derived reagents and their potential derived affinity use, an overview of production, Ph.D., Abcam, presented Alejandra Solache, Clewell, polyclonal antibodies. Rebecca of monoclonal and and characteristics group of a review by a working Tox Consulting, summarized Ph.D., 21st Century Laboratory European Union Reference Advisory Committee of the the Scientific on the scientific validity and benefits of animal- for Alternatives to Animal Testing affinity reagents. Presentations from the webinar free technologies to produce The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The 24 The Science of Substance | 3. Publishing, Collaboration, and Science Communications 27 .6 3 The Division of NTP (DNTP) at NIEHS offers a limited number of postdoctoral trainingThe Division of NTP (DNTP) at for careers in pharmaceutical and chemicalfellowships to prepare trainees and academia. In FY 2020, NIEHS/DNTP staffindustries, regulatory agencies, at NIEHS in seven focal areas. Full details onmentored 20 postdoctoral fellows . the application process is on the NIEHS training website opportunities, benefits, and NIST cell study of the experimental to support agreement this interagency Through of Institute and the National exposure, NIEHS radiation radiofrequency phone exposure system a new to develop collaborated and Technology Standards systems. the limitations in existing that addresses Training Opportunities

EPA/NCEA EPA National Center for This interagency agreement between NIEHS and the and coordination to Environmental Assessment facilitates communication and to minimize the foster common practices in human health assessments exchange of scientific differences in hazard assessment methodologies. Through increase the quality expertise and peer review of work products, the agencies efficient use of federal and integrity of toxicological assessments while ensuring resources and avoiding duplicative effort. EPA/NCCT the EPA National Center for Computational Under this interagency agreement, the development and use of advanced Toxicology works to accelerate and qualitative risk assessments of methods and models for quantitative 2020 provided products environmental chemicals. Specifically, research in FY understanding on for standardization across transcriptomic screening assays, methods, and a how diverse biology affects results using our current screening collaborative chemical management to reduce redundancies. DOE/ORNL NIEHS is working with the Department Under this interagency agreement, Laboratory (ORNL) to develop tools for of Energy’s Oak Ridge National of its work. For example, ORNL is developing evaluating the use and outcomes help to evaluate the effectiveness of NTP’s work publication mining tools that the work is disseminated. ORNL is also developing with stakeholders, to whom processing and machine learning methods to tools to apply natural language in the full text of scientific publications. determine specific data elements NIH/NCATS/DPI studies and anticipated ongoing supports agreement This interagency for Advancing Center of Health/National Institutes at the National conducted (DPI) to Innovation of PreClinical (NCATS)/Division Sciences Translational of screening assays in support and high-content evaluate high-throughput federal is a collaboration among the 21st Century (Tox21). Tox21 Toxicology in isolated chemicals by using cells and the toxicity of agencies to characterize animals. This interagency agreement instead of laboratory molecular targets substances data for information-poor and NCATS/DPI produces between NIEHS including toxicological evaluation, them for further studies, to help prioritize modeling development of predictive action investigation, and mechanisms of response. for biological The Science of Substance | 3. Publishing, Collaboration, and Science Communications Science and Collaboration, Publishing, 3. | Substance of Science The 26 The Science of Substance | 4. Science, Innovation, and Methods Development 29

.0 4 Science, Science, Innovation, and Methods and Methods Development

The National Toxicology Program Program The National Toxicology (NTP) develops and tests innovative methodologies that advance toxicological science while also decreasing reliance on animal studies. By providing leadership in the state of the science, NTP and its partners are addressing today’s public health challenges and preparing to meet the emerging challenges of tomorrow. The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 28 The Science of Substance | 4. Science, Innovation, and Methods Development 31

Methods (ICCVAM) and NIEHS/DNTP projects, especially those related to Toxicology in the related to Toxicology in the (ICCVAM) and NIEHS/DNTP projects, especially those 21st Century (Tox21). NICEATM Activities NICEATM Toxicological The NTP Interagency Center for the Evaluation of Alternative evaluation of alternatives Methods (NICEATM) focuses on the development and activities of the to animal use for chemical safety testing that supports of Alternative Interagency Coordinating Committee on the Validation Integrated Chemical Environment on freely available, Successful computational toxicology projects depend workflows. The NICEATM high-quality data formatted for use in computational its partners ICE resource, launched in 2017, provides data from NICEATM and designed to support as well as other resources and tools in an environment and evaluation. chemical safety assessment and new method development Stem cells in developmental toxicology and the developmental and the developmental toxicology in developmental Stem cells of adult disease basis Erik Tokar role of environmental chemical are investigating the NIEHS/DNTP scientists predicting screening system for defects. A rapid and accurate exposures in birth test chemicals environment is needed to toxicants to humans in the developmental 3D bodies are free-floating little to no safety testing. Embryoid that have had three germ layers that recapitulate differentiate into spheres that spontaneously high-throughput processes. A transcriptomic-based, many early embryogenic developed bodies is being human embryonic stem cells/embryoid platform using to gain developmental toxicants teratogens and to screen for environmental on pluripotency and early linage commitment.insights into the chemical effects screening high-throughput for model liver human vitro in optimizing Time Ferguson Katelyn Lavrich, Stephen of 1,000 HepaRG human liver cells can Hepatocyte spheroids composed and show liver functions better than regular, efficiently metabolize chemicals work seeks to optimize the maturation time of flat cell cultures. Ongoing functionality over time to optimally spheroids for their “adult” hepatocyte determine changes in media environment assess chemical toxicity screening, the reversibility of chemical changes. Time and and composition, and estimate of reference chemicals for toxicity will establish the concentration dependence to pathological cellular toxicity. relationship of molecular changes

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4 NTP is a leader in the development of new approach development of new approach NTP is a leader in the that use human-based methodologies (NAMs) approaches to assess harmful systems and computational without using animals. effects of substances Approach Methodologies Approach Development of New Development The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 30 The Science of Substance | 4. Science, Innovation, and Methods Development 33 Dermal absorption cause systemic substances to the potential of key factor in absorption is a Dermal inin vitro rat, and in vivo rat, data from EPA combines upon skin exposure. toxicity to the “triple pack,” referred to as commonly studies, dermal absorption vitro human of using factor. To assess the feasibility dermal absorption calculate a chemical-specific conducted this value, NICEATM and EPA studies alone to estimate data from in vitro in vitro and compared in vivo with analysis of triple pack reports a retrospective in vitro in in vitro data alone or human that using either human results. Results suggest absorption that an estimate of dermal rat in vitro data would provide combination with pack data. an estimate derived from triple or more conservative than is either close to Additivity approaches of formulations toxicity to predicting NICEATM evaluated the use of the GloballyUsing data obtained from EPA, and Labeling of Chemicals additivity equationHarmonized System of Classification mixtures. NICEATM compared toxicityto predict acute toxicity of chemical additivity equation for antimicrobial cleaningpredictions generated by the formulations with experimental measurements fromproducts and agrochemical that toxicity of these mixtures was not wellin vivo data. The analysis suggested ingredients’ toxicity, either because interactionsrepresented by the sum of their the toxicity of the mixtures or because the additivitybetween components increase account for variability of the animal studies.calculation does not adequately Acute fish toxicity species, EPA currently requires testing of each of threeTo assess hazards to wild fish cold water, and marine/estuarine. NICEATM anddifferent fish types: warm water, and Toxics evaluated acute fish toxicity datathe EPA Office of Pollution Prevention to the EPA office to determine whether fewerfrom pesticide safety data submitted testing. The analysis revealed that cold water speciesspecies could be used for this species, and thus it may be possible to run thesewere most often the most sensitive the number of fish neededtests in only one species. This would substantially reduce goals. to meet this testing requirement while still meeting risk protection data acute toxicity analysis of in vivo Variability lethality test using aNICEATM assessed the reproducibility of the rat acute oral chemicals. It conductedcomprehensively curated data set of more than 2,000 a global confidence intervalquantitative and categorical analyses and established These analyses revealedto characterize the inherent variability of the test method. orders of magnitude apart,that independent studies can yield LD50 values that are perspective due to thewhich can have significant implications from a regulatory NICEATM did not find anyresulting impact on hazard classification and labeling. that the observedevaluated parameter to correlate with variability, suggesting publicly available datavariability may be inherent to the animal test. The resulting interval can beset can be used for modeling, and the calculated confidence methodologies (NAMs).applied when assessing performance of new approach

Ability for users to upload their own data for IVIVE analyses. data for IVIVE their own for users to upload Ability model. based toxicokinetic physiologically A new action on mode of results based filtering of of assays and Selection or toxicity endpoint. graphics. Improved output provide additional analysis plots that Principal component tool. in the Chemical Characterization visualization options the Environmental send search query results to The ability to Chemicals Dashboard. Protection Agency (EPA) CompTox and additional metadata provided in User interface improvements results downloads. screening assays based on Organizing curated high-throughput of action and mapping these mechanistic targets and modes from the National Cancer Institute’s assays to controlled terms Metathesaurus. lists, including chemical lists relevant New data sets and chemical to carcinogenesis.

f f f f f f f f f In silico models Workgroup organized a global project in The ICCVAM Acute Toxicity of acute oral systemic toxicity that FY 2018 to develop in silico models agencies. NICEATM predict five specific endpoints needed by regulatory and used models developed for the project that met qualitative for the quantitative review criteria to generate consensus predictions these models using acute oral toxicity endpoints of interest. It combined The consensus a weight-of-evidence approach into a consensus model. Toxicity Modeling predictions are available in the Collaborative Acute chemicals for Suite (CATMoS), a free resource for screening organic the Open Structure- acute oral toxicity. CATMoS is implemented in v2.0 of in more detail Activity/Property Relationship App (OPERA), described predictions below under “Computational Toxicology.” The consensus with generated for the project are also available in ICE. Partnerships a presentation atfederal agencies to apply CATMoS were described in on Alternative the 2020 meeting of the Scientific Advisory Committee Toxicological Methods. Acute Systemic Toxicity Improvements in FY 2020 to the ICE in vivo extrapolation (IVIVE) tool included tool (IVIVE) vivo extrapolation ICE in to the FY 2020 in Improvements Other updates to ICE in FY 2020 included Other updates The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 32 The Science of Substance | 4. Science, Innovation, and Methods Development 35 SEAZIT useful vertebrate make it a of zebrafish development size and rapid The small using and development on growth effects of substances for assessing model adoption of the broader To enable methods. screening high-throughput Evaluation established the Systematic screening, NTP zebrafish for toxicological led by (SEAZIT) program, jointly of Zebrafish in Toxicology of the Application NICEATM scientists. NIEHS/DNTP and of ontologies (standardized the establishment NICEATM is coordinating tested the Three laboratories systems) for zebrafish screening. nomenclature assessments. designs and morphology set using similar study same 90-chemical to evaluate researchers were asked were posted online, and Zebrafish images terminology. Results were collected and compiled, the phenotypes using in-lab the Zebrafish Phenotype Ontology. A second online and terms were mapped to in FY 2020 and employed controlled vocabulary. evaluation was completed during the initial information gathering The interlaboratory study conceived The study is designed to determine the effect of phase of SEAZIT began in 2019. media renewal on study outcomes. Participating chorion removal and exposure dose range-finding experiments. laboratories are conducting data reference toxicity identificationdevelopmental of Automating approaches, it is critical to determine andTo establish confidence in alternative full text of scientific publications when developingextract information from the manually extracting protocol details such asreference databases. However, and dosing regimen is labor-intensive and canspecies, route of administration, collaborating with FDA and the Department ofintroduce errors. NICEATM is Laboratory to apply natural language processingEnergy’s Oak Ridge National to determine specific data elements in the full textand machine learning methods trained on a curatedof scientific publications. NICEATM is applying an approach ) toset of data from the rodent uterotrophic database (Kleinstreuer et al. 2016 developmental toxicity studies from the published literature. study data toxicity developmental Extraction and annotation of legacy developmentalTo support the evaluation of nonanimal approaches for from approximatelytoxicity assessment, NICEATM scientists extracted information studies and a subset of250 NTP legacy prenatal developmental toxicity animal Agency that were deemedabout 50 studies submitted to the European Chemicals details extractedhigh quality by NIEHS/DNTP subject matter experts. Study and treatmentincluded species, strain, administration route, dosing duration, effects extractions byrelated effects. Efforts are underway to standardize the computationalapplying controlled vocabularies and ontologies to facilitate such as EPA’s ToxRefDB.analyses and integration with other structured databases , now maintained

by NICEATM. OPERA, a free and open-source/open-data suite of QSAR modelsby NICEATM. OPERA, a free and open-source/open-data available on the NIEHS GitHub repository, is an ongoing collaboration between and physicochemicalNICEATM and EPA. OPERA predictions of toxicity endpoints Chemicals Dashboard. properties are available through ICE and the EPA CompTox predictions on tools such In FY 2020, OPERA added a JAVA library to enable live as ICE and the EPA Chemicals Dashboard. Computational Toxicology toxicology group uses mathematics, informatics, data NICEATM’s computational to better understand toxicity mechanisms and analytics, and computer models predict toxic effects. OPERA of chemical activity that can augmentQSAR models provide predictions robust QSAR modelsnonanimal approaches for predicting toxicity. To provide National Center forfor chemical properties of environmental interest, the EPA ) Computational Toxicology created OPERA (Mansouri et al. 2018 Cardiotoxicity approaches test new nonanimal build, and to design, is underway An initiative human and develop goal is to identify hazard. The cardiotoxicity to assess structure-activity quantitative well as in silico assays, as -based cell- and efficiently and chemicals more models, to screen relationship (QSAR) initiative is or circulatory system. The to be toxic to the heart for their potential the Food and Drug Administration by NIEHS/DNTP, NICEATM, supported jointly and the Health and for Drug Evaluation and Research, (FDA) Center focus on projects within this initiative Sciences Institutes. Two NTP Environmental safety. The NICEATM for assessing cardiovascular in silico screening approaches compile a reference cardiotoxicant public data sources to project is mining human-relevant data. It is also analyzing tool compound list and determine chemicals on the basis of their activity against the Tox21 data to prioritize This work is intended to help build and test cardiotoxicity-related endpoints. and integrated testing strategies for cardiovascular the capabilities of models molecular and cellular events contributing to hazards. Work in FY 2020 identified which may be measurable by assays as part of a cardiovascular failure modes, translational toxicology pipeline. The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 34 The Science of Substance | 4. Science, Innovation, and Methods Development 37

Braga et al. 2017). Pred-Skin Web Portal for Predicting Human Human Portal Predicting for Web Pred-Skin Skin Sensitizers of North Carolina In FY 2020, NICEATM collaborated with scientists at the University update a web tool at Chapel Hill and the Universidade Federal de Goiás to implemented in Pred-Skin version 3.0, an application based on externally predictive QSAR models of skin sensitization (Borba et al. 2020). The updates integrate multiple human ex vivo data into a QSAR models developed with in vitro, animal in vivo, and ( consensus naïve Bayes model that predicts human effects Nonanimal Risk Assessment for Isothiazolinones Isothiazolinones for Assessment Risk Nonanimal on the request for input to an EPA responded NTP and NICEATM the NIEHS/D In FY 2020, to evaluate defined approach network-based artificial neural in vitro and use of an plastics, are used in which isothiazolinones, products containing risks for skin sensitization conducted NIEHS/DNTP and NICEATM and laundry detergents. household cleaners, beforeWorkgroup reviewed the reports ICCVAM Skin Sensitization studies, and the this type of in It was the first use of to EPA to develop risk assessments. delivering them a regulatory risk assessment. defined approaches to support vitro data and to Identify Skin Sensitizers Approaches Expanding ICCVAM chemicals nominated by tested more than 200 In FY 2020, NIEHS/DNTP industrial chemicals, and others, using three in vitro agencies, including pesticides, the direct peptide reactivity assay, and the methods: the KeratinoSens method, NICEATM coordinated the testing, which began in human cell line activation test. early FY 2020. The study data will allow NICEATM and 2017 and was completed in it’s appropriate to use these three in vitro methods for ICCVAM to evaluate whether various regulatory applications. Skin Sensitization DataHuman Reference for of the American Society for Cellular and Computational At the 2020 annual meeting work by NICEATM, the German Federal Institute for Toxicology, presenters described to evaluate nonanimal approaches for skin Risk Assessment, and other collaborators group had collected data for more than 2,300 human sensitization assessment. The than 1,700 publications. Human predictive patch predictive patch tests from more single doses make determining thresholds uncertain and test data that are based on reference data. To overcome this challenge and help these data difficult to use as using a decision tree resolve ambiguity in results, classification criteria were extended of defined approaches to a reference list of 200 substances to support the evaluation to help develop for skin sensitization. This database will be publicly available additional methods and models. established a Skin Sensitization established a Skin Sensitization conducts many of these research activities, conducts many of these research activities, ICCVAM Regulatory agencies use skin sensitization test data sensitization test data Regulatory agencies use skin to chemicals and to assess risks from exposure labeling to protect to develop product hazard these toxicology consumers and workers. While animals, scientists are tests are often performed on increasingly using biochemical or cell-based (in vitro) systems to determine skin-sensitizing substances. Under U.S. laws and policies, scientists must consider alternative methods before using animals for toxicology research and testing. In some parts of the world, including Europe, testing cosmetic products and their ingredients on animals is banned. These efforts have led to increased development of in vitro tests for skin sensitization. The Workgroup to help develop and validate nonanimal methodologies for skin sensitization tests. The NICEATM including the following work during FY 2020.

Before marketing, manufacturers test pesticides, cosmetics, household cleaners, and other chemical products to identify substances that can cause allergic reactions, known as skin sensitizers. HIGHLIGHT Skin Sensitization The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 36 38

The Science of Substance | 4. Science, Innovation, and Methods Development IV (donotrequireeyeprotection). protection whenhandling)fromchemicals fallingintohazardcategoriesIIIand differentiated chemicalsfallingintoEPA hazardcategoriesIandII(requireeye corrosives ornonirritants/corrosivesusing aninsilicoapproach.Thisapproach NICEATM developedstatisticalmodelstoclassifychemicalsaseyeirritants/ modelsforStatistical eye classification irritant Document 220onanintegratedapproachtotestingandassessingeyeirritation. Organisation forEconomicCo-operationandDevelopment(OECD)Guidance other information,suchasphysicochemicalproperties,consistentwiththe complete spectrumofeyeirritationeffects.Thisreviewalsoconsidersincluding make suggestionsforoptimizingthemostpromisingmethodstodistinguish Consortium (PETA-ISC)draftedareviewarticletodetailtheseperspectivesand and PeoplefortheEthicalTreatmentofAnimalsInternationalScience Laboratory forAlternativestoAnimalTesting,theInstituteInVitroSciences, NICEATM, inconjunctionwithscientistsfromEPA,theEuropeanUnionReference more reproduciblethanthecurrentlyusedinvivorabbiteyetest.Accordingly, In vitroandexvivomethodsareatleastasreflectiveofhumanbiology agrochemical formulations Human-relevant toassesseye potentialof approaches corrosion/irritation Eye Irritation published inFY2020. stand-alone OPERAapplication.ApaperdescribingtheCoMPARAprojectwas Dashboard. TheconsensusmodelsfromCoMPARAwerealsoaddedtothe Predictions fromthesemodelsareavailablethroughICEandtheEPAChemistry Collaborative ModelingProjectforAndrogenReceptorActivity(CoMPARA). the worldwidemodelingcommunitytopredictandrogenicactivityin NICEATM andEPAranaglobalcollaborationtoleveragetheexpertiseof In silicopredictions ofandrogenreceptor pathway activity Endocrine Disruption performance ofnewapproachmethodologies. the referencetestmethodbutalsotoaidinsettingexpectationsfor These analyseshelpprovidemuchneededcontextnotonlytoassess are morelikelytobeclassifiedinadifferentcategorywhentestedagain. reproducible, whereassubstancesclassifiedasmildormoderateirritants extremes oftheclassificationscales(corrosiveornonirritant)aremost The resultsshowthat,regardlessoftheclassificationsystemused, records toassessvariabilityinresultsfromchemicalstestedmultipletimes. compiled andcuratedadatasetofmorethan3,000invivoassaytest To bettercharacterizethereproducibilityofinvivoassay,NICEATM responses intherabbittestisaconfoundingfactorforsuchcomparisons. variability inherenttothesubjectivescoringoferythemaandedema alternatives forskinirritationtestingarecompared.However,the The invivorabbitskintestisthebenchmarkagainstwhichnonanimal Variability data analysisofinvivo skinirritation Skin Irritation

39 The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The The Science of Substance | 4. Science, Innovation, and Methods Development 41 Imbue Tox21 assays with metabolic capability Screen the Tox21 10,000-compound collection using these new methods to identify chemicals that are either bioactivated or detoxified by liver cytochrome P450s and cofactors Identify the particular cytochromes P450 responsible for observable shifts in bioactivity Developed a computational and cellular testing framework Developed a computational and to chemical agents for assessing genetic susceptibility neurotoxicity suspected of causing developmental outbred mouse neural Used approximately 200 diversity chemical test battery progenitor cell lines exposed to a using high-content imaging Assessed cellular effect potency techniques effects from exposures to Improved the prediction of adverse drugs and chemicals toxicologically important Used additional assays to probe in current Tox21 testing targets and pathways not captured Refined existing methods to: • • • Developed a data-driven approach to choose cell lines to maximize biological diversity Picked a diverse set of cell types based on publicly available gene expression data and baseline gene expression profiling

ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ Details Using an in vitro pipeline to assess population toxicodynamic variability for chemicals suspected to cause developmental neurotoxicity Evaluating expansion of pathway coverage by Tox21 qHTS assays for better prediction of adverse effects from exposures Retrofitting existing Tox21 high-throughput screening assays with metabolic capability Selecting cell lines for Tox21 screening Test Method Test

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4 the National Center for Advancing Translational Sciences, EPA, Translational Sciences, Center for Advancing the National to rapidly test whether develops new methods and FDA. It affect human health. Tox21 uses newchemicals adversely high-throughput and high-contentapproaches, including large numbers of chemicals quicklymethods, to evaluate insight into human health effects.and efficiently to provide toxicology support for Tox21Bioinformatics and computational members.is provided by Tox21 Tox21 among NIEHS/NTP, federal collaboration Tox21 is a unique The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 40 The Science of Substance | 4. Science, Innovation, and Methods Development 43 EpiAirway™ EpiAirway™ Electrophilic allergen screening assay allergenElectrophilic screening OptiSafe Test Method Evaluations Test NICEATM coordinated a multi-laboratory Status: NICEATM coordinated a multi-laboratory Recommendations/Agency ICCVAM reliability and relevance of the OptiSafe test method. In this validation study to determine the to a semi-permeable membrane to assess the substance’s method, a test substance is applied 2018, and a report on the study potential to cause eye irritation. The study was completed in FY was published in FY 2020 (Choksi et al. 2020). for identifying nonsurfactant The study demonstrated that the OptiSafe test method is useful can reduce the use of animals substances not requiring classification for ocular irritancy and thus for this type of testing. Status: A cooperative agreement under the NIEHS Phase Recommendations/Agency ICCVAM Corporation to validate its IIb Small Business Innovation Research provides funding to MatTek of inhaled chemicals. Testing EpiAirway in vitro human bronchial tissue model to predict toxicity of EpiAirway for this purpose of reference chemicals to determine the usefulness and limitations are members of the cooperative has been completed. Several ICCVAM agency representatives agreement steering committee. ICCVAM Test Method Evaluation Activities Evaluation Method Test ICCVAM might be methods that innovative test of welcomes submissions ICCVAM studies validation adequate use and for which regulatory for specific acceptable methods of new test effective implementation To maximize completed. have been those test and recommends only however, ICCVAM evaluates or approaches, agencies’ align with ICCVAM member for regulatory uses that methods proposed submissions. see ICCVAM test method For more information, needs and priorities. Status: National Institute This test method, nominated by the Recommendations/Agency ICCVAM is an in chemico assay intended to identify skin sensitizers.for Occupational Safety and Health, began in FY 2017 with four ICCVAM agencies participating A validation study of the method the study, and members of the ICCVAM Skin Sensitization in the study. NICEATM is coordinating management team. Workgroup are serving on the study 2018 showed that the method had sufficiently good Testing of 10 chemicals during FY to support further evaluation. In FY 2019, the Consumer reproducibility and accuracy rates the National Institute of Standards and Technology modified Product Safety Commission and increase throughput and accessibility. Planned testing of 20 the assay to a 96-well format to has been delayed due to lab closures during the COVID-19 chemicals using the 96-well assay completed in FY 2021. pandemic but is expected to be

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4 In vitro testing of agrochemical formulations formulations In vitro testing of agrochemical CropLife America member companies areNICEATM, PETA-ISC, EPA, and in vitro-defined approach for hazard classification ofcollaborating to develop an formulations. A three-phased prospectiveeye irritation potential of agrochemical (1) assess the applicability of seven in vitro eyeevaluation was designed to to agrochemical formulations, and (2) developirritation/corrosion protocols formulations testing for prediction ofa defined approach for agrochemical classifications. In Phase 1, completed in FY 2018,U.S. and international irritancy in seven different eye irritation test protocols. Tensix formulations were tested with in vivo data representing a wider rangeadditional agrochemical formulations were evaluated in Phase 2 in FY 2019. Although noneof eye irritation classifications several methods showedof the methods directly correlated with the in vivo results, formulations.potential for use in a defined approach to assess agrochemical next steps of the study.Analysis of the data from these phases will determine the assay of an in vitro botulinum neurotoxin Optimization and validation required by multiple Tests to detect and measure botulinum neurotoxins are toxins in federal agencies for a variety of purposes, such as measuring food or wildlife. drug formulations or detecting toxins in possibly contaminated lethality assay that Currently, the standard test for these endpoints is a mouse the optimization and may use a large number of animals. NICEATM supported replace animal-based validation of -linked immunosorbent assay that Methods were methods for diagnosing suspected avian botulism samples. of botulinum neurotoxin developed for determining the presence or absence wide range of bird species. serotypes C, D, and E in field-collected samples from a requirements for the These methods may be used in the future to support testing U.S. Geological Survey’s National Wildlife Health Center. of NAMs federal that provide U.S. recent projects ICCVAM coordinated with new nonanimal approaches to agencies and stakeholders of chemicals and medical products. safety and risk assessment Validation and Adoption Adoption and Validation The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 42 The Science of Substance | 4. Science, Innovation, and Methods Development 45 ICCVAM agency scientists served on the peer-review panel for a study for a study panel on the peer-review served agency scientists ICCVAM The study skin irritants. Skin SIT in classifying the LbL-3D the use of evaluating performance standards that the LbL-3D Skin SIT meets will help ensure human for reconstructed Document 220 described in OECD Guidance methods. epidermis test study team for a validation served on the management A NICEATM scientist which identifies immunotoxic screening assay, for the multi-immunotox and dendritic cells. assaying the activity of T cells chemicals by

f f In FY 2020, ICCVAM and NICEATM scientists participated in two validation studies studies validation in two participated scientists NICEATM and ICCVAM 2020, In FY Methods: of Alternative for the Validation Japanese Center by the coordinated a validation the peer-review panel for NICEATM scientist served on In addition, a Center for the Validation of Alternative study coordinated by the Korean the use of KeraSkin™ SIT, an in-vitro skin irritation Methods. The study is evaluating it meets performance standards described in OECD test method, to ensure that Guidance Document 220. defined a guideline for defined expert group developing Serving on an with respect to hazard approaches for skin sensitization, or potency subcategorization; identification various guidelines related Serving on expert groups reviewing and eye irritation; to skin sensitization and skin for an OECD-coordinated Serving on a peer-review panel the kinetic direct peptide reactivity study evaluating the use of for skin sensitization potency; assay for classifying substances and review of available information Contributing to a retrospective guideline for a reconstructed to support a draft OECD test phototoxicity testing. human epidermis model for

f f f f ICCVAM Activities InternationalICCVAM Validation test method in international participate and ICCVAM NICEATM with They also collaborate through OECD. activities validation Cooperation in the International participating and regions countries the European Union, Test Methods (ICATM), including on Alternative Canada, Brazil, and China. Japan, Korea, staff supported the members and NICEATM In FY 2020, ICCVAM FY 2020 by: Programme during OECD Test Guidelines three critical areas of cooperation: ICATM collaborations address independent peer review of test method validation studies, of formal recommendations validation studies, and development In October 2019, representatives of on alternative testing methods. a workshop to celebrate ICATM’s NICEATM and ICCVAM attended future of 10th anniversary. Workshop participants discussed the ICATM alternatives in the different jurisdictions represented by Union regulatory partners. Representatives from a range of European perspectives agencies also attended the workshop and shared their on the role of alternatives in future activities. At the subsequent move forward coordination meeting, attendees discussed ways to the with new validation approaches. Specifically, they discussed setting need to move away from validating protocols and toward performance standards. and the ICATM partners are also collaborating with NICEATM paper Physicians Committee for Responsible Medicine on a of regulatory summarizing the acute systemic toxicity testing needs ICATM. authorities in the countries and regions represented by The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The 44 46

The Science of Substance | 4. Science, Innovation, and Methods Development following areas: The NTPwebsitedescribesactivitiesunderway toaddressroadmapgoalsinthe to addressthreestrategicgoals: and implementationofthesenewapproacheswillrequirecoordinatedefforts reduce oreliminatetheneedfortestinginanimals.Thesuccessfuldevelopment new approachestotoxicitytestingthatimprovehumanhealthrelevanceand The roadmapcreatedaframeworkforenablingtechnologiesandpromoting NTP website. Products intheUnitedStates,”publishedJanuary2018andavailableon Establishing NewApproachestoEvaluatetheSafetyofChemicalsandMedical Methods (ICCVAM)coordinatedthedevelopmentof“AStrategicRoadmapfor The InteragencyCoordinatingCommitteeontheValidationofAlternative Progress toward Goals Roadmap Strategic toxicity Acute systemic

eye irritation Skin and

UTILIZATION and regulatedindustries. approaches byfederalagencies and useofnewmethods 3. TECHNOLOGY methodologies. developers ofnewapproach 1. Connectuserswith Encourage theadoption confidence innewmethods. practices toestablish flexible, androbust 2. Fostertheuseofefficient, CONFIDENCE sensitization Skin

compile, andreportmetricsthattrackprogressinreducinganimaluse. recommendations onhowmemberagenciescandetermine, receives supportfromNICEATM.Theworkgroupiscurrentlydeveloping The workgroupincludesmembersfromnineICCVAMagenciesand In response,ICCVAMestablisheditsMetricsWorkgroupinFY2020. incorporated intoICCVAMbiennialprogressreports. testing. Furthermore,thereportrecommendedthatsuchmetricsbe agencies couldusetohelpreduce,refine,orreplaceanimalin that ICCVAMestablishaworkgrouptodevelopmetricsmember alternative methodsaffectanimaluse.Thereportrecommended metrics thatdemonstratehowtheireffortstoencouragetheuseof that federalagencieshavenotroutinelydevelopedorreported the useofalternativemethods;however,GAOalsofound its ensure thatalternativemethodsarebeingconsideredandused.In Accountability Office(GAO)investigatedhowfederalagencies In responsetoarequestbyCongress,theGovernment andReducing Alternatives Animal Use ProgressMeasuring in Adopting report, theGAOconfirmedthatfederalagenciesactivelypromote

47 The Science of Substance | 4. Science, Innovation, and Methods Development Methods and Innovation, Science, 4. | Substance of Science The The Science of Substance | 5. Testing and Assessment 49

.0 5 Assessment Testing and Testing

health-related effects using health-related effects using well-established rodent models and study protocols as well as new, nonanimal methods. In addition, NTP evaluates scientific literature to identify conditions that could pose a hazard to human health. and circumstances for a variety and circumstances for a variety of cancer and noncancer The National Toxicology Program Program Toxicology National The (NTP) evaluates substances The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 48 The Science of Substance | 5. Testing and Assessment 51 NTP Studies NTP of a variety utilizing of UV filters the safety assessing NTP is reported findings from its In FY 2020, NTP model systems. studies on 2-hydroxy-4-methoxybenzophenone long-term test UV filters—to of the most common (2H4MBP)—one to of chronic exposure and carcinogenicity the toxicity studies Also included were in rats and mice. this chemical that activity. NTP concluded endocrine that characterized activity in mice and the 2H4MBP had no carcinogenic equivocal, or uncertain, as to whether findings in rats were 2H4MBP. NTP could not conclude if they were due to the activity. 2H4MBP alters endocrine next? What’s NTP is assessing and reproductive the documenting data toxicity and developmental on ethylhexylmetoxycinnimate, is It also UV filter. another on a reportworking summarizing cell-based and short-term animal additional on studies conducted ensulizole, , filters: UV padimate-o,, octylmethoxycinnamate, octylsalate, and . O OH 3 CH Chemical structure of 2H4MBP Chemical structure

Millions of people use Millions of people use lotions, creams, and sprays on their skin to prevent and resulting skin damage. Ingredients in these products, known as (UV) filters, are added to sunscreens to absorb or block UV radiation from the sun. UV filters have been found in urine, indicating they make their way into the body. HIGHLIGHT Filters Sunscreen/Ultraviolet The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 50 The Science of Substance | 5. Testing and Assessment 53 Toxicogenomic Studies Toxicogenomic technologies to solve Toxicogenomics involves the use of advanced genomic of chemical exposures problems in toxicology and to gain insights into the effects has grown from on human health. Over the last two decades, toxicogenomics sequencing and microarrays or “gene chips” to now include next-generation chemical effects metabolomic and proteomic analyses to determine adverse toxicants. and better understand an organism’s response to environmental is daunting, but Measuring the entire genetic information of a cell or organism genetic information to advances in technology have allowed production of in vitro models with increase in speed and breadth at a rapid pace. NTP uses chemicals for toxicity animal and human cells to rapidly screen environmental toxicity occurs. Animal and to better understand the mechanism of how chemical to toxicity and then models are used when needed to relate chemical exposure that the public might extrapolate these findings to chemical exposure conditions face in the environment or workplace. Assessing cardiovascular liabilities associated HIV therapeutics with cardiovascular Assessing Santos Janine and HIV treatment important in are HIV therapeutics Combination development on early these therapeutics effects of The health prophylaxis. effects that extend into as are longer-term are being investigated being studied molecular changes are outcomes and adulthood. Cardiovascular to combination HIV therapeutics. of pregnant rodents exposed in the offspring this work for will be developed from vitro cardiovascular assays Appropriate in chemicals of NTP interest. of these and other use in rapid screening of aqueous film-forming screening In vitro toxicity foams Stephen Ferguson being conducted to evaluate qualified aqueous In vitro screening efforts are and comparator per- and polyfluoroalkyl substances film-forming foam products chemicals for binning and estimations of liver (PFAS) constituents to reference concentration modeling. injury potential through benchmark and reproductive on developmental and GenX exposures Study of PFOA effects in mice Suzanne Fenton is a PFAS associated with adverse pregnancyPerfluorooctanoic acid (PFOA) The potential for developmental andoutcomes in mice and humans. widely detected PFAS contaminants in thereproductive effects of two perfluoro-2-proposypropionate) and PFOSenvironment—GenX (ammonium being studied in pregnant mice and their offspring.(perfluorooctanesulfonate)—are

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5 NIEHS/DNTP scientists apply tools of modern toxicology to screen individualNIEHS/DNTP scientists apply and evaluate their effects on human health. Resultschemicals or chemical classes be used to prioritize testing of hazardous chemicals orfrom these approaches can to help make public health decisions. Seedocumented in published literature NIEHS/DNTP Investigative Projects in Appendix III for a complete list of chemical screening project summaries in FY 2020. 5.1.1 Chemical Screening Scientists with the Division of NTP (DNTP) at the National of NTP (DNTP) at the National Scientists with the Division Health Sciences (NIEHS) use a Institute of Environmental to better understand how factors in variety of approaches impact our health. our environment may NTP at NIEHS/DNTP NTP at The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 52 The Science of Substance | 5. Testing and Assessment 55

Highlights Exploring informatics and semi-automation workflow in analysis Vickie Walker, Andrew Rooney to the literature-evaluationNIEHS/DNTP is implementing novel informatics methods and otherprocess by developing, refining, or adapting machine-learning Current effortsinformatics approaches to its project workflow and visualizations. steps (i.e., identifyingare focused on the labor-intensive literature screening and datarelevant studies among tens of thousands of relevant publications) chemical,or exposure the as such details, study key capturing including extraction, An innovativeexperimental animal species, and health outcomes studied. and testing. semi-automated method for data extraction is under development 5.1.2. Chemical Testing Testing Chemical 5.1.2. generally effects, health-related for substances evaluates program testing NTP The an appropriate team develops article, a study For each test models. using rodent a project review needs, and research the identified strategy to address testing studies, and summaries of NTP toxicity the strategy. Reports committee evaluates are development and reproduction, and effects on including carcinogenicity list of testing and for a complete NTP website. See Appendix II available on the toxicology in FY 2020. Testing and completed or in progress toxicology studies included in Appendix I. published in FY 2020 are studies with results 5.1.3 Noncancer Research Research 5.1.3 Noncancer literature-based evaluations using systematic NIEHS/DNTP scientists conduct evidence that environmental substances may be review methods to assess the health effects. NTP provides opinions on associated with adverse, noncancer might be of concern because of what is known about whether these substances current human exposure levels. were published (see Appendix I) that In FY 2020, three scoping reviews limitations of the evidence base on challenging determine the extent and on future research or analyses. These reviews scientific topics to inform decisions review methods and present interactive evidence apply transparent systematic knowledge about the extent of the literature maps that advance scientific explore the relevant health effects data that are and enable researchers to literature analysis for a complete list of noncancer identified. See Appendix III projects in FY 2020.

NTP continues to conduct chemical exposure experiments that that experiments exposure chemical conduct to continues NTP for methods tissue variability, animal and expression, differential genetic affect toxicogenomic conducting conditions for standardized and tissue sampling, ongoing NTP Planned or conditions. laboratory under high-quality studies below. are listed from FY 2020 studies toxicogenomic neurotoxicants developmental for In vitro screen Alison Harrill life exposure neurotoxicity within early produce developmental Chemicals that health that aim to predict human for in vitro test batteries pose a challenge use a to address this issue is to disease. One approach effects and adult which the DO diversity outbred, mouse model called genetically diverse of people. Neural progenitor cells, as early nerve simulates the complex genetics mice for screening with six known developmental cells, were isolated from DO concentrations at which molecular transcript neurotoxicants to find benchmark toxicity observed using cell-based changes. The changes occur relative to that are activated prior to traditional cell-based program hopes to find genes help identify the underlying mechanisms of measures of toxicity and thereby screen for developmental toxicity. neurotoxicants and more effectively hepatocarcinogens by genetic changes in liver Tracking Julie Foley, B. Alex Merrick liver cancer-causing chemical (hepatocarcinogen) Aflatoxin B1 is a widespread that occur on grains, corn, peanuts, and many that is a byproduct of molds humans and animals. After absorption, the liver other foods consumed by DNA-damaging forms, causing mutations that, can metabolize aflatoxin into cancer. Rats fed an experimental diet containing over time, can lead to liver DNA sequenced by a specialized method aflatoxin developed liver cancer. regions of DNA to find called Exome-Seq was used to target the most critical mutations that might lead to cancer. Extrapolation data in vitro high-throughput screening to animal toxicity William Gwinn, Nancy Urbano extrapolation of The parallelogram approach to toxicity assessment involves variation and in vitro data to an in vivo model that accounts for interspecies Studies are underway to exposure levels between animal models and humans. from human and animal evaluate high-throughput transcriptomic in vitro data relationships to in liver spheroid cell models to establish in vitro dose-response vivo extrapolation for vivo rat toxicity data and use the ICE database in in toxicity. estimation of human chemical exposure and potential The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 54 56

The Science of Substance | 5. Testing and Assessment and releasethereport. Department ofHealth andHumanServicesSecretary’sapproval ofnewlistings 15th RoC,andthereport isinthefourthstepofRoCprocess toseekthe NTP hascompleted the reviewsofeightsubstancesforpotential listinginthe established listing criteria, with several opportunities for scientific and public input. public health.Substancesarelistedinthe reportafteramulti-stepprocessusing step incancerprevention,publicationof thereportisimportanttoimprove anticipated humancarcinogens.Asthe identificationofcarcinogensisakey public healthreportthatprovidesacumulative listofknownorreasonably The Identifying cancerhazards: ReportonCarcinogens 5.1.4 CancerHealthEffects Research future projectsandresearcherswithinoutsideNIEHS/DNTP. evidence mapthatisafoundationforevidence-baseddecisionsandinforms cardiovascular toxicityandeffectstodevelopaninteractive to identifypublishedliteratureonenvironmentalexposuresassociatedwith of pregnancyandcardiovasculardisease).Thegoalstheseevaluationsare potential environmentalfactorsonhumandiseases(e.g.,hypertensivedisorders evaluations, broad-basedscopingreviewsarebeingconductedthatexamine As partofNIEHS/DNTP’sapproachtodevelopingfit-for-purposeliterature-based Brandy Beverly,VickieWalker decisions ondisease-focused environmental healthquestions Scoping reviews evidence-based tosupport research andanalysis uncertainty thatcouldbetargetsforfurtherresearch. more evidenceorwidespreadexposure,aswelldeterminingareasof in-depth analysis,suchasselectingachemicalorsubsetofchemicalswith outcomes. Thesereviewswillserveasafoundationalstepininformingmore products orpatternsofpersonalcareproductuseandkeyreproductive of associationsbetweenthewiderangechemicalsfoundinpersonalcare Two scopingreviewsareunderwaytodetermineandcategorizetheevidence especially forsusceptiblelifestages,includingpregnantwomenandchildren. fragrances) isanimportantclose-contactexposuretoenvironmentalchemicals, The useofpersonalcareproducts(e.g.,soaps,lotions,cosmetics,and Kyla Taylor,KembraHowdeshell of exposures Personal care products: An example ofevaluations ofbroadclasses Report onCarcinogens (RoC)isacongressionallymandated,science-based,

complement othercross-DNTP projectsonPAHsandPACs. and nitro-PAHliterature-basedcancerhazard evaluations).Theseprojects others areevaluatingclasses,subclasses, orindividualPACs(e.g.,PAH mapping andliterature-basedcancerhazard evaluations),whereas PAHs mixtures(e.g.,theepidemiological studyandwoodsmokeevidence literature-based cancerhazardevaluations. Someprojectsareevaluating epidemiological study,evidencemapping andscopingactivities, NIEHS/DNTP isconductingresearchonPAHs andPACs,includingan smoke, andcontaminatedair. sources, includingthroughcertainoccupationsandfromfoods,tobacco attached toaPAH.PeopleareexposedmixturesofPACsfrommultiple part. Nitro-PAHsarePACswithatleastoneormorenitro-functionalgroups -only chemicalswithatleasttwoaromaticringsinthefused Polycyclic aromatichydrocarbons(PAHs)arePACswithcarbon-and are oftenformedasaresultofincompletecombustionororganicmatter. ubiquitous contaminantsthatexistincomplexanddynamicmixtures Polycyclic aromaticcompounds(PACs)areastructurallydiversegroupof hydrocarbons andothertypesofpolycyclic compounds aromatic Evaluating evidence for thecarcinogenicity ofpolycyclic aromatic Highlights in FY2020. Appendix IIIforacompletelistingofcancerliteratureanalysisprojects and isconductinghazardassessmentsforseveralsubstances.See NIEHS/DNTP hascompletedscopingandproblemformulationactivities whether asubstanceiscancerhazard. across manydifferenttypesofstudiestoreachconclusionsabout use that areindependentoftheRoC.Theseliterature-basedassessments an recommended forlistingintheRoC.Theassessmentiscaptured cancer studiestodeterminewhetherasubstanceshouldbe NTP conductsanextensiveassessmentofpubliclyavailable,relevant literature-based reviews, andstudies Evaluating cancerhazards: Scoping, evidencemapping, RoC monograph.NTPmayalsoconductcancerhazardevaluations systematic reviewmethodsthatintegratetherelevantevidence

57 The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The The Science of Substance | 5. Testing and Assessment 59

the animal cancer studies; the protocol and cancer hazard assessment are the protocol and cancer hazard assessment are the animal cancer studies; under development. is conducting a cancer hazard evaluation that includes assessment of cancer of cancer includes assessment that hazard evaluation a cancer is conducting and is using data and mechanistic human studies in animal and studies class, of PAHs (individual, carcinogenicity to predict models computational and animal of the human a scoping report completed NIEHS/DNTP subclass). 59th Annual Society of Toxicology the results at the studies and presented the human of the science review of It also completed a state Meeting in 2020. the results cancer and presented studies of PAHs and breast epidemiological Society of Environmental Conference of the International at the 32nd Annual assessment, and computational The protocol, cancer hazard Epidemiologists. development. models are under Nitro-PAHs Gloria Jahnke (NPAHs) pose public health problems Nitro-polycyclic aromatic hydrocarbons Information on specific or classes of NPAHs is due to widespread exposure. to decrease exposure and prevent health effects. important for monitoring ways RoC; however, new cancer data related to other Five NPAHs are listed in the To identify cancer hazards for approximately 100 NPAHs are now available. an evaluation that includes an assessment of NPAHs, NIEHS/DNTP is conducting physio-chemical properties and mechanistic data animal cancer data and uses as a class or subclass(es). to potentially group NPAH the scoping and study quality assessment of NIEHS/DNTP has completed To determine a more comprehensive list of carcinogenic PAHs, NIEHS/DNTP NIEHS/DNTP PAHs, carcinogenic list of comprehensive a more To determine

A prospective analysis A prospective Suril Mehta studies, although disease in epidemiologic with chronic associated PAHs are States outcomes in the United of PAH exposure on fatal the contribution a collaborators conducted NIEHS/DNTP and other is largely unknown. metabolites urinary hydroxylated PAH analysis of prospective epidemiological deaths and cardiovascular-specific all-cause, cancer-specific, (OH-PAHs) with Health and Nutrition Examination sample (i.e., National in a representative using the Mortality was ascertained of the U.S. population. Survey 2001–2014) detected Metabolites of commonly Index, followed up to 2015. National Death as naphthalene) were evaluated phenanthrene, fluorene, PAHs (pyrene, complex mixtures. NIEHS/DNTP presented the aggregate exposure and Annual Conference of the International Society of study’s findings at the 32nd Environmental Epidemiologists. woodsmoke of Evidence mapping evaluation and cancer hazard Ruth Lunn is use wood as a primary heat fuel, and there More than 2 million U.S. households exposure to woodsmoke from increasing numbers an emerging concern about natural substance, which is often perceived as safe, of wildfires. Because it is a between the public’s perception and that of the there may be a disconnect health effects of woodsmoke. scientific community on the concerns, NIEHS/DNTP is developing an evidence To address and translate these hazard evaluation of woodsmoke and will map and conducting a cancer hazard concerns. communicate its findings about and have largely NIEHS/DNTP scientists completed the literature scoping and mechanistic studies. completed a detailed evidence map of human cancer development). (The protocol and cancer hazard evaluations are under PAHs Amy Wang for exposure Among hundreds of PAHs, only 16 are commonly monitored has identified more than assessment and 13 are listed in the RoC. NIEHS/DNTP a small number of these 250 animal studies on at least 35 PAHs. However, only classification (e.g., have an adequate database of studies for carcinogen of PAHs of varying multiple species or tumor sites). An expanded database use of structure- and toxicities would provide an opportunity for increased prediction. bioactivity-based read across for toxicity and carcinogenicity Urinary PAH metabolites and mortality States: the United in Urinary PAH The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 58 The Science of Substance | 5. Testing and Assessment 61 Binding activity of estrogen receptors alpha and beta Binding activity of estrogen receptors Androgen receptor Rat alpha-fetoprotein Human sex hormone binding globulin of estrogen Agonistic and antagonistic activity receptor alpha Develop a method for error-corrected sequencing of for error-corrected sequencing Develop a method in somatic hotspot cancer-driver amplicons enriched mutations levels data on cancer-driver mutation Establish how the a and mice can be used to derive from humans, rats, risk, and expansion, predict future cancer metric of clonal studies sub-chronic, repeat-dose rodent extrapolate from risk to human cancer and methadone Investigate the effects of morphine when administered exposure on neural tube development to mice during early pregnancy hypoxia as a Focus on maternal toxicity, specifically defect development contributing factor to neural tube defects, fetal Assess incidence rate of neural tube clinical blood gas and maternal drug levels, maternal uterine artery blood flow parameters, and measurement of endocrine-related Develop models integrating diverse endpoints for chemicals, including: • • • • • Quantitatively assess the neurotoxicity associated with developmental exposure to cannabidiol in rats Perform behavioral assessments, including measures of complex cognition, neuropathological and neurochemical endpoints, among many others Develop an in vitro model to determine the effects of drugs and chemicals on spermatogenesis Investigate whether the in vitro testes culture system can produce sperm and is responsive to known male reproductive toxicants, as assessed by steroid levels, immunohistochemistry, and metabolomic markers of injury

Objectives ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ ƒ Research Somatic oncomutations as biomarkers translating for safety datapreclinical to riskhuman cancer Barbara Parsons of opioid- Investigation induced neural tube defects in a mouse model Amy Inselman of Enhance prediction potential endocrine activity integrating of chemicals by data endpoints multiple Huixiao Hong Assessing the neurotoxicity developmental in of cannabidiol exposure rats Sprague Dawley Tim Flanigan of an in vitro Evaluation testis organ system as an alternative male model for toxicology reproductive Noriko Nakamura Highlights

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5 NTP at FDA/NCTR NTP at role in (NCTR) plays a critical Center for Toxicological Research The National (FDA) and the Department the Food and Drug Administration the missions of NCTR and protect public health. Human Services to promote of Health and in FDA, other government with researchers from elsewhere collaborates industry to provide innovative technology, methods agencies, academia, and training, technical expertise, and data generation development, vital scientific unique scientific expertise and infrastructure of for FDA decision-making. The FDA product centers and their regulatory roles. NCTR is critical in supporting designated NCTR as FDA’s principal partner to In 1979, the FDA Commissioner interface between FDA’s regulatory mission and NTP, enabling a science-based NTP projects provides brief summaries of NTP’s research mission. Appendix III FDA/NCTR in FY 2020. completed or underway at The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 60 62

The Science of Substance | 5. Testing and Assessment additional research studies. microbial growth,and adverserespiratoryhealtheffectsand havecalledfor that identifiedassociationsbetweenexposure todampindoorenvironments, and theWorldHealthOrganization(WHO) havepublishedconsensusreports Academy ofSciences(NAS)Committee onDampIndoorSpacesandHealth including allergicrhinitis,asthma,andhypersensitivity pneumonitis.TheNational can besignificantlyelevatedandlead torespiratoryhealtheffects, environments (e.g.,agriculture,forestry, service,manufacturing)whereexposure across theUnitedStates.Thisexposureis ofparticularconcerninoccupational derived frombiologicalsourcessuchas fungi,continuestocauseconcern Inhalation exposureandassociatedhealtheffectsofaerosols,particularlythose Fungal Exposures Immunotoxicology Research toStudy CDC/NIOSH inFY2020. Appendix IIIprovidesbriefsummariesofNTPprojectscompletedorunderwayat and determinestheapproximatenumberofworkersexposedtothesechemicals. dermal exposurestohigh-prioritychemicalsatworkplacesintheUnitedStates Through asetofexposureassessmentstudies,NIOSHcharacterizesinhalationand agenda areinfluencedby by theNationalOccupationalResearchAgenda.Prioritiesforthisresearch National InstituteforOccupationalSafetyandHealth(NIOSH)isprimarilydriven and thousandsofnewchemicalsareintroducedeachyear.Researchatthe Approximately 84,000chemicalcompoundsarefoundintheworkplace, Occupationally RelevantExposureResearch:OverviewoftheNTPProgram NTP atNIOSH f f f 5

.3 The chancethatnewdataorapproachescanmakeadifference. The seriousnessofahazardorproblem,and The numberofworkersatriskforaparticularinjuryorillness,

Recent projectsincludethefollowing: strengthen thescientificbaseforfungalriskassessment. quantification ofenvironmentalfungi,aswellgeneratedatathatwill projects willassistindevelopingimprovedtestingmethodsforthe with sub-chronicfungalexposures.Theknowledgegainedfromthese identification andbettercharacterizethehealtheffectsassociated assessment methodologiestoimprovespecies-specificfungalhazard WHO reports.Theyalsosupportthedevelopmentofinnovativeexposure Projects directlyaddresstheknowledgegapshighlightedinNASand workplace. Theagreementfocusedontwoareasofstudy: occupationally relevantaerosolsencounteredinthebuiltenvironmentor exposed toquantifiableconcentrationsoffungalbioaerosolsandother supported toxicologicalevaluationsofpotentialhealtheffectsinhumans In 2019,aninteragencyagreementbetweenNIEHSandNIOSH understanding healthimpacts,severalknowledgegapsremain,including: receive requestsforinformationonthesubject.Despiteprogressmadein NIEHS andtheNIOSHHealthHazardEvaluationProgramfrequently Because ofthepublichealthburdenassociatedwithfungalexposures, f f f f f f

underway towarddevelopment offinalreportsforpublication. chartarum exposurehavebeencompleted anddataanalysisis repeated Aspergillusfumigatusexposure andrepeatedStachybotrys Studies toexaminetoxicologicalresponses inrodentsto encounter inafungal-contaminatedoccupational environment. studies moreaccuratelymodelsthenatural exposureaworkerwould An AcousticalGeneratorSystemdeveloped byNIOSHforthese the NTPin2004 Pulmonary immunologyandfungaltoxicologystudiesnominatedto exposure sources andpotentialbiomarkersofpersonaloccupational The developmentofimprovedmethodologiestodetectmicrobial cardiac systemiceffects The effectofrepeatedfungalexposureonneurologicaland such asrespiratorymorbidity the mechanismsbywhichfungimodulateadversehealtheffects, The assemblageoffungithatcontributetopersonalexposureand

63 The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The The Science of Substance | 5. Testing and Assessment 65 Nail salons installation Rigid polyiso board Roofing foam Spray polyurethane installation

f f f f Carpet installation Chemical manufacturing Electronic scrap Foam manufacturing Gymnastics studios Findings peer- Using results from the study, publishedreviewed journal articles were on (1) polyurethane spray foam worker exposures to tris(1-chloro-2-propyl) phosphate (TCPP), (2) nail technicians’ (TPhP), exposures to triphenyl phosphate and (3) all-industries, air- and hand-wipe . sampling results and comparisons The study’s results showed high TCPP exposure in spray polyurethane foam installation, foam manufacturing, chemical manufacturing, roofing, and rigid board installation relative to exposure in other industries. TPhP air concentrations were very high for chemical manufacturing workers compared with other industries. PBDE exposures were present among carpet installers and electronic scrap workers and in gymnastics studios, but they were lower than in previous studies as a result of the phaseout of PBDEs.

f f f f f NTP Study on Occupational Exposure OccupationalStudy on NTP Exposure that measured assessment a comprehensive NIOSH conducted In FY 2020, brominated and non-PBDE PBDEs, organophosphate, exposures to workers’ settings: industries and following in the flame retardants workers’ personal breathing zone, hand-wipe The exposure assessment included collection. sampling, and urine and serum

Flame retardants are often added added Flame retardants are often or applied to furnishings, electronics, materials, building and construction and transportation products. Many flame retardants have been removed from the market or are no longer produced. The most common type, polybrominated diphenyl ethers (PBDEs), were phased out in or before 2013 due to evidence of liver and thyroid effects, developmental changes, and neurotoxicity in humans. However, because they do not break down easily, these chemicals can remain in the environment for years. They can also build up in people and animals over time, a process known as bioaccumulation.

Flame retardants are Flame retardants are chemicals that are applied to materials or to prevent the start slow the growth of fire. These chemicals have been used in many consumer and industrial products since the 1970s to decrease the ability of materials to ignite. HIGHLIGHT Flame Retardants The Science of Substance | 5. Testing and Assessment Assessment and Testing 5. | Substance of Science The 64 66

The Science of Substance | 2. About NTP For generalinquiries, contact: Research TrianglePark, NC 27709 [email protected] P.O. Box12233,MDK2-05 NTP WebTeam 984-287-3211