From Lab to Legislation: NAMs research is evolving, but is it ready to drive change in Global Regulations?

Introduction

New Approach Methods (NAMs) have always been a valuable tool to predict product safety.? NAMs are being routinely used by the R&D labs of the Indian Industry, as well as the Academia.? It is being performed at the initial discovery, lead identification as well as lead optimization stages. Thus, the Indian industry, interestingly, was never in inertia w.r.t. acceptability of NAMs! It has been a routine process at all the R&D centers of the top 5 pharma and Cropcare corporations in India to use different NAM-based tools for screening their new products as well as developing safer options.? in vitro ADME-driven assessments have been routinely undertaken by these R&D centers, as well as some of the Indian CROs engaged in regulatory/discovery safety evaluations, for more than two decades.? The application of in silico tools in lead identification has been in place for several years, with either their in house efforts or outsourced to the in silico experts in the Indian IT services corporations. These efforts were led by several experts at the top five Indian IT corporations, as well as niche, in silico-focused CROs.?? A large number of the top industrial R&D centers, as well as CROs, have been working on NAMs-based regulatory as well as discovery solutions for a period exceeding 12 years (Reference: unpublished observation).

Regulators have opened their doors for NAMs-driven Hazard Assessment, Globally as well as in India.

NAMs have a long history beyond two decades, in India. With the regulators formalizing the application of NAMs through the 27 well-defined guidelines and six Test Methods (*Reference:1), At least three of the six acute tests have been released free from animal research in the EU. The EU, OECD, NTP-US, EURL-ECVAM, NTP-ICCVAM, JaCVAM, NICEATM, IIVS, and allied organizations have been committed for the past two to three decades to develop test methods that would be reliable alternatives to animal toxicity tests.

Several Regulators have mandated the elimination of animal testing for cosmetics, and a range of animal tests have been implemented.

Back home in India, the Indian Government, released a GSR 346E of 2014, clearly mandated no use of animals in testing cosmetics. It states "148 C: prohibition of testing of cosmetics on animals - No person shall use any animal for testing of cosmetics"

CIB-RC, in 2017, published its guidance document (*Reference:2) on chemical pesticides, allowing for the acceptance of alternate approaches. More recently, the CIB&RC approved new guidelines for chemical pesticides and biopesticides amended as per 450th RC minutes (*Reference:3), which states the following:

"The replacement alternatives not involving experiments on animals would be considered and accepted in those cases where data from valid 3Rs (Replacement, Reduction and Refinement) methods recognized by OECD and other renowned Regulatory Authorities is provided wherever applicable, case to case basis."

Dr. Swaminathan et al., (*Reference:4) Chief Scientist, WHO published a thought-triggering article.? It states,

"Although India made some advances in regenerative medicine technology (primarily by academic and government institutions) using stem cells derived from induced pluripotent cells and mesenchymal stem cells, such research has not been integrated or applied for drug discovery and development (DD&D) research of private sector. If India is to emerge as the innovation centre for DD&D research, the current state of the art should be embraced by both public and private sectors."

The Indian Drug Regulator DCGI "Drug Controller General of India"? released The Gazette of India Notification no 136, dated March 13, 2023 (*Reference:5), proclaiming acceptance of the NAMs data.

It states as below;

"The general requirements of non-clinical studies have been specified in the Second Schedule. (II) The non-clinical testing methods to assess the safety and efficacy of a new drug or investigational new drug include the following, namely: (i) Cell-based assay; (ii) Organ chips and micro physiological systems; (iii) Sophisticated computer modeling; (iv) Other human biology-based test methods; (v) Animal Studies."

Harmonization and Quality definition efforts

The OECD went on to release the concept of IATA (*Reference:9)? (Integrated Approach to Testing and Assessment). It states,

"Integrated Approaches to Testing and Assessment (IATA) combines multiple sources of information to conclude on the toxicity of chemicals. IATAs may include existing information from the scientific literature or other resources, along with newly generated data resulting from new or traditional toxicity testing methods to fill data gaps. These approaches are developed to address a specific regulatory scenario or decision context."

Quality Assurance

The Success of NAMs will depend upon perfect validation data of high quality, assuring reproducibility, ruggedness, and robustness of the tests being applied for safety prediction.? While the OECD, the EU, and US agencies are working on harmonized quality standards, as of 2023, there are no harmonized and globally acceptable quality standards w.r.t. NAMs.? Three major efforts, which have laid a foundation for harmonized quality, have laid down the principle framework for future quality.? These are fairly elaborate and are quite a challenge to implement.?

The first effort was OECD Guidance Document no. 34 (*Reference:7), which elaborated parameters for validation and international acceptance of test methods for hazard identification in August 2005!

Several groups and agencies worked in close coordination to establish the quality norms. The first guidance for in vitro methods to come was GIVIMP (*Reference:8) (for defining the quality parameters applicable to the in vitro practices), released by the OECD in 2018.

GIVIMP is divided into ten sections covering:

1. Roles and Responsibilities
2. Quality considerations
3. Facilities
4. Apparatus, material, and reagents
5. Test systems
6. Test and reference/control items
7. Standard operating procedures
8. Performance of the method
9. Reporting of results
10. Storage and retention of records and materials

The GIVIMP appears to piggyback on OECD GD 34(*Reference 5), which defines the following

1. Test definition (including purpose, need, and scientific basis);
2. Intra-laboratory repeatability and reproducibility;
3. Inter-laboratory transferability;
4. Inter-laboratory reproducibility;
5. Predictive capacity (accuracy);
6. Applicability domain; and
7. Performance standards.

GCCP-2 for the in vitro assays (*Reference:9):

The third successful effort to establish The principles of Good Cell Culture Practices (GCCP) was based upon the following operational principles:

1. Understanding the cell culture system and factors that affect the performance of the said system

2. Assurance of the quality in order to maintain the integrity, validity, and reproducibility of any work conducted, by:

• Assurance of the quality of all reagents, materials, equipment, cells, and tissues, and
• Application of quantitative suitability criteria, validated with performance standards.
• Documentation and reporting of the information necessary to track the materials and methods used, to permit the repetition of the work, and to enable the target audience to understand and evaluate the work.
• Establishment and maintenance of adequate measures to protect individuals and the environment from any potential hazards
• Compliance with relevant laws and regulations, and with ethical principles
• Provision of relevant and adequate education and training for all personnel, and promote high-quality work and safety

One of the thoughts under consideration is carrying out the tests of the test chemical with the defined positive and negative controls (affecting diverse AOPs) and submitting the full raw data and report! Thus, to assess one compound, one may end up using several positive and negative reference compounds to assess one test chemical in each test! The CRO/sponsor has to bear the expenses in such a case!

All these efforts and defined standards would be very valuable and useful if made mandatory to be followed across the globe by global regulators.

Operational difficulties

We provide specific cases below.

In this case, the challenge emanates from the availability of equipment, cells, and consumables for MPS, on the following counts;

1. Availability of certified primaries for experimental use.

2. These primaries, when available, are certified with the characterization data to establish their lineage with suitable IHC-based tests/gene expression analysis for the specific biomarkers for each lot/batch of cells.? This cost loads on the user of the systems.

3.???? Availability of "certified Test systems".?

• We recently encountered three different innovation vendors of MPS systems using primary hepatocytes. They claimed to provide the "validated primary hepatocytes," which perform well in their systems! However, they also claimed that the "applicability of the specific cell lot" sourced from different human subjects may not behave "as expected" in their system, hence the need for procurement of the "Vendor Certified lot."?
• Most of the innovation vendors also suggested mandatory "procurement of the equipment and cells" from them for reliable output. This situation provides the innovation vendors of MPS systems and cells with a platform for a monopolistic business environment. Such wasn't the case with the lab animals! The only consideration was a "healthy SPF" animal!

4.???? Each innovation vendor has its unique selling points. Many times, certain endpoints are well resolved in one system, requiring the use of another system (with different sources of cells) to arrive at coverage of maximal endpoints, thereby putting the cost back on the assay-performing labs.

The Regulatory Conundrum

The regulators, for several decades, have been comfortable granting registrations based on the animal studies submitted in compliance with the specific, globally harmonized guidelines with defined endpoints.? Unfortunately, by the very design of the experiments, it is almost impossible to generate well-defined guidelines for NAMs bases tests, following the process, as per OECD GD 34 (*Reference:5). An attempt was made to develop such guidelines for certain in vitro endocrine disruption tests by EPA with their OPPTS 890 series guidelines (*Reference:10).? These guidelines were very useful and exacting, but almost appeared like the SOPs!?

Such a scenario tends to force the regulator to be cautiously optimistic in granting blanket approvals for the NAMs driven studies.

The current challenge is the lack of clear agreement by all global regulatory bodies about the global acceptance criteria for harmonized consideration of NAM-based solutions. The industries and CROs have been and will continue to conduct NAM tests for screening / early discovery phase studies.?

The US Senate bill has been a driving force for the implementation of NAMs.? The FDA Modernization Act 2.0 (*Reference:11).? The act states;

NONCLINICAL TEST DEFINED.—For purposes of this section, the term ‘nonclinical test’ means a test con- ducted in vitro, in silico, or in chemico, or a non-human in vivo test that occurs before or during the clinical trial phase of the investigation of the safety and effectiveness of a drug, and may include animal tests, or non-animal or human biology-based test methods, such as cell-based assays, microphysiological systems, or bio-printed or computer models.’’.

The option appears in the US FDA Guidance to Industry 2020, about "in vitro Drug Interaction Studies, Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (*Reference:12),?the methodology defined could become a good landmark for developing guidance about the conduct of the NAMs-driven test methods.

Another concept gaining approval is the "Context of Use" (CoU) principle, which gives liberty to develop and use proprietary method/s, as long as the principle of the test clearly defines the CoU.? This may open the doors, proviso it receives global acceptance.

However, Strauss et. al. (*Reference:13), state;

The point to note is "there are opportunities for alternative methods to make additional inroads in addressing the 3Rs for specific contexts of use"

Way forward

It would be desirable to enforce a Global regulatory agreement regarding blanket refusal to accept animal studies for a set of commonly used tests, e.g., Acute-6-pack and a few others. Such a mandate is the need of the hour to ensure rapid progress in the development of new NAM-based solutions.

Representing the industry, we advocate the following measures to effectively implement NAMs worldwide.

1.???? Make all the NAMs mandatory for all the OECD/OPPTS guideline-driven studies with a blanket ban on the usage of Animals, across the globe to all the regulators.        

2.     Facilitate the supply chain of the cells, consumables, and equipment at competitive prices across the globe.        

3.???? Establish global quality and acceptability standards for the supplies of cells, consumables, and equipment.        

4.???? Establish global performance standards for the NAMs-driven studies.        

5.???? Establish global acceptance standards for the NAMs data.        

6.???? Rigorous implementation of IATA across the globe.        

End note:

NAMs have been, and will help reducing animals in the near future!? Moreover, the testing would be human-relevant if we move towards NAMs-driven approaches using human AOPs for in silico and in vitro safety prediction.

That said, the focused approach to resolving the challenges listed above should help the rapid progress of NAMs as reliable, reproducible, robust, and globally acceptable solutions for establishing human safety.

Questions? - [email protected]

References:

1. https://www.epa.gov/sites/default/files/2019-12/documents/alternative_testing_nams_list_first_update_final.pdf; (Pg 242, general recommendations, paragraph 1)
2. Indian Pesticide Guidelines: https://ppqs.gov.in/sites/default/files/toxguidancedocsept2017.pdf;
3. https://ppqs.gov.in/sites/default/files/450_rc_minutes.pdf Agenda item no 10.32
4. GSR 346 E; https://cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2018/UploadGazette_NotificationsFiles/GSR_346(E)animal_testing_ban.pdf
5. Need for alternatives to animals in experimentation: An Indian perspective" Indian J Med Res 149, May 2019, pp 584-592 Quick Response Code: DOI: 10.4103/ijmr.IJMR_2047_17? Soumya Swaminathan?, Vijay Kumar1 & Rajni Kaul1,*
6. IATA: https://www.oecd.org/chemicalsafety/risk-assessment/iata/
7. OECD Guidance Document No 34: GUIDANCE DOCUMENT ON THE VALIDATION AND INTERNATIONAL ACCEPTANCE OF NEWOR UPDATED TEST METHODS FOR HAZARD ASSESSMENT https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/oecd/oecd-gd34.pdf
8. GIVIMP: OECD Series on Testing and Assessment Guidance Document on Good In Vitro Method Practices (GIVIMP) no.286, 2018, 201 pages
9. Guidance Document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0), ALTEX 39(1), 2022, 30-70; David Pamies, Marcel Leist, Sandra Coecke, Gerard Bowe, David G. Allen, Gerhard Gstraunthaler, Anna Bal-Price, Francesca Pistollato, Rob B. M. de Vries, Helena T. Hogberg, Thomas Hartung & Glyn Stacey
10. https://www.regulations.gov/docket/EPA-HQ-OPPT-2009-0576/document
11. ‘‘FDA Modernization Act 2.0’’; https://www.congress.gov/117/bills/s5002/BILLS-117s5002cps.pdf; S. 5002 "To allow for alternatives to animal testing for purposes of drug and biological product applications"
12. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/in-vitro-drug-interaction-studies-cytochrome-p450-enzyme-and-transporter-mediated-drug-interactions.
13. David Strauss, MD, PhD "FDA New Alternative Methods Group Members – June 14, 2022"; https://www.fda.gov/media/159235/download.

Suggested Reading

1.???? EPA (2021). List of alternative test methods and strategies (or new approach methodologies [NAMs]). Update-2: February 4th, 2021. Office of Pollution Prevention and Toxics, U.S. Environmental Protection Agency . Available at: https://www.epa.gov/sites/default/files/2021-02/documents/nams_list_second_update_ 2-4-21_final.pdf Sited on Jan 26, 2024.

2.???? Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of industrial chemicals and pesticides for effects on human health Andreas O. Stucki*, Tara S. Barton-Maclaren, Yadvinder Bhuller, Joseph E. Henriquez, Tala R. Henry, Carole Hirn, Jacqueline Miller-Holt, Edith G. Nagy, Monique M. Perron, Deborah E. Ratzlaff, Todd J. Stedeford and Amy J. Clippinger , Frontiers in Toxicology, ALTEX, Sept 01, 2022; DOI 10.3389/ftox.2022.964553)

3.???? https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32009R1107, Sited: Jan 26, 2024

4.???? https://www.federalregister.gov/documents/2019/04/05/2019-06624/significant-new-use-rules-on-certain-chemical-substances, sited Jan 26, 2024

5.???? Judy Strickland, Esther Haugabrooks, David G. Allen, Luciene B. Balottin, Yoko Hirabayashi,Nicole C. Kleinstreuer, Hajime Kojima, Claudio Nishizawa, Pilar Prieto, Deborah E. Ratzlaff, Jayoung Jeong, JinHee Lee, Ying Yang, Pinpin Lin, Kristie Sullivan and Warren Casey, International regulatory uses of acute systemic toxicity data and integration of new approach methodologies, CRITICAL REVIEWS IN TOXICOLOGY, 2023, VOL. 53, NO. 7, 385–411 https://doi.org/10.1080/10408444.2023.2240852 ,

6.???? https://www.epa.gov/reviewing-new-chemicals-under-toxic-substances-control-act-tsca/framework-assess-eye-irritation-or sited on Jan 26, 2024

7.???? oppt-ncd-eye-irritation-framework-frn-final-12-13-2023.pdf, New Chemicals Program Decision Framework for Hazard Identification of Eye Irritation and Corrosion, Dec 13, 2023.

8.???? Sebastian Schmeisser, Andrea Miccoli, Martin von Bergenc, Elisabet Berggren, Albert Braeuning, Wibke Busch, Christian Desaintes, Anne Gourmelon, Roland Grafstrom, Joshua Harrill, Thomas Hartung, Matthias Herzler, George E.N. Kass, Nicole Kleinstreuer, Marcel Leist, Philip Marx-Stoeltinga, Oliver Poet. Bennard van Ravenzwaay, Rob Roggeband, Vera Rogiers, Adrian Roth, Pascal Sanders, Russell S. Thomas, Anne Marie Vinggaard, Mathieu Vinken, Bob van de Water, Andreas Luch, Tewes Tralau, New approach methodologies in human regulatory toxicology – Not if, but how and when!, Environment International 178 (2023) 108082

9.???? Mohammad A. Akbarsha &) and Benedict Mascarenhas, Cosmetic Regulation and Alternatives to Animal Experimentation in India, www.cosmeticsinfo.org/Regulation-in-eu. ? The Author(s) 2019 H. Kojima et al. (Eds.): Alternatives to Animal Testing, pp. 57–62, 2019. https://doi.org/10.1007/978-981-13-2447-5_7

10.? OECD ENVIRONMENT DIRECTORATE JOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY GUIDANCE DOCUMENT ON THE VALIDATION OF (QUANTITATIVE)STRUCTURE-ACTIVITY RELATIONSHIPS [(Q)SAR] MODELS, ENV/JM/MONO(2007)2 , Organisation de Coopération et de Développement Economiques Organisation for Economic Co-operation and Development 30-Mar-2007

11.? MINUTES OF THE 442nd REGISTRATION COMMITTEE MEETING HELD ON 18.11.2022.