The ARRIVE 2.0 Guidelines: Importance and Full Adoption by AALAS Journals

Introduction

A recent editorial discussed the pending merger of the AALAS Comparative Medicine journal into the Journal of the American Association for Laboratory Animal Science (JAALAS), including the rationale supporting the decision and the March 2024 survey results that endorsed this change.³¹ In that editorial, we also briefly noted that the survey results supported full adoption of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) 2.0 guidelines, and we announced that AALAS journals would fully adopt the guidelines starting on January 1, 2025. The purpose of this editorial is to document support for the full adoption of ARRIVE guidelines; provide some background on scientific reproducibility issues and the potential role of ARRIVE in addressing concerns about reproducibility, replicability, and generalizability; briefly summarize the guidelines; and describe steps to be taken to assist authors once ARRIVE guidelines are fully adopted for new manuscripts submitted starting January 1, 2025.

Support for Adoption of ARRIVE Guidelines

As part of the March 2024 survey, respondents were asked 3 questions related to adopting ARRIVE 2.0 guidelines for AALAS journals (Table 1).

Table 1.

Responses to ARRIVE 2.0 survey questions

	A. Are you aware of the ARRIVE 2.0 Guidelines?	B. Does a requirement to address the ARRIVE 2.0 Guidelines make you LESS likely to submit a manuscript to a scientific journal?	C. Does a requirement to address the ARRIVE 2.0 Guidelines make you MORE likely to submit a manuscript to a scientific journal?
Yes	153 (67.4%)	4 (2.6%)	62 (40.8%)
No	56 (24.7%)	132 (86.8%)	66 (43.4%)
I am aware but not familiar	18 (7.9%)
I don’t know		16 (10.6%)	24 (15.8%)
Total	227 (100%)	152 (100%)	152 (100%)

The responses suggest that most respondents were aware of the ARRIVE guidelines, although question A did not capture the depth of respondent familiarity with the guidelines. We cannot be certain, but it is likely that those respondents who were aware of the guidelines (“yes” to question A, n = 153) were the ones who elected to answer questions B and C (n = 152). The responses to questions B and C suggest that an author requirement to address the ARRIVE 2.0 guidelines would not discourage manuscript submission (86.8%).

In summary, the survey results reflected broad support for the full adoption of the ARRIVE 2.0 guidelines by AALAS journals.

ARRIVE Guidelines: Background and Role in Addressing Reproducibility Issues

The ARRIVE guidelines were originally developed as a 20-item checklist in 2010 by an international working group organized by the National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)²¹ and were then updated in 2020 as the ARRIVE Guidelines 2.0.^28,29 Before discussing the individual guidelines and why they are important, it is critical to understand that their development has been driven by the need for improved reporting of animal research, a factor contributing to concerns about scientific reproducibility and translation of animal research to human clinical application.^6,13,14 Emphasizing such concerns was a 2009 survey of 271 animal research articles that reported that only 59% of articles stated a hypothesis, less than 15% used randomization to assign groups or blinding to reduce observation bias, and only 70% of the publications that used statistical methods reported any measure of variability or error.¹⁴

Since then, there has been growing awareness of scientific reproducibility issues. A PubMed search using the search term “scientific reproducibility” returned 102,318 hits on July 26 of this year, encompassing all areas of science and technology, including animal research.^{5,9,15–19,32} AAALAC International notes that “programs should consider scientific rigor and experimental reproducibility when evaluating animal research activities” and cites the ARRIVE guidelines as a means to encourage both when experimental design methods are reported in peer-reviewed journals.² Some other key relevant publications addressing reproducibility (all of which reference the ARRIVE guidelines) include a 2011 publication by the U.S. National Academies,²³ 2016 reproducibility recommendations by the Federation of American Societies for Experimental Biology (FASEB),¹⁰ and a second detailed 2019 report by the U.S. National Academies.²⁰ In this 2019 publication, consensus definitions were provided for key terms:

• • Reproducibility is taken to mean computational reproducibility—obtaining consistent computational results using the same input data, computational steps, methods, code, and conditions of analysis and replicability to mean obtaining consistent results across studies aimed at answering the same scientific question, each of which has obtained its own data. In short, reproducibility involves the original data and code.

• • Replicability involves new data collection and similar methods used by previous studies.

• • Generalizability refers to the extent that the results of a study can be applied in other contexts or populations that differ from the original one.

In response to the expanded public and research community discussion of reproducibility concerns, the U.S. Congress included Section 2039, “Enhancing the Rigor and Reproducibility of Scientific Research,” in the 21st Century Cures Act passed in late 2016.¹ This section directed the NIH Director to assemble an advisory committee, which published a final report on “Enhancing Rigor, Transparency, and Translatability in Animal Research” in 2021.³ The report covers a variety of topics, and a detailed summary is beyond the scope of this manuscript, but a key recommendation was for NIH to “…improve reporting of results by setting expectations for the use of the ARRIVE 2.0 Essential 10 checklist and the inclusion of statistical measures of uncertainly and effect size.” In early 2023, NIH released Notice NOT-OD-23-057 that encouraged the use of the ARRIVE 2.0 Essential 10 checklist in all scientific reports involving NIH-supported animal research using vertebrates and cephalopods.²⁵ Note that the ARRIVE “Essential 10” items are a subset of the full ARRIVE Guidelines, which are described next.

What Are the ARRIVE Guidelines?

Simply put, the guidelines are a checklist of principles to help in all stages of animal study design and results reporting, that is, when planning, conducting, and reporting the study and for reviewers when reviewing manuscripts. An online checklist is available listing and describing the 10 items in the Essential 10 (Figure 1) plus the 11 additional items in the Recommended Set for a total of 21 items (Figure 2), as well as detailed explanations of each of the items in both checklists.²⁸

Figure 1.

The ARRIVE 2.0 Essential 10 with descriptions. Adapted from Table 1 in Percie du Sert and colleagues²⁹; used per Creative Commons CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/) and with the permission of the National Centre for the Replacement, Refinement and Reduction of Animals in Research.

Figure 2.

ARRIVE 2.0 Recommended Set with descriptions. Adapted from Table 2 in Percie du Sert and colleagues²⁹; used per Creative Commons CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/) and with the permission of the National Centre for the Replacement, Refinement and Reduction of Animals in Research.

In a sense, the ARRIVE Guidelines assist researchers with organizing and reporting the experimental details in a way that facilitates reproducibility, and it should be noted that they do not prescribe specific experimental approaches. Most of the guidelines have been required by AALAS journals for many years (Table 2), but some discussion of specific items is provided below.

Table 2.

Status of ARRIVE 2.0 item adoption by AALAS journals for hypothesis-driven manuscripts

Essential 10/Recommended Set	Current policy	New policy as of January 1, 2025
Essential 10
1. Study design	Required somewhere in manuscript	Required in new “Study Design” section in Materials and Methods
2. Sample size	Partially required: a priori sample size calculations (for example, power analysis) analyses to determine group sizes not always described	Required in new “Sample Size” section in Materials and Methods
3. Inclusion and exclusion criteria	Optional: reviewers can request at their discretion	Required in new “Inclusion and Exclusion” section in Materials and Methods
4. Randomization	Optional: reviewers can request at their discretion	Required in new “Randomization” section in Materials and Methods
5. Blinding	Optional: reviewers can request at their discretion	Required in new “Blinding” section in Materials and Methods
6. Outcome measures	Required somewhere in the manuscript	Required in new “Outcome Measures” section in Materials and Methods
7. Statistical methods	Required somewhere in the manuscript	Required in new “Statistical Methods” section in Materials and Methods
8. Experimental animals	Required in Materials and Methods	Required in new “Experimental Animals” section in Materials and Methods
9. Experimental procedures	Required somewhere in manuscript	Required in new “Experimental Procedures” section in Materials and Methods
10. Results	Required in Results section	Required in Results section
Recommended Set
11. Abstract	Required in Abstract	Required in Abstract
12. Background	Required in Introduction section	Required in Introduction section
13. Objectives	Required in Abstract and Introduction sections	Required in Abstract and Introduction sections
14. Ethical statement	Required	Required in new “Ethical Statement” section
15. Housing and husbandry	Required in Materials and Methods	Required in new “Housing and Husbandry” section in Materials and Methods
16. Animal care and monitoring	Required in Materials and Methods	Required in new “Animal Care and Monitoring” section in Materials and Methods
17. Interpretation/scientific implications	Required in Discussion	Required in Discussion
18. Generalizability/translation	Optional	Required in new “Generalizability/Translation” section
19. Protocol registration	Optional	Optional: prompted it applies
20. Data access	Optional	Response required but data access not required
21. Declaration of interests (conflicts of interest, funding source(s))	Required in separate “Conflict of Interest” and “Funding” sections	Required in separate “Conflict of Interest” and “Funding” sections

Item 2b (sample size).

“Explain how the sample size was decided. Provide details of any a priori sample size calculation, if done.”

Commentary: AALAS journals have required a description of experimental groups and the number of animals in each group for many years. Although many individual reviewers ask for a justification of group sizes by power analysis or other methods, this has not been required in manuscripts upon submission. Of note, institutions with a U.S. PHS Assurance or accreditation by AAALAC, International must follow recommendations as noted in the Guide.²⁴ Specifically, it is stated on page 25 of the Guide:

“The animal use protocol is a detailed description of the proposed use of laboratory animals. The following topics should be considered in the preparation of the protocol by the researcher and its review by the IACUC: … justification of the species and number of animals proposed; whenever possible, the number of animals and experimental group sizes should be statistically justified (e.g., provision of a power analysis; …).”

Some may view the need for sample size calculation as a means of preventing the use of excessive numbers of animals, but the reality is that using too few animals can just as wasteful as using too many. As quoted from Percie du Sert et al²⁷:

“Sample sizes that are too small (i.e., underpowered studies) produce inconclusive results, whereas sample sizes that are too large (i.e., overpowered studies) raise ethical issues over unnecessary use of animals and may produce trivial findings that are statistically significant but not biologically relevant. Low power has three effects: first, within the experiment, real effects are more likely to be missed; second, when an effect is detected, this will often be an overestimation of the true effect size; and finally, when low power is combined with publication bias, there is an increase in the false positive rate in the published literature. Consequently, low-powered studies contribute to the poor internal validity of research and risk wasting animals used in inconclusive research.”

Item 3.b (Inclusion/Exclusion Criteria).

“For each experimental group, report any animals, experimental units or data points not included in the analysis and explain why. If there were no exclusions, state so.”

Commentary: This has been requested by many reviewers in the past but has not been a general requirement for submission. There are statistical overtones to this issue that should be considered by authors during manuscript preparation.

Items 4 (Randomization) and 5 (Blinding).

Although frequently addressed by reviewers, addressing these items in the manuscript at submission has not been required previously.

Item 14 (Ethical Statement).

AALAS has required proof of IACUC or similar ethical committee review and approval for decades, but the location of this information currently varies widely between manuscripts. The location of this information within manuscripts will be standardized.

Item 16c (Animal Care and Monitoring, Endpoints).

This information has not been required in submitted manuscripts but is a commonly required protocol component by IACUCs and, therefore, should be readily available for authors.

Item 18 (Generalizability/Translation).

These concepts have not been regularly addressed in animal research, but it is important to describe the applicability of the findings to other species or research circumstances and how the results might impact, directly or indirectly, human or animal health.

Item 19 (Protocol Registration).

A small number of animal research manuscripts are registered with sites such as the Open Science Framework website.²⁶ Authors may optionally provide any protocol registrations in this section.

Item 20 (Data Access).

Authors will be asked to state if any data from the study can be shared; however, data sharing is not required by the AALAS journals. Note that NIH has issued a number of data-sharing requirements, including Notice NOT-OD-21-013, which applies to research funded or conducted by NIH that results in the generation of scientific data.²² Many other countries have similar requirements for federal funds.

Helping Authors Provide the ARRIVE Information

As shown in Table 2, of the 21 ARRIVE items, 14 are currently required. Of the 14 items currently required, 4 (items 1, 6, 7, and 9) are required but the location in the manuscript is not specified, which leads to some searching by the reviewers. In addition, the 3 items currently expected in the Materials and Methods (items 8, 15, and 16) can appear anywhere in the Materials and Methods, leading to additional searching by the reviewers.

To assist authors and reviewers, a new manuscript template will be released for use starting January 1, 2025, which will organize the manuscript so that all ARRIVE guidelines are addressed in the template. This will benefit both authors and reviewers and most likely save time for many authors because following the template helps ensure that information needed by reviewers will be present. It will also make the reviews more consistent because reviewers will not have to search for information requested by the reviewer form and all reviewers will see the information laid out in a more organized manner.

Only hypothesis-driven studies will need to use the new manuscript template. Authors of other types of manuscripts, such as case study/case series manuscripts, will be able to choose which sections and items are applicable to their data and organize the manuscript as needed.

The editors will monitor any author and reviewer concerns and make changes as needed.

Conclusions

Potential issues with respect to reproducibility, replicability, and translatability are now widely recognized in research involving animal subjects and are greatly concerning. The stakes are compounded by the waning confidence of legislative bodies and the public that science is performed ethically and carefully and that published results can be trusted. Groups intent on ending animal research have leveraged these concerns into false narratives that claim animal research is neither needed for human health nor done in an ethical manner.

These are complex problems, and there are no easy solutions. AALAS journals have consistently published relevant manuscripts on enhancing reproducibility, including a special topic emphasis on extrinsic factors affecting research divided between the March (63:2) and May (63:3) 2024 issues of JAALAS.^{4,7,8,12,30,33–35} We will always welcome new manuscripts addressing these areas.

One strategy to address all these concerns is the full adoption of the ARRIVE guidelines, which AALAS journals will do starting January 1, 2025. Use of the ARRIVE guidelines is no panacea,¹¹ but it is a step in the right direction. The 2011 NRC document noted that all stakeholders have an important role in improving reproducibility and emphasized the responsibility of journal editors to “actively promote effective and ethical research by encouraging the provision of sufficient information to enable assessment and interpretation of research findings and advancement of knowledge based on reproducible results.”²³ The editorial staff looks forward to partnering effectively with authors and reviewers to ensure that AALAS journals continue to serve our community and continue to make a strong case that animal research is critical to improving both human and animal health.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

Preparation of this manuscript was funded by AALAS.