Validation is largely determined by the purpose of the test method, making it adaptable (Ref9). It serves as a cornerstone for gaining international acceptance of a testing method. Typically, a validation study takes around two years to complete; however, it may take longer if any issues arise (Ref20).

Overview of important pre-validation steps

Ensure that you can clearly explain the relevance of your new test method (Ref6,Ref13,Ref21,Ref22). Outline what your new test method is designed to measure and, if applicable, how it compares to existing tests. Consider questions such as: What motivated the development of this new test? What specific insights can the test provide? These considerations are essential (Ref6,Ref13,Ref14) It is important to distinguish between biological and regulatory relevance, as both are critical to address.

For pharmaceuticals, the EMA refers to this as the "context of use." This term outlines the specific situations in which the testing approach can be applied to evaluate human or veterinary medicinal products. It should also detail the limitations within which existing data sufficiently support the use of the 3Rs approach (Reduce, Refine, Replace). For instance, it should be demonstrated that the new or alternative testing method or strategy either provides new data that fill a known gap or offers data that are at least as valuable, if not more so, than those obtained through current methods (Ref22).

Another important aspect, referred to as the "rationale," can be based on several factors (Ref7). These may include:

• ​the absence of an existing test that addresses the hazard endpoint of interest,
• ​the availability of a test that yields more reliable or better information,
• ​a method that is safer and uses fewer or no animals,
• ​or a test that is more cost-effective.

Additionally, describing the relationship between the test's endpoint and the biological phenomenon of interest can be particularly helpful, especially for in vitro tests (Ref7, Ref23). While these principles are primarily written for chemicals, they can also be relevant to other subjects.

Ensure that your new test is reliable, meaning it should be repeatable (within-lab reproducibility), reproducible, and robust. (Ref1, Ref6, Ref13, Ref21, Ref23). One of the best ways to demonstrate this is by making your test transferable, which means that other laboratories can achieve the same results as you based on your documentation or Standard Operating Procedure (SOP) (Ref5, Ref13). Keep in mind, an SOP is not (yet) a Test Guideline or a standard. EURL ECVAM provides a list of EU NETVAL laboratories you can use, but any adequately equipped laboratory can be utilized as a naïve lab, as long as they follow an agreed-upon SOP and a detailed protocol before starting testing to minimize the risk of failure (Ref13, Ref7). It is advisable to systematically gather and standardize all this data.

Additionally, ensure that your new test is accurate. Accuracy refers to how close your test results are to a standard or known value (Ref13, Ref23). Whenever possible, compare your results to human data rather than relying solely on data from animal experiments (Ref16). However, some researchers suggest using data collected from proxies or surrogates, such as animal models (Ref6). Keep in mind that not all animal models have been validated for their reliability or repeatability in humans (Ref6, Ref19).

l'Association PEPPER is a public-private platform for the validation characterization methods that can help accelerate facilitate the transition from academic research to regulatory-compliant methods for assessing endocrine disrupters of endocrine related test methods. PEPPER’s mission is to be the missing link to fast-forward the validation process and to organize and fund scientific research and testing in order to obtain the proofs required by the authorities in charge of international validation (OECD, ECVAM, ISO). PEPPER also plays a role in connecting to the OECD process, responding to peer reviews, and developing test guidelines (Ref18).  Every year Pepper selects test methods on endocrine disruption, with a high level of technological maturity, developed by private or public laboratories, for which it will support validation. This action aims to diminish the lack of validated methods (Ref68).

The goal of the PEPPER Association is to identify test methods that are currently under development and to facilitate the transition from academic research to regulatory-compliant methods for assessing endocrine disrupters. Initially, an overview of available methods was created (Ref24) which would be suitable to enter the validation process. The next step involved conducting transferability studies inanother lab, where some weaknesses in procedures were identified. A blinded ring test was performed to evaluate the reproducibility, reliability, and applicability of these methods. Throughout this process, PEPPER received assistance from the international Validation Management Group (Ref18).

When designing your tests, remember that a research hypothesis is tested through these evaluations. Consider whether your test design reflects the objectives of the hypothesis and whether the test parameters are optimized to either accept or reject it (Ref1).

The literature mentions four partially overlapping packages related to test methodology:

1) Test method description
2) Technical test procedure (including labware used and steps taken during research; this can also be found in the SOP)
3) Characterization of test and reference materials/chemicals
4) Issues concerning data processing and archiving.
 
Additionally, there is a fifth package that addresses the purpose and limitations of the test. It is also important to acknowledge any known scientific limitations of your test (Ref23).

Data Translation 
 
The following steps are outlined to translate data into information (Ref25 ):

1) What is being modeled?
2) Which hypothesis is being tested?
3) Are there additional (often overarching) hypotheses that the test information helps to clarify?
 

The Role of Applicability Domains and Context-Dependent Validation
The importance of human relevance and the concept of "applicability domains" in validation studies is discussed, noting this aspect is often underestimated (Ref23). It emphasizes the need to clearly define the domain of application, especially in "borderline domains" that require evaluations on a case-by-case basis, as well as applications that are physically feasible but typically yield unsatisfactory results.

A common misconception about validation is the belief that a new test should serve as a direct replacement for an existing traditional test. However, validation is context-dependent and purpose-driven, encompassing a variety of assays (Ref6). According to sources, a test should be evaluated in relation to relevant information from the species of concern and existing toxicity data (Ref7).

Scientific Rigor and Peer Review in the Validation Process
It is crucial to recognize that merely summarizing and analyzing test data is insufficient for a validation study. The validation process is a scientific endeavor, requiring objectivity and appropriate methodology, including sound study design (Ref6). It is crucial to specify the domain to which the new test applies (Ref13).

Modeling mechanisms in alternative test methods is vital for making robust and relevant predictions, but it does not guarantee the accuracy of those predictions (Ref6). Following the completion of the validation study, an evaluation of its performance should be conducted (Ref13).

Upon concluding the validation study, it is possible to initiate an independent peer-review process. The sponsor has the option to select peer reviewers for the study's results, or they can choose to submit the test without peer review. In the latter scenario, if submitted to the OECD, they will organize their own review panel (Ref13).

Transparency and Regulatory Acceptance
Some regulatory agencies require transparency in the validation process. If this transparency is lacking, it could result in delays, suspensions, or cancellations of the validation (Ref5).

It is essential that the validation report includes all relevant data and potentially provides access to this data to facilitate the process (Ref7). The EURL ECVAM maintains a tracking system to monitor the test methods that have been submitted to them (Ref13).

Regulatory acceptance is aided by having a clear scope and realistic goals. A Validation Management Team (VMT) is appointed to oversee the validation study. The VMT's responsibilities include coordination, independent selection of reference chemicals, expert review, and independent statistical analysis of results. An independent statistical analysis is necessary to evaluate the test method's variability within and between laboratories and its predictivity (Ref20).

In December 2022, the OECD held a WNT workshop where participants discussed varying interpretations of what constitutes readiness for a test method. They identified three possible stages of readiness:

1. Readiness for validation,
2. Readiness for inclusion in the Programme for Test Guideline development,
3. Readiness for regulatory application.

These different contexts determine the level of supporting information expected on a given test method. Since each stage requires different types of support, it was recommended to establish minimum readiness criteria (Ref27).

• ​The number of test laboratories to be included in a validation study is specific to each test method, but there is a general consensus that at least three laboratories should be involved (Ref20).
• ​On-site training for laboratory staff is crucial when transferring a test method for the validation study (Ref10).
• ​It is important to evaluate the test "in relation to relevant information from the species of concern and existing relevant toxicity data." This consideration is particularly crucial if the test aims to replace or substitute an existing one. The goal should be to maximize predictive range while minimizing false negatives.
• ​A significant challenge is the availability of good data due to possible poor or unknown quality of in vivo animal data to which in vitro test methods are validated, or limited human data available (Ref9).
• ​Limited resources, changes in staff, and a lack of commitment from the validation sponsor can increase the duration of a validation study. (Ref20).
• ​A test method that addresses an identified endpoint gap is likely to gain more international acceptance (Ref20)
• ​When creating a test to detect toxicity, it's essential to draft a proposal that outlines the number and identity of reference chemicals (positive and negative), or the specific toxins the test is expected to identify (Ref7).