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3 Experience at OECD with the Validation of Various Types of Test Methods

3 Experience at OECD with the Validation of Various Types of Test Methods

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Validation in Support of Internationally Harmonised OECD Test Guidelines…



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new and updated test methods for hazard assessment (OECD 2005). This was a

challenge for those involved in validation studies: while validation studies for

in vivo and in vitro assays were being designed, countries were building consensus around important principles of validation in parallel, and setting good practice for how to conduct validation. The resulting guidance was generalised across

new and updated in vitro and in vivo test methods. The area of endocrine disruption testing and assessment has succeeded in bringing together toxicologists and

ecotoxicolgists to organise validation studies following the same principles, and

testing the same chemicals. Three validation management groups (VMGs) were

established approximately at the same time at OECD under the Test Guidelines

Programme: the VMG-mammalian, the VMG-eco (for ecotoxicity testing) and

the VMG-non animal (for in vitro assays). Practical challenges arose in some

areas; for instance, it was not a common practice in aquatic toxicity testing to

use coded chemicals. Also, some disciplines of toxicology have been required to

provide clear guidance and formalise best practice through consensus OECD

guidance document in areas such as histopathology for various types of organs

and taxa.

Differences between types of studies (e.g. oral administration of a dose to a rat

or mouse versus waterborne exposure system for fish) made it difficult for aquatic

toxicity studies to show as low coefficients of variation as rodent studies. The chemical delivery to the test system in aquatic toxicity studies and the ability of the laboratory to maintain the exposure level over an extended period of time are major

challenge for the success of validation studies assessing the inter-laboratory reproducibility. As a result, the inter-laboratory variability is typically higher in aquatic

toxicity studies.

Furthermore, experience in laboratories and level of standardisation of test procedures varied substantially between assays that had a history of 50-years of use in

the pharmaceutical industry when they entered validation studies at OECD (e.g.

uterotrophic bioassay), and assays in fish measuring vitellogenin as a biomarker for

estrogenicity of chemicals, which had been performed for a maximum of five years

in the most advanced laboratories.

Finally, to conclude on differences between ecotoxicity and toxicology, the

diversity of environmental species used in regulatory testing in OECD countries is

intended to represent the biological diversity of ecosystems. This diversity makes

it challenging to develop a harmonised Test Guideline that can accommodate all

species using the same test procedure, but is essential for the regulatory acceptance

of the Test Guideline when the goal is to protect indigenous fauna. This requirement to use countries preferred species in OECD validation studies created additional constraint on the design of the validation. Nowadays a posteriori, other

approaches would be pursued, e.g. Performance-Based Test Guidelines, which tend

to simplify the emergence of additional similar and alternative methods by setting

essential components of the test method, clear goals and expected performance of

the given method.



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2.3.4



A. Gourmelon and N. Delrue



Test Methods Describing In Vitro Alternatives to Animal Testing



There is now more experience in the validation and regulatory acceptance of in vitro

procedures, and certainly the OECD GD 34 (2005) has been beneficial in that

respect, as well as all the experience gained by validation centres such as ICCVAM

in the United States, ZEBET in Germany, ECVAM in the European Union and

JaCVAM in Japan. Several OECD Test Guidelines have been published in the last

10 years that witness progress made in the conduct of validation studies, leading to

their regulatory acceptance. Challenges are often different from in vivo studies, for

one part because purposes are different. By providing clear mechanistic information, in vitro methods may pave the way to Integrated Approaches to Testing and

Assessment (IATA), where data from various in vitro tests combined with other

source of information, may lead to a reduction in the use of animals and ultimate

replacement of animal testing.

Performance standards (PS) have been developed for some Test Guidelines (e.g.

TG 435, TG 439, TG 455) to address two issues relating to in vitro test methods: (1)

in vitro test methods often use proprietary components such as cell lines, and abuse of

monopoly situations should be avoided, and (2) the emergence of similar test methods

is expected to be frequent due to innovation in this area. The concept of PS was

already elaborated in the OECD Guidance Document 34 on validation (OECD 2005).

Indeed, several existing in vitro Test Guidelines contain elements that are covered by patents and/or licensing agreements that cannot be reproduced or reengineered, and for which fees have to be paid by the user. In the validation study,

this is not an issue as such, as everyone can be requested to use the same cell line or

commercial kit in order to minimise sources of variability in the results. However,

the OECD policy is to enable a broad and unrestricted use of the test method at

reasonable expenses for the purpose of protecting human health and the environment; situations of abuse of a monopoly for a given test method, where a single

commercial provider could take a disproportionate financial advantage, are therefore avoided. For that purpose, performance standards are developed facilitating the

validation of other similar test methods.

Additionally, PS can also be developed for proposed test methods that are mechanistically and functionally similar to each other. The PS include the following three

elements:

– Essential test method components,

– A minimum list of reference chemicals, and

– The level of accuracy and reliability that a similar test method should

demonstrate.

They are developed for the validation of future alternative or “me-too” test methods that will have to be adopted by OECD in order to be covered by the Mutual

Acceptance of Data. The performance standards are based on one or several validated test methods. Any other similar “me-too” test method, whether it contains

intellectual property elements or not, should meet the minimum criteria set in these

PS in order to be considered for inclusion in an existing OECD Test Guideline.



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Validation in Support of Internationally Harmonised OECD Test Guidelines…



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The concept of Performance-Based Test Guideline (or PBTG) was developed as

an elaboration of PS, in view of the variety of methods that could address the same

endpoint through the same mode of action (e.g. binding to the estrogen receptor).

However, test systems are not necessarily strictly similar (e.g. systems using radiolabeled elements versus non-radiolabeled systems). A PBTG (e.g. TG 455) is a TG

that only provides a generic description of how the test method operates and is

based on at least two validated and accepted test methods (designated the Validated

Reference Method (VRM), or just “reference test method”). The test methods themselves are described in further details in annexes.

The PBTG concept has also been promoted to prevent the duplication of similar

Test Guidelines covering similar test methods; it should allow faster validation of

test methods addressing the same endpoint. There is still limited experience at

OECD on the implementation of these new approaches that offer greater flexibility

vis-à-vis innovative methods, provided they are well described, characterised, communicated, and used appropriately.

As a new test method is used, the usefulness of the test method may be expanded.

It is appropriate from time to time to review and reassess the performance characteristics of established test methods. Data generated could be subjected to the same

validation principles as described for a new test method if the proposed changes are

significant, but it may also be appropriate to undertake a more limited assessment or

review of reliability and accuracy using the established PS. The extent of the validation study or type of review that would be appropriate should be commensurate to

the extent of changes proposed. In recent updates in 2013 and 2014 of OECD TG

431 on in vitro skin corrosion using reconstituted human epidermis, amendments

have been proposed to enable the use of the test methods included in the TG for the

sub-categorisation of corrosive chemicals. A statistical performance analysis

(OECD 2013) has been carried out to define the predictive capacity of the methods

for this purpose, without impacting the rest of the TG.



3



OECD Guidance Document on the Validation Principles

and Regulatory Acceptance of New and Updated Test

Methods



The development of the OECD Guidance Document 34 started in 1998 as a followup to the 1996 Solna Workshop on “Harmonisation of Validation and Acceptance

Criteria for Alternative Toxicological Test Methods”. Whereas the principles and

criteria for validation and regulatory acceptance of new and revised test methods,

agreed in Solna (OECD 1996) were generally accepted, the principles needed to be

expanded and additional guidance provided.

The principles of the OECD Guidance Document 34 apply generally to new and

updated in vivo or in vitro test methods, for effects on human health or the environment; however, some principles are more sound in the context of in vitro test methods that are intended as alternatives or replacement of an existing in vivo test. The



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A. Gourmelon and N. Delrue



OECD Guidance Document 34 principles include the following points as described

below: (1) the availability of a rationale for the test method; (2) description of the

relationship between the test method’s endpoint(s) and the biological phenomenon

of interest; (3) the availability of a detailed protocol for the test method; (4) demonstration of the intra-, and inter-laboratory reproducibility of the test method; (5)

demonstration of the test method’s performance based on the testing of reference

chemicals representative of the types of substances for which the test method will

be used; 6) evaluation of the performance of the test method in relation to relevant

information from the species of concern, and existing relevant toxicity data; (7) the

data supporting the validity of a test method should be obtained in accordance with

the principles of GLP; and (8) all data supporting the assessment of the validity of

the test method should be available for expert review.



3.1



Rationale for the Test Method



A rationale for the test method should be available, and should include a clear statement on the regulatory needs in one or more countries, and the scientific justification supporting the method. The rationale can be: (1) the absence of an existing test

method to address the hazard endpoint of interest, (2) the possibility to have an

alternative test method that can be safer or provide better, more reliable information, or use fewer or no animals or be more cost-effective for the same level of

human health or environmental protection. Here, considerations of the 3Rs (replacement, reduction, refinement) principles should be addressed.



3.2



Relationship Between the Test method’s Endpoint(s)

and the Biological Phenomenon of Interest



The relationship between the test method’s endpoint(s) and the biological phenomenon of interest should be described. This second principle of validation is especially relevant for in vitro test methods intended to replace or predict an effect

in vivo. For in vivo methods, the relationship is usually more direct, although in the

case of biomarker endpoints, a justification based on mechanistic considerations

leading to an adverse outcome is expected. It is not always possible, nor essential,

for further regulatory acceptance of the test method being validated to have a deep

understanding of all possible chemical interactions to their targets at various levels

of biological organisation; however, existing knowledge of the relationship linking

the test system being validated and response measured to the in vivo adverse effect

should be described (e.g. similarity between the in vitro test system and the target

tissue in vivo, associative or correlative relationship between the endpoint measured

in the system being validated and the biological effect it intends to predict).

Integrative test systems being validated (e.g. organ-level test systems such as ex vivo



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eye test) typically require less justification about their biological relevance to the

biological effect of interest measured in vivo, while more simple in chemico or

in vitro systems will require greater justification of their relationship to the target

biological effect of interest. For simpler test systems, based on e.g. a cell line, a very

clear understanding of their applicability and limitations (e.g. absence of metabolism) is necessary to reach regulatory acceptance.

For in vivo test methods, the relationship of the endpoint measured in the test

system being validated (e.g. egg numbers in a fish test to predict reproductive fitness, hepatocyte enlargement via histopathology evaluation to predict liver toxicity)

is often more implicit and intuitive for the determination of the toxicity in vivo.

As science and techniques progress, regulators may be faced with test systems

that are quite sophisticated (e.g. reconstituted 3D tissue or organ) and resemble or

mimic biological processes in the target organ, including its metabolic capacity. In

that case, the biological relationship will be relatively straightforward to demonstrate. In other cases, as progress is made in the understanding of mechanisms of

action, future test systems may be simplified to such extent that a demonstration of

the biological relevance will be as critical as the demonstration of the reproducibility of test results obtained using that particular test system. This issue is easily

conceivable in the case of in chemico test systems for which a well-calibrated experiment will be reliable over time and between laboratories due to limited number of

sources of biological variability, but the demonstration of the relationship of the

response measured to an effect in vivo will be the main challenge of the validation.

In these cases, the test system will not likely be a stand-alone method, and the context of use, the applicability, limitations and possible combinations with other test

systems in a more complex framework, will require careful consideration.



3.3



Detailed Test Method Protocol



A detailed protocol for the test method should be available. This principle calls for

transparency in the test procedure proposed, as a pre-requisite to the success of the

validation. In order for laboratories to participate in the validation, a detailed protocol including a description of the material needed, a description of what is measured

and how it is measured, a description of how data need to be recorded and analysed,

a description of the criteria for the acceptance of results, a template to record data

are essential to enable the user to adhere to the protocol and to have means to control

deviations from the prescribed procedures and report them. For the validation studies, it is important for participating laboratories to have the agreed standard operating procedures in hand prior to starting the study in order to minimise the sources of

variation in the conduct of the study. Changes to the protocol that occur in the

middle of the experiments will systematically lead to failure of the validation. If

certain aspects of the protocol are flexible, these needs to be indicated as such in the

protocol ahead of the validation studies. Problems encountered in validation studies

sometimes resulted from a lack of standardisation of the protocol, leaving choice to



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