EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

12345678910111213141516171819202122232425262728293031323334353637383940414243STATEMENT ON THE SCIENTIFIC VALIDITY OF CYTOTOXICITY/CELLFUNCTION BASED IN VITRO ASSAYS FOR EYE IRRITATION TESTING
At its 31st meeting, held on 7 and 8 July, 2009 at the European Centre for the Validation ofAlternative Methods (ECVAM), Ispra, Italy, the non-Commission members of the ECVAM
Scientific Advisory Committee (ESAC)1 unanimously endorsed the following statement:
The replacement of traditional animal-based test methods by alternative ones should ideallybe obtained by one-to-one replacements: to keep the testing regime simple and economical
one single alternative method should, wherever feasible, be sufficient to generate data ofequal or better quality than the traditional test.
However, in the case of eye irritation it is currently generally accepted that, in the foreseeable
future, no single in vitro eye irritation test will be able to replace the in vivo Draize eye test topredict across the full range of irritation for different chemical classes. However, strategic
combinations of several alternative test methods within a (tiered) testing strategy may be able
to replace the Draize eye test.
A possible conceptual framework for such a (tiered) testing strategy has been developed
within an ECVAM workshop (Ref. 1). The framework is based on alternative eye irritation
methods that vary in their capacity to detect either severe irritant substances (EU R41; GHS'Category 1') or substances considered non-irritant (EU 'Non-Classified'; GHS 'No Category').
According to this framework the entire range of irritancy may be resolved by arranging tests
in a tiered strategy that may be operated from either end: to detect first severe irritants andresolve absence of irritancy ("Top-Down Approach") or to proceed inversely, starting with theidentification of non-irritants first ("Bottom-Up Approach"). Mild irritancy will be resolved ina last tier in both approaches.
To evaluate the scientific validity of possible building blocks of such a test strategy and toassess their possible placement within a Bottom-Up and Top-Down Approach, ECVAM hasundertaken a retrospective validation study of four cell-based in vitro methods.
The test methods evaluated were:
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Cytosensor Microphysiometer (INVITTOX Protocols 97 and 102 modified)2
Fluorescein Leakage (INVITTOX Protocols 71, 82, 86 and120);
Neutral Red Release (INVITTOX Protocol 54 and PREDISAFETM);
Red Blood Cell haemolysis (INVITTOX Protocols 37 and 99),

The four test methods, including ten protocol variations, were subjected to independent,
expert review with respect to their use to either
1Details can be found in the PRP report
Invittox protocols can be downloaded from ECVAM's database service on Alternative Methods to Animal
Experimentation, DBALM: http://ecvam-dbalm.jrc.ec.europa.eu
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In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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8687888990a) initiate a Bottom-Up Approach, for consideration for regulatory use to identify nonirritants (EU: 'Non Classified'; GSH: 'No Category'; EPA: 'Category IV') from all otherclasses as part of a tiered testing strategy, orb) to initiate a Top-Down Approach, to identify ocular corrosives and severe irritants(EU R41, GHS 'Category 1', and EPA 'Category I') from all other classes as part of atiered testing strategy.
In the absence of internationally agreed performance criteria for either approach, the PRP of
the ESAC applied the following criteria:• any test used to initiate a Top-Down Approach must balance specificity and sensitivity
to correctly identify a substantial proportion of severe irritants, with a false positive
rate that would not lead to the over-classification of an unreasonable number ofmaterials of lower ocular irritancy potential – an over-classification rate (falsepositives) of <10% was considered acceptable• any test used to initiate a Bottom-Up Approach should ideally give no false negatives
with respect to human safety, and no false negative should be produced by highmoderate or severe irritants.
Following independent ESAC peer review of this retrospective validation study andconsidering the potential test strategies in which the tests may be used, the ESAC concluded
the following:
1. CYTOSENSOR MICROPHYSIOMETER TEST METHODThe Cytosensor Microphysiometer test method can be used for two of the three EU and GHS
classification categories used for the endpoint of ocular irritation:
A. The Cytosensor Microphysiometer test method (INVITTOX Protocol 102 modified) is
considered to have been scientifically validated and to be ready for consideration forregulatory use as an initial step within a Top-Down Approach to identify ocular corrosives
and severe irritants (EU R41, GHS Category 1, and EPA Category I) from all other classes forthe chemical applicability domain of water-soluble chemicals (substances and mixtures).
B. Furthermore, the Cytosensor Microphysiometer test method (INVITTOX Protocol 102modified) is considered to have been scientifically validated and to be ready for consideration
for regulatory use as an initial step within a Bottom-Up Approach to identify non-irritants(EU:NC; GHS: NC; EPA: cat IV) from all other classes only for water-soluble surfactants andwater-soluble surfactant-containing mixtures.
C. On the basis of a thorough evaluation of the data compiled in the course of the ECVAM
validation study, the ESAC concludes that the Cytosensor Microphysiometer test method
does NOT correctly identify moderate and mild ocular irritants (EU: R36; GHS: Cat 2A/B;
EPA: Cat II/III). Therefore, the test method can only be employed to make decisions on twoof the three categories of the eye irritation classification scheme (see A and B). Consequently,
ESAC does NOT recommend this test method as a full replacement method. It should be
noted in this context that the Top-Down and Bottom-Up Approach foresees the theoreticalpossibility of a default mild/moderate categorization (e.g. EU R36 or GHS Cat 2) of all those
substances neither identified as ocular corrosives and severe irritants (see A) nor as "nonPage 2 of 6
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Institute for Health and Consumer Protection
In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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classified" substances (see B) in the first two tiers of the strategy. However, the test method's
high false negative rate (9-55%) when initiating a top-down approach and high false positiverate (50-69%) when initiating a bottom-up approach exclude the possibility to use the methodfor default categorization. The test methods can thus not be considered a full-replacement
method on its own using the Top-Down and Bottom-Up approach.
Although these recommendations are based on the evaluation of data sets obtained using
specific hard- and software, it is anticipated that other Cytosensor Microphysiometerequipment and software may become available with either equivalent or better performanceand will need to be efficiently validated. Depending on the similarity of new equipment withrespect to the validated one, this may be performed as a Similar Method Validation ('me-too')or an Update Validation. ESAC therefore recommends the development of PerformanceStandards for the Cytosensor Microphysiometer test method.
The current chemical applicability domain is limited: whilst in some cases this might beincreased by expanding the data set of studied compounds, the test method is not amenable totesting non-water soluble solids, suspensions, or viscous materials.

2. FLUORESCEIN LEAKAGE TEST METHODThe Fluorescein Leakage test method (INVITTOX Protocol 71) is considered to have beenscientifically validated and to be ready for consideration for regulatory use as an initial step
within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41,GSH Category 1, and EPA Category I) from all other classes for water-soluble chemicals(substances and mixtures).
Additional testing and further refinement, in particular with respect to variability anddefinition of the applicability domain, by expanding the dataset of tested chemicals and directcomparison with in vivo data is recommended and should be kept under review.
With regard to the• Neutral Red Release (INVITTOX Protocol 54 and PREDISAFETM);• Fluorescein Leakage (INVITTOX Protocols 82, 86 and120);• Red Blood Cell haemolysis (INVITTOX Protocols 37 and 99),
ESAC considers that the available evidence is insufficient3 to support a recommendation thatthey are ready for consideration for regulatory use.
Similarly, the available evidence for Fluorescein Leakage INVITTOX Protocol 71 does notsupport a recommendation for its use to initiate a Bottom-Up Approach for regulatory use.

3Details can be found in the PRP report
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EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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This statement takes account of the dossiers prepared for peer review; the views of
independent experts of the ESAC Peer Review Panel (PRP) who evaluated the dossiers
against defined validation criteria as well as supplementary submissions made by theValidation Management Group.

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In agreement with common practice upon completion of a validation study, ESAC
recommends the development of Performance Standards for the Cytosensor
Microphysiometer and the Fluorescein Leakage assays to allow the validation of similar test
methods or modifications of the validated test methods based on pre-defined evaluation andacceptance criteria.

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Joachim Kreysa

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Head of Unit

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In vitro methods Unit

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European Centre for the Validation of Alternative Methods

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Ispra, 10th July 2009

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In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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REFERENCECS

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1.

Scott, L. et al. (2009) A proposed eye irritation testing strategy to reduce and replacein vivo studies using Bottom-Up and Top-Down approaches. Toxicol In Vitro. May
31. [Epub ahead of print]

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In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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162The ESAC was established by the European Commission, and is composed of nominees fromthe EU Member States, industry, academia and animal welfare organisations, together withrepresentatives of the relevant Commission services.

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This statement was endorsed by the following members of the ESAC:

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Ms Argelia Castaño(Spain)
Ms Maija Dambrova (Latvia)
Ms Alison Gray (ESTIV)
Ms Katalin Horvath (Hungary)
Ms Dagmar Jírová (Czech Republic)
Mr Roman Kolar (Eurogroup for Animals)
Ms Elisabeth Knudsen (Denmark - acting as moderator at the meeting)
Mr Manfred Liebsch (Germany)
Mr Gianni Dal Negro (EFPIA)
Mr. Walter Pfaller (Austria)
Mr Tõnu Püssa (Estonia)
Mr Dariusz Sladowski (Poland)
Mr Jon Richmond (UK)Ms Vera Rogiers (ECOPA)
Mr Michael Ryan (Ireland)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Carl Westmoreland (COLIPA)
Mr Timo Ylikomi (Finland)

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The following Commission Services and Observer Organisations were involved in theconsultation process, but not in the endorsement process itself:

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Commission services
Mr Joachim Kreysa (DG JRC, Head of In vitro methods Unit/ECVAM, chairman)
Mr Claudius Griesinger (DG JRC, ESAC secretariat)
Ms Susanne Hoke (DG ENTR)
Ms Susanna Louhimies (DG ENV)
Mr Juan Riego Sintes (DG JRC)
The following observers were present
Mr Hajime Kojima (JaCVAM)
Mr William Stokes (NICEATM)
Ms Marilyn Wind (ICCVAM)

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