EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

PEER REVIEW PANEL REPORT ON THE RETROSPECTIVE VALIDATION OFCYTOTOXICITY/CELL-FUNCTION BASED IN VITRO ASSAYS (EYE IRRITATION)
BackgroundTo satisfy in-house decision making and statutory requirements, a range of substances thatmight either by intention or accidentally come into contact with the eye (for example
pharmaceuticals, cosmetics, agrochemicals) have to be evaluated to determine and manage
any potential human ocular hazard. Where the testing of substances is undertaken to informthe regulatory assessment of ocular hazard studies involving the in vivo rabbit eye test
(Draize eye test) data is generally required.
As yet, no full in vitro replacement for the rabbit Draize eye test is available. Two ex vivoorganotypic test methods, the Bovine Corneal Opacity and Permeability (BCOP) and theIsolated Chicken Eye (ICE) test methods1 have been scientifically validated at international
level and can be accepted for regulatory purposes to identify severe ocular irritants andcorrosive materials.
This validation study report evaluates the existing evidence and analysis with respect to fourcell-based assays to either screen-out severe irritants in a Top-Down approach, and/or nonirritants in a Bottom-Up approach as part of a weight of evidence approach in a tiered testing
strategy.
The test methods, and protocols, evaluated are:-Neutral red release test (INVITTOX 54 and Predisafe),
Fluorescence leakage (INVITOX 71, 82, 86 and120),
Cytosensor microphysiometer (INVITOX 97 and 102), andRed blood cell haemolysis test (INVITOX 37 and 99).

A retrospective validation study has been undertaken, based mainly on data obtained frompeer reviewed literature. As a consequence, as concurrent animal studies were not generally
undertaken or reported, few direct contemporary in vivo/ in vitro result comparisons can be
made. Only in a few instances is the original in vivo data available, but information on
reproducibility of the in vivo test method with the relevant range of test materials is scarce.
These alternative cell-based test methods were devised, developed and evaluated withspecific types of test material in mind: as a result, the test database contains results
predominantly about test performance with mainly liquid, water soluble, surfactant materials
and multi-component test materials weighted towards low irritancy potential. The materials
referenced in the database do not represent a balanced cross-section of all types of material
which are evaluated for eye irritancy potential, or the full range of mechanisms andresponses that might be seen.
The predictive capacities of each test method and prediction model were assessed according
to their ability to categorise test materials for eye irritancy potential based on the EU, GHS
and EPA classifications systems, with test performance being evaluated against
classification using ocular irritation data generated using the in vivo rabbit test.

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DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

The Peer Review Panel
Before the ECVAM Peer Review Panel (PRP) was established, relevant information was
gathered and evaluated by an ECVAM-appointed Validation Management Group (VMG)
which produced a report and recommendations for further consideration by the PRP.
ECVAM produced a background review document for each of the four tests, and establishedthe PRP to advise ESAC on whether and on what terms these tests might be considered tohave been scientifically validated.
The PRP comprised two members from the ECVAM Scientific Advisory Committee (ESAC),
one European expert nominated by the ESAC, and two US experts nominated by ICCVAM.
JacVam was invited to nominate experts but declined.
The PRP held one teleconference, communicated by e-mail to produce independent
individual reports, and then held a two-day face-to-face meeting to review the available
evidence and analysis, with a view to preparing a consensus report with recommendations
on the validation status of the test methods for submission to ESAC for further consideration.
There are no published Performance Standards for the evaluation of this class of in vitro
replacement tests, and no guidance has been provided to the Peer Review Panel (PRP) on
the performance outcomes required to consider the in vitro tests to have been scientificallyvalidated. The PRP considers that to ensure international acceptance of the resulting ESAC
statement there should have been international agreement on these between ECVAM,ICCVAM and JacVAM before the PRP was asked to offer its advice.
In evaluating the available information in the absence of an internationally agreed
Performance Standard the PRP applied the following general principles:•••even accepting that the rabbit test is imperfect, in the absence of authoritative humandata significant discordance from the available in vivo data cannot be considered abenefit:any test used in a Bottom-Up approach should give no false negatives, and no false
negative should be produced by high-moderate or strong irritants;
any test used in a Top-Down approach must balance specificity and sensitivity tocorrectly identify a substantial proportion of severe irritants, with a false negative ratethat would not lead to the over-classification of an unreasonable number of materialsof lower ocular irritancy potential – an over-classification rate of <10% wasconsidered acceptable.

General Comments
The PRP evaluated the test methods with a view to their being incorporated into weight ofevidence and tiered-testing strategies: the individual tests were evaluated both with a view totheir relevance for use in a Bottom-Up approach (to reliably identify ocular non-irritants)
and/or a Top-Down approach (to reliably identify strong ocular irritants).
The PRP gave due weight to both the ECVAM Background Review Documents and the VMG
report.

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DIRECTORATE GENERAL JRC
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European Centre for the Validation of Alternative Methods (ECVAM)

The interpretation of the Background Review Document was hampered by the fact that,
although these followed the modular validation approach, several protocols were considered
for some of the test methods and the BRD text (and in some cases the conclusions) did notalways clearly or sufficiently distinguish between the different protocols.
The VMG made recommendations on the readiness for regulatory acceptance of the models
largely on the basis of the frequency of false positives in the Top-Down approach and false
negatives in the Bottom-Up approach, and the range of test materials for which data wasavailable. The VMG report did not discuss in detail other relevant elements such variability,transferability, applicability domain and it is not clear how these were taken into account in
the final VMG recommendations.
Due to the number of protocols to be assessed, and the short time stipulated by ECVAM for
their evaluation, the PRP, consistent with the VMG findings, did not fully re-evaluate the Red
Blood Cell haemolysis test due to established discrepancies in specificity and sensitivity
confirmed in the studies reviewed; the INVITOX 82 and 86 (Fluorescence Leakage) due toproven inadequacies of the datasets; the NRR IIVS due to the very limited dataset; and
INVITOX 97 since it is technically obsolete.

GENERAL RECOMMENDATIONSThe PRP wishes to draw two general points relating to ECVAM validation studies and PRP
review to the attention of ESAC and ECVAM.
1. Several of the test methods on which the PRP was asked to advise were known inadvance not to be supported by data relating to all of the essential components of modular
validation. In the view of the PRP under these circumstances, as the outcome of any test
method PRP review must be “not scientifically validated to the standards required forregulatory consideration”, there no PRP opinion is required.
2. No internationally agreed and harmonised performance standards for the performance of
these tests in bottom-up and top-down approached were developed before the PRP review.
The PRP has therefore determined and displayed elements of its own standard. As theintention is to evaluate alternative methods with a view to their being international agreementon their status, such performance standards should be internationally agreed andharmonised before peer review.

1

http://ecvam.jrc.it/ft_doc/ESAC26_statement_Organotypic_20070510_C.pdf

3EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

FLUORESCEIN LEAKAGE ASSAY: INVITTOX120 BOTTOM UP APPROACH

1. Data Collection
Data were collected by retrospective compilation of publicly available information andinformation submitted by ECVAM and COLIPA for in vivo animal ocular irritation tests and invitro data generated for previous validation studies.
For INVITTOX 120 there were data for 11 surfactants and 23 surfactant-based formulations,the tests having been conducted in two laboratories.
2. GoalThe goal of the validation study was to evaluate whether and on what terms Protocol
INVITTOX 120 can be considered for use as part of a weight of evidence or tiered-testing
strategy in a Bottom-Up approach to correctly identify and classify ocular non-irritants (i.e.,
EPA Category IV, GHS Not Classified, EU Not Labelled) with little to no likelihood for false
negatives caused by materials proving to be more than very mild irritants. No PerformanceStandard was developed before the PRP review.
3. Test definition
The Fluorescein Leakage (FL) assay measures leakage of a fluorescein dye across anepithelial monolayer using cells that form tight and desmosomal junctions.
The assay as described is performed using a monolayer of non-proliferating Madin-Darby
Canine renal tubular epithelial cells that form tight and desmosomal junctions similar to thoseon the apical side of conjunctival and corneal epithelium. After a short exposure period, thetest material is removed and a fluorescent dye is added. Test chemicals that disrupt tightjunctions allow the dye to leak through the monolayer into the well below where it can bemeasured spectrophotometrically. FL20 is measured, the concentration that causes 20%
fluorescein leakage relative to untreated controls.
Although this is the only one of the four test systems evaluated in the validation study andpreviously considered by the VMG that may be capable of assessing recovery, its potential to
do this was not fully explored in the information provided to the PRP. The PRP therefore
offers no opinion as to its usefulness for this purpose.
The FL release method is technically straightforward and reasonably and practicably
available: relying largely on commonly available skills and equipment. There are some
performance issues relating to potential challenges in culturing this specific cell type; andsome unanswered questions related to exposure times, potential reversibility, and doseresponse considerations.
A range of factors have been identified that may compromise the performance of the test:
these include the relationship of medium calcium concentration to tight junction integrity; celldensity requirements; potential read-out interference from highly-coloured test materials;
chemical binding to the monolayer or the insert membrane; the effects of multiple rinsing
steps; and serum protein binding. Chemical binding to the monolayer was reported to be
more likely for cationic surfactants.

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DIRECTORATE GENERAL JRC
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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

The nature of the inert inserts used for the assay should be more clearly defined.
There is a VMG recommendation to test tight junction integrity before performing FL assay;
but no explanation as to how this would be done, or what the pass-fail criteria might be.
It is the opinion of the PRP that the test method should also allow users to establish before
and after testing that there is a confluent monolayer and an intact membrane. The
information supplied to the PRP does not explain how this might be done.
4. Data quality
Original data were available from the ECVAM prevalidation study, but only summarised data
were available from the COLIPA study.
The BRD evaluated four protocols: in places the text did not clearly distinguish between
them.
No reliable human data were available.
The majority of animal data were obtained in non-GLP studies, reported in the peer-reviewed
literature.
The PRP could not confirm for INVITTOX 120 that the in vivo and in vitro data were obtained
simultaneously, and with the same batch and lot of test materials.
5. Test materials
INVITTOX 120 evolved from INVITTOX 71 (previously evaluated in the HO/EC study).
Using INVITTOX 120, a COLIPA study was previously performed with 11 surfactants and 18
surfactant formulations in two laboratories. This protocol was also used for two phases of aprevious ECVAM study (Southee, 1998); Phase II ( 4 chemicals and a shampoo formulation
tested in 4 laboratories) and Phase III (10 surfactants, 3 laboratories).
The prediction model was only for surfactants, and was devised, empirically based on thefindings, during or after the studies.
The VMG restricted its evaluation of test performance to the COLIPA data. The reasons forthis were not clear in the VMG document.
6. Within-laboratory variability
The within-laboratory variability was high for INVITTOX 120 with a CV range of 28-36%, andvery high variability for the positive control ranging from 17-603%.
7. Transferability
The protocol did not transfer well.

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DIRECTORATE GENERAL JRC
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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

The CV values varied considerably. For the COLIPA study, the CV mean was 40.1% andthe range was 0.9-133.9%.
8. Between-lab variability
The COLIPA study in two laboratories evaluating test performance with surfactants andformulations had a CV of 47% for surfactants, and was slightly better for formulations(33.6%). 89% of the chemicals would have been identically classified on the basis of theinformation obtained by both laboratories.
The two ECVAM studies (Phase II and III) had CV range of 32-57%; possibly due to poor
protocol adherence or SOP ambiguities (Southee, 1998). Original data were not included in
the BRD; only mean values were provided.
9. Predictive capacity
The prediction model was restricted to the domain of water-soluble surfactants or surfactantbased formulations.
This is insufficient to evaluate or determine applicability to a broader domain of test
materials.
The prediction model was developed post-hoc.
The predictive capacity was evaluated on the basis of test material classification using in vivo
data from the peer-reviewed literature; no original in vivo data produced on the same batchand lots of test materials used for the in vitro studies were available.
10.

Applicability domain

In view of the findings of the ECVAM study, consideration of the applicability domain must be
limited to water-soluble surfactants or surfactant-based formulations on the basis of the
COLIPA study.
There are other potential limitations related to test material colour, viscosity, solids, andability to bind to the cell layers and inserts.
11.

Performance standards

No Performance Standards are available.
INVITTOX 120 uses standard tissue culture equipment and competencies.
12.

Readiness for regulatory purposes

In the view of the PRP INVITTOX 120 is not considered ready for regulatory acceptance in a
Bottom-Up approach for the reasons set out above.
Nor is it recommended as a full replacement method for the in vivo Draize test.

6EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

Although the false negative rate for the three classification systems were low, ranging from 05% in the Background Review Documents, there are other compelling reasons for notconsidering this test to have been scientifically validated and shown to be ready for
regulatory consideration on the basis of the information available.
The intra- and inter-laboratory variability and poor transferability are causes for concern.
For the purposes of GSH classification, one significant irritant was misclassified. Although
the false negative rate was low, one of the ocular irritants produced irritation that cleared only
by seven days and would have been misclassified under GHS. This would present anunacceptable risk to humans if such results could occur with other products intended forwidespread human exposure.
The prediction model and protocol only applied to surfactants and surfactant-based
formulations.
In addition, in the view of the PRP there is a need for a secondary means to perform aqualitative assessment of any negative prediction. Consideration was given as to whether amicroscopic visual inspection of the cells would suffice. However, given that a Bottom-Up
approach would be the approach where no or mild irritancy would be expected and strong
irritancy would not expected, many compounds selected for evaluation in a Bottom-Up
approach would be expected to have at most only very subtle effects, thus there may be little
likelihood of this being an adequate safeguard in practice.
Expansion of the range of compounds and formulations considered in the applicability
domain is needed. INVITTOX 120 should be considered for further validation by testing of abalanced data set of clearly classified chemicals in at least three laboratories in order tofurther evaluate the variability and limitations of the assay.
Further evaluation would benefit from additional data, preferably acquired by further testing,including recovery up to 72 hr. Performance standards and any positive and negative
controls should be clearly defined and agreed at international level.

7

EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

FLUORESCEIN LEAKAGE ASSAY, TOP-DOWN APPROACH INVITTOX PROTOCOL 71
1. Data collection
Data were collected by a retrospective compilation of data from the peer-reviewed literature.
An extensive literature search was undertaken, and the relevant publications were taken into
account.
For the INVITTOX 71 protocol, the key reference is Balls et al., 1995 paper (EC/HO study).
2. Goal of the study
The goal of the validation study was to evaluate whether and on what terms Protocol
INVITTOX 71 can be considered for use in a weight of evidence or tiered-testing strategy in a
Bottom-up approach to correctly identify and classify ocular non-irritants, with little or no
likelihood for false negatives caused by materials proving to be more than mild irritantsand/or in Top-Down approach to identify severe irritants.
On the basis of the available evidence the VMG focused on the validity of the test method fora Top-Down approach, discriminating severe irritants from all other classes, or discriminating
non-irritants from all other classes in a bottom-up approach, based on EU, GSH and EPA
classifications.
3. Test definition
The Fluorescein Leakage (FL) assay measures leakage of a fluorescein dye across anepithelial monolayer using cells that form tight and desmosomal junctions. The BRD confirmsthat disruption of such inter-cellular junctions, increasing permeability of the cell membrane,
is relevant to the mode of action of some ocular irritants.
Although this is the only one of the four test systems evaluated in the validation study andpreviously considered by the VMG that may be capable of assessing recovery, its potential to
do this was not fully explored in the information provided to the PRP. The PRP therefore
offers no opinion as to its usefulness for this purpose.
The FL assay was designed and developed initially to identify possible irritating surfactants
and surfactant-based formulations.
The assay as described is performed using a monolayer of non-proliferating Madin-Darby
Canine renal tubular epithelial cells that form tight and desmosomal junctions similar to thoseon the apical side of conjunctival and corneal epithelium. After a short exposure period, thetest material is removed and a fluorescent fluorescein dye is added. Test chemicals that
disrupt tight junctions allow the dye to leak through the monolayer into the well below where
it can be measured spectrophotometrically. FL20 is measured, the concentration that causes
20% fluorescein leakage relative to untreated controls.
A prediction model (pm) for protocol 71 was devised based on the HO/EC study. Based on invitro and in vivo data a prediction model (pm) was then constructed to facilitate and evaluate
EU, GHS and EPA classification for the 60 chemicals for which data was available. These
represent a number of different chemical classes, including surfactants, and in varying
physical states (solids, and liquids of unknown viscosity).

8

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DIRECTORATE GENERAL JRC
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European Centre for the Validation of Alternative Methods (ECVAM)

The FL release method is technically straightforward and reasonably and practicably
available: relying largely on commonly available skills and equipment. There are some
performance issues relating to potential challenges in culturing this specific cell type; andsome unanswered questions related to exposure times, potential reversibility, and doseresponse considerations.
A range of factors have been identified that may compromise the performance of the test:
these include the relationship of medium calcium concentration to tight junction integrity; celldensity requirements; potential read-out interference from highly-coloured test materials;
chemical binding to monolayer or the insert membrane; the effects of multiple rinsing steps;
and serum protein binding. Chemical binding to the monolayer was reported to be more
likely for cationic surfactants.
The nature of the inert inserts used for the assay should be more clearly defined.
There is a VMG recommendation to test tight junction integrity before performing FL assay;
but no explanation as to how this would be done, or what the pass-fail criteria might be.
It is the opinion of the PRP that the test method should also allow users to establish before
and after testing that there is a confluent monolayer and an intact membrane. The
information supplied to the PRP does not explain how this might be done.
4. Data quality
Data for 60 test materials were obtained from the peer-reviewed literature.
For various reasons the quality of the data is regarded by the PRP as not sufficiently robust,and there is concern about protocol breaches.
5. Test materials
Data from 60 chemicals is available (mainly Ball et al, 1995).
These represent a number of different chemical classes, including surfactants, and in varying
physical states (solids, and liquids of unknown viscosity).
There is no data on test performance with multi-component formulations.
6. Within-laboratory variability
There was a large variation in results for this protocol (CV 56.5 – 63.2%).
The variation between operators ranged from 0% to 50% and did not seem to improve overtime or with experience.
7. Transferability)
The PRP was provided with the results of only one study relevant assessing transferability.
In that publication the protocol did not transfer well (consequently, refinements were made to
the protocol which was later accepted as INVITTOX 120).
9

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DIRECTORATE GENERAL JRC
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European Centre for the Validation of Alternative Methods (ECVAM)

The VMG recommended training for technicians to improve operator reproducibility if this test
method is further developed: but as it not clear what caused the problems in practice, the
PRP cannot comment on whether or to what extent this might improve test performance.
8. Between-laboratory variability
The PRP was provided with data from only one study where between-laboratory variability of
protocol 71 was assessed and all laboratories fully adhered to the test protocol.
An agreement/concordance score of only 52% was obtained.
9. Predictive capacity
The predictive capacity was evaluated mainly against rabbit in vivo data from the literatureand obtained with the LVET. Although it is accepted that the LVET does not provide resultsthat are in complete agreement with the standard Draize eye test, the significance of this is
not fully evaluated in the BRD or VMG report.
No original contemporary in vivo data for the same test material dataset was available.
For protocol 71 no prediction model (pm) was available and therefore a new pm using thedata from the EC/HO study was constructed for chemicals only.
Since no data were available for formulations its suitability for these could not be assessed.
The concordance for non-irritants versus the rest was 62%-70% and 78%-83% for theirritants versus the rest. Specificity was in both situations higher than sensitivity indicating agreater predictive capacity for the non-irritants.
Concordance did not improve when only surfactants and surfactant based formulations weretaken into account.
In general, the predictive capacity was better for surfactants than for alcohols. Also, thepredictive capacity was better for the non-irritants than for the irritants.
10. Applicability domain
Due to short exposure times, relatively high chemical concentrations are used.
As the test materials must be removed from the test system after exposure, the retention of,or damage caused by the remove of, viscous substances might therefore affect the test
performance and predictive capacity. However, as there was no information on viscosity ofchemicals used in protocol 71 these effects could not be evaluated by the PRP.
The tested chemicals generally were of low irritancy potential. Strong acids and bases,
fixatives and highly volatile chemicals should be excluded from the applicability domain as
they are incompatible with the cell mono-layers used.
Although INVITOX 71 was tested with chemicals from different chemical classes, in the viewof the PRP too few chemicals of each class were tested and therefore no predictive capacity
for each class could be ascertained.
10EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
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European Centre for the Validation of Alternative Methods (ECVAM)

If the test is further developed the PRP recommends that more chemicals in different classes
should be tested to improve information on the applicability domain.
It is hypothesized that some highly- coloured substances may affect the results of protocol
71.
Furthermore, the fact that the some test materials may bind to the insert and affect thepredictive capacity is a point of concern.
In the view of the PRP, on the basis of the available evidence, other factors potentiallyaffecting test performance include the relationship of medium calcium concentration to tightjunction integrity; cell density requirements; coloured test materials; and serum protein
binding. A further potential confounding factor was chemical binding to the insert membranes
despite multiple rinsing steps, as well as damage to the insert membranes because ofmultiple washing steps. Chemical binding to the membranes was found to be more likely forcationic surfactants.
The PRP could assess the potential use of the test to evaluate recovery on the basis theinformation provided.
11. Performance standards
Performance standards are not provided in the material presented to the PRP for
consideration.
12. Readiness for regulatory purposes
The available evidence is inclusive.
The evidence available has not satisfied the PRP that the test can be considered to havebeen scientifically validated and to be ready for regulatory consideration.
However, the available information has, in the view of the PRP, not demonstrated
conclusively that the test method does not have the potential to be further developed forconsideration for use in a Top-Down approach.
Protocol 71 may have potential for a Top-Down approach (the BRD data suggest a false
positive rate of only 7-9%), as part of a tiered testing strategy but needs additional testing
and further refinement, in particular with respect to variability and definition of the applicability
domain by expanding the dataset of tested chemicals and direct comparison with in vivo data
before it can be regarded ready for consideration for regulatory purposes.
Recovery data might improve the predictive capacity of the test.

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European Centre for the Validation of Alternative Methods (ECVAM)

NEUTRAL RED RELEASE ASSAY PROTOCOLS (PROTOCOL 54)
1. Data Collection
Data from a number of sources have been identified, obtained and evaluated.
The best dataset is from the study carried out by CTFA in their Phase I, II, and III studies,and includes quality in vitro and in vivo data.
Even with this dataset, it is the view of the PRP that additional data are needed to fully
evaluate Protocol 54. Currently the dataset is limited to 10 alcohol formulations, 18 oil/water
emulsions and 25 surfactant-based formulations.
2. Goal of the study
The goal of the study was to consider the scientific validity in the context of a testing strategy
for a Bottom-Up approach. The goal was clearly understandable and an acceptable scientificrationale was given in the BRD. No agreed performance standard was provided to the PRP.
3. Test definition
The test and its purpose are well-defined.
The test has mechanistically relevant features for modelling eye irritation, namely cell
membrane damage. The test was not designed to assess reversibility or recovery.
Originally there was no prediction model for using the NRR assay (Protocol 54) to predict theocular irritancy of chemicals according to the EU, GHS or EPA classifications. Various
prediction models have been empirically devised and evaluated in order to use NRR assay
data to predict ocular irritation potentials of the test materials
4. Data Quality
In general, the data comprised results from peer-reviewed articles, unpublished data (withhand-written corrections of results in the case of the FRAME submission (1995)) and inhouse data.
Only one set of original high quality data (CTFA) is provided in the BRD. The other data areof varying quality.
All of the in vitro data and the majority of the in vivo data were obtained in non-GLP
compliant studies. The in vivo data in the CTFA study was GLP compliant. Both the in vivo
and in vitro data in the CTFA study were obtained concurrently.
5. Test materials
The assay was designed and developed for the in-house evaluation of the ocular irritancy
potential of cosmetic ingredients and formulations with expected mild or no irritancy.

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DIRECTORATE GENERAL JRC
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European Centre for the Validation of Alternative Methods (ECVAM)

Consequently test materials represented in the testing database are predominantly
surfactants and surfactant based cosmetic formulations, and two pesticides.
Materials tested included 10 hydro-alcoholic formulations, 25 surfactant based formulations,and 18 oil/water emulsions.
A broader range of materials needs to be tested using Protocol 54 to better establish its
applicability domain.
6. Within-laboratory variability
Seven reported studies based on INVITTOX Protocol 54 had appropriate data-sets for withinlaboratory reproducibility analyses. The test materials were restricted to surfactants or
surfactant-based formulations, with one study also containing data obtained with 2
pesticides.
The number of test runs varied for each test material, with the tendency of higher repeats forchemicals giving variable results: thus in interpreting the test performance it is necessary tokeep in mind that the data-sets are biased in favour of such materials.
Other data limitations include the absence of “greater-than” values; and in some cases only asingle value was used for analyses, which is not sufficient.
The within-laboratory variability could be calculated only for eight data-sets and all but one
were generated by testing surfactants or surfactant-based formulations without detained
information on their chemical composition (and one by testing two pesticides).
Overall, the within–laboratory variability for INVITTOX Protocol 54 was high, with mean CVs
ranging from 22% - 85%.
Only the data for INVITTOX Protocol 54, provided by FRAME Alternatives Laboratory,
enabled analyses of time and operator variability. The operator variability was found to be
7% - 32%, independent of the number of experiments performed. The data indicated that the
INVITTOX Protocol 54 has different levels of variability according to the type of test material;
specifically variability was particularly high with surfactants (see Table 3.2.21, page 78-81 of
the BRD).
A thorough study on a wide range of chemicals regarding their type and irritancy potential isrecommended by the PRP if additional studies are undertaken for a further validation study.
7. Transferability
No information is available regarding transferability. This is an essential component of themodular validation requirements.
8. Between laboratory variability
No information is available regarding between laboratory variability. This is an essential
component of the modular validation requirements.

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9. Predictive capacity
The capacity of the NRR assay to predict ocular irritation in vivo was assessed against invivo data from rabbits, as data for humans are virtually nonexistent.
Only the CTFA included original in vivo data. In the other studies historical in vivo data wereused. All available in vivo data were compiled and entered into ECVAM template v6, whichconverted them into EU, GHS and EPA classifications.
In the case of the CTFA Phase I-III studies, there was no prediction model before the study.
Threshold values were empirically assigned in the BRD for the CTFA study (cut-off values
250 and 600 mg/ml).
With the prediction model used the assay is highly sensitive (100% sensitivity) with poor
specificity as it has a tendency to over predict the irritancy of materials (20-44% false
positives). It showed better predictive capacity for severe irritants rather than non-irritants ormild irritants. The predictive capacity for oil in water emulsions could not be assessed as
actual NRR values could not be obtained for 83% of materials. The dataset for hydroalcoholic formulations (10 materials) was too small to draw any conclusions; however, thepredictive capacity for hydro-alcoholic formulations seemed to be better than for surfactantbased cosmetic formulations.
A compilation of data on chemicals (5 surfactants, 24 non-surfactants) was performed fromthe literature and the in vivo and in vitro results were paired purely on chemical name andconcentration. The sensitivity of the assay was confirmed as very high (92.3 – 100%) with alower specificity (56.3 – 80%) with slightly better specificity for evaluation of severe irritants
versus the rest (71.4-72.7% for all classification systems). For the limited number ofsurfactants (5), 100% sensitivity and 50% specificity was observed. However, in the case of
29 non-surfactants, the sensitivity was 80-100% and specificity 70-76.9% when analyzed forsevere irritants versus the rest.
However, due to the lower quality of the data, these results should be treated with caution.
10.

Applicability domain

The INVITTOX Protocol 54 was the only NRR assay protocol for which original in vitro and invivo data were available to assist in the determination of the applicability domain. The
available data were on hydro-alcoholic, oil-in-water and surfactant-based formulations.
The PRP concluded that the assay was not sensitive enough to measure the toxicity of oil-inwater emulsions. Only formulations were tested in the CTFA study and it is not possible toassume that the predictivity for chemicals would be similar.
On the basis of the evidence available to the PRP the applicability domain of this assay,
based on high quality data, is restricted to hydro-alcoholic and surfactant-based formulations.
The compilation of lower quality data on chemicals from the literature suggested that the
protocol had 100% sensitivity and better predictive capacity for severe irritants than nonirritants and mild irritants. The protocol had varying predictive capacities for different types offormulations with overall tendency to over predict non-irritants as irritants.

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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

Data were lacking on the potential effects of pH, colour or viscosity. Colour did not appear toaffect the result, while viscosity may play a role.
The EC Collaborative Study (1991) identified problems using this assay to evaluate certain
chemicals (silver nitrate, acetaldehyde and mercury chloride were misclassified).
Too few solids were tested to determine the predictive capacity with this type of material.
11.

Performance standards

No performance standards have been defined.
12.

Readiness for regulatory purposes

In the opinion of the PRP the INVITTOX Protocol 54 is not ready for consideration of
acceptance for regulatory purposes either for a Bottom-Up or Top-Down approach within atiered-testing strategy without further testing because of the limited number and classes ofchemicals/formulations tested, lack of information on transferability and interlaboratory
variability.
Conclusions and recommendation
It is important to note that the opinions of the PRP are premised on issues which cannot be
resolved on the basis of the available information, rather than the available evidence having
established conclusively that the test method should not be further developed and isincapable of being developed for regulatory use.
The INVITTOX Protocol 54 utilizes a commercially available and easily cultured cell line
routinely used for other methods worldwide.
Refinement of protocols including modifications and different prediction models designed for
different types of materials may be necessary, e.g. reduction of exposure time in case of
surfactants may increase specificity, mindful that longer exposure may decrease variability.
Positive and negative controls and the acceptance criteria should be defined in order toensure that the cells are responding in a predictable and reproducible manner and the results
are valid.
Without additional studies using a better defined and balanced set of chemicals andformulations in at least three laboratories, the reproducibility of results, applicability domain
and limitations of the assay cannot be determined.
Some false negatives may be the result of the oil/water matrix trapping the neutral red dye
and this should be evaluated. The protocol should be optimized to ensure that this trapping
does not occur by evaluating all negative samples under the microscope to determine thelocation of the neutral red dye.
The SOP needs refinement to include details and specific procedures for rinsing and forverifying that the neutral red is not bound by the oil/water matrix but actually inside of thecells.

15

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European Centre for the Validation of Alternative Methods (ECVAM)

Dilutions of the sample including the neat material should be tested.
In the opinion of the PRP this method may have potential to be further developed for use in a
Bottom-Up approach as part of a tiered-testing strategy or as a screen prior to in vivo Draize
testing, but not as a full replacement for the Draize eye-test.
However, in the view of the PRP the current BRD provides insufficient evidence to justify its
acceptance for consideration for regulatory use.

16

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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

PREDISAFE KIT (NEUTRAL RED RELEASE ASSAY)
1. Data collection
In the view of the PRP the data collection procedure was described adequately in the
Background Review Document and VMG report.
Although the test method is approved in France as an official regulatory method (Methode
Officiel Francaise), surprisingly little data were provided on the use of the PREDISAFE kit by
its manufacturer or users.
2. Goal of the study
The BRD and VMG papers were prepared with a view to considering the scientific validity
and readiness for regulatory consideration of the test method protocol as part of a weight ofevidence and tiered-testing strategy to minimise animal use to establish the eye irritancy
potential of test materials.
3. Test definition
The test definition is adequately defined in the view of the PRP.
It relies on mechanistically relevant features for modelling eye irritation, namely membrane
damage, and is not designed to assess reversibility or recovery.
In the view of the PRP the SOP for Predisafe needs to be improved, in part because ofvariation in use of the kit, possibly impacting on test performance, has been attributed toambiguity in the instructions.
The original prediction model was only qualitative and not applicable for predicting ocular
irritancy of chemicals according to the EU, GHS and EPA classifications.
For the purposes of the COLIPA study an alternative prediction model was developed inorder to convert the IC50 values into predictive MMAS DET scores.
4. Data quality
Surprisingly, in the view of the PRP, although the test method has been accepted by the
French Authorities for testing cosmetics, the data on use and performance of this protocol
were scarce (one COLIPA study with quality data).
5. Test materials
The assay was originally designed and developed for the testing of cosmetic ingredients andformulations with expected mild or no ocular irritancy potential.
Consequently, the data-base of test material contains predominantly surfactants andsurfactant-based formulations.

17EUROPEAN COMMISSION
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European Centre for the Validation of Alternative Methods (ECVAM)

The COLIPA study contained 22 ingredients of which 13 were surfactants, 9 chemicals and
32 formulations. The majority of the materials were water soluble or at least miscible inwater.
6. Within-laboratory variability
The PRP could not evaluate within-laboratory variability because of a lack of relevant
performance data for the PREDISAFE kit.
7. Transferability
There was no available documentation which could be used by the PRP to evaluate
transferability.
Although the PREDISAFE Protocol was conducted in more than one laboratory with thesame set of materials, there was no information on whether one of the laboratories was naive
and so the transferability could not be determined.
No information on PREDISAFE kit storage, shelf life, or overseas shipment was available to
the PRP.
The assay uses commercially available cell culture equipment; however, the Predisafe
protocol is based on a kit that contains live-cell cultures and reagents for which stability andperformance after long-distance shipping and storage is unknown.
8. Between-laboratory variability
The COLIPA study based on the PREDISAFE Protocol investigated the same set ofchemicals in three laboratories; however, no detailed information on potential sources ofvariability, such as the time-frame, lot and identical use of protocol were available to the
PRP.
Comparison of IC50 values of 22 ingredients (13 surfactants) and 32 formulations showed
CV of 45% (for ingredients) and 22% (for formulations). The use of different batches of test
materials might explain the higher variability.
Moreover, the PREDISAFE SOP did not contain detailed information on storage, rinsing anddelay between arrival and usage, which might increase between-laboratory variability.
All laboratories classified the irritants and slight irritants similarly with greater variation formoderate irritants. Good agreement in classification was observed, 85.5% of materials wereclassified identically in all three laboratories.
Further work should be done with a wider range of chemicals to evaluate its predictive
capacity for other types of test materials.
9. Predictive capacity
The capacity of the NRR assays to predict ocular irritation in vitro was assessed against invivo data from rabbits, as data for humans are virtually nonexistent.

18

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European Centre for the Validation of Alternative Methods (ECVAM)

The level of false negatives in the evidence provided to the PRP was up to 16% (EPA
classification) and the assay was not able to reliably distinguish ocular irritants from nonirritants sufficiently well to be endorsed by the PRP as a replacement or partial replacement
for the rabbit Draize eye-test. This opinion is reinforced by the severity and number of ocularlesions that would have been caused by some of the test materials that the test would havemiscategorised as ocular non-irritants.
The COLIPA study provided the only original data available for this assay (i.e. mean in vitro
data and raw in vivo data on 22 chemicals and 32 formulations). The PM was established
before the results were known (an algorithm converting IC50 values into predictive MMAS
DET scores). The sensitivity was high (92.9- 95.5%) but the specificity was much lower(60.6-70.4%).
Six different surfactants in multiple concentrations confirmed 100% sensitivity, but very poor
specificity (0% when analyzed non-irritants versus the rest, 28.6-33.3% when analyzed
severe irritants versus the rest). The PREDISAFE Protocol has a good predictive capacity forsevere irritants, but tends to over predict non-irritants and mild irritants (non-surfactant
chemicals only (9) showed 100% sensitivity and specificity 42.9-75% for non-irritants versus
the rest and decreased sensitivity 50-66% and specificity 42.9% for severe irritants versus
the rest). In the case of formulations, the best predictive capacity was found for severe
irritants versus the rest (sensitivity 100%, specificity 84%).
In the view of the PRP, none of the prediction models used for the purposes of this validation
study adequately distinguished between test materials falling into any of the principal
regulatory categories.
In the view of the PRP, on the basis of the information provided for consideration, the method
can be used for screening to identify likely potential ocular non-irritants prior to in vivo Draize
testing but not as a replacement for regulatory purposes.
10. Applicability domain
The potential applicability domain discussed in the BRD is restricted.
The PREDISAFE kit did not correctly classify any of the non irritant surfactants and therefore,
the test should not be used to evaluate and classify surfactants in a Bottom-Up approach.
The predictive capacity of PREDISAFE was calculated on the basis its performance in the
COLIPA study. Its sensitivity was consistently higher than specificity and the concordance
was better for non-surfactants than surfactants. There was insufficient data for coloured,solid or viscous chemicals for the PRP to determine the effects of physical features on thepredictive capacity of this protocol. The best predictive capacity was found for formulations(100% sensitivity, 84% specificity) when analyzed as severe irritants versus the rest. The
applicability domain comprises both chemicals (based on data on 13 surfactants and 10
alcohols and preservatives) and formulations, with better predictive capacity for nonsurfactants.
In general, the NRR test requires the use of high concentrations of chemicals which may
react with the plastic of wells and will be difficult to remove, thus increasing the apparent
toxicity of the tested samples reacting with the cells uncontrollably for prolonged time. The
solubility in water seems to be critical. No results are available on materials tested in mineral

19EUROPEAN COMMISSION
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European Centre for the Validation of Alternative Methods (ECVAM)oil or other solvents. The assay is not suitable for materials with fixative properties or forstrong acids and bases and highly volatile materials.
11. Performance standards
No performance standards were provided to the PRP.
12. Readiness for regulatory purposes
In the view of the PRP, on the basis of the available evidence, this test is not ready for
acceptance for regulatory purposes without further testing, including evaluation of
performance using a broader range of chemicals in at least three laboratories to establish
acceptable intra-laboratory variability and transferability.
Conclusions and recommendation
It is the view of the PRP that further testing should be undertaken to determine if thePredisafe kit can be recommended for screening as part of a battery of tests in a Bottom-Up
approach for non-surfactant cosmetics but not for EPA classification and not for Top-Down
due to the high number of false positives.
The test has a false negative rate of 6% with a false positive rate greater than 35% (verypoor with non-irritant surfactants but better with multi-component mixtures).
The test seems promising, but it tends from the available dataset to over predict the irritancy
of materials.
PREDISAFE is supplied in the form of a standardized kit including reagents and cells;
however, no information on performance after prolonged shipment e.g. overseas is available.
In the view of the PRP the time between delivery and use of the PREDISAFE kit may play arole in its performance, and this is another factor requiring additional evidence to evaluate.
Refinement of the protocol including modifications and different prediction models designed
for different types of materials may be necessary, e.g. reduction of exposure time in case of
surfactants may increase specificity, on the other hand longer exposure may decrease
variability.
The acceptance criteria should be defined in order to ensure that the cells are responding ina predictable and reproducible manner and the results are valid.
In the view of the PRP without additional studies of a defined and balanced set of chemicals
and formulations in at least three laboratories, the reproducibility of results, application
domain and limitations of the assay cannot be determined.

20

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DIRECTORATE GENERAL JRC
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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

RED BLOOD CELL ASSAY
1. General Comments:
The VMG and PRP reviews of the data and analysis of INVITTOX protocols 37 and 99
confirmed significant shortcomings and inconsistencies with respect to specificity andsensitivity, protocol deviations, and problems with the prediction model.
These shortcomings, and the failure on the basis of the available information to satisfy otherrequired validation modules, in the view of the PRP currently preclude consideration of thesetest methods for consideration for regulatory use, even as part of a tiered-testing strategy, todetermine the eye irritancy potential of test materials.
2. Data collection:
Reasonable efforts have been made to obtain relevant, robust data, and three studies arereviewed in the BRD.
3. Goal of the study:
The BRD and VMG papers were prepared with a view to considering the scientific validity
and readiness for regulatory consideration of the test method protocols as part of a testing
strategy to minimise animal use to establish the eye irritancy potential of test materials.
4. Test definition:
Test definition is adequately described. The mechanistic basis and its relevance to eye
irritancy are explained.
5. Data quality:
Data quality is variable.
In the view of the PRP there may be a case for discarding the least reliable data if any further
or supplementary evaluation is made of these test methods and better quality information
becomes available.
6. Test materials:
As described in the BRD.
7. Intra-laboratory variability:
The intra-laboratory variability was difficult for the PRP to asses, with one study showing CVs
of up to 67%.
A wide range of potential variables were indentified, but cannot be fully evaluated on thebasis of the material contained in the reference documents.

21

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European Centre for the Validation of Alternative Methods (ECVAM)

8. Transferability:
There was no evidence for transferability available.
9. Inter-laboratory variability:
The reported CVS are unacceptably high.
10.

Predictive capacity:

The predictive capacity was poor when judged against the standard classification systems.
The method is likely to over-classify 20% of test materials if used for Top-Down evaluation:and to misclassify more than 10% of irritants with a Bottom-Up approach.
11.

Applicability domain:

The applicability domain was evaluated primarily with water-soluble test materials.
The method is known not to be suitable for assessing ketones, alcohols, fixatives, dyes andviscous materials.
12.

Minimum performance standards:

Performance standards were not provided to the PRP.
13.

Readiness for regulatory purposes:

In the view of the PRP although it may be suitable for in-house decision making, on the basisof the available evidence these protocols have not been shown to be ready for consideration
either as a Top-Down or a Bottom-Up screen for regulatory purposes.

22EUROPEAN COMMISSION
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European Centre for the Validation of Alternative Methods (ECVAM)

CM ASSAY – INVITTOX PROTOCOL 102 – FOR TOP-DOWN APPROACH
1. General Comments:
INVITTOX Protocol 97 is considered by the PRP to be redundant, and is not consideredfurther.
INVITTOX Protocol 102 has been used as in-house screen by the test-developer.
Essential equipment and consumables are no longer manufactured, and the original software
now obsolete.
2

Data collection:

In the view of the PRP reasonable efforts have been made to obtain relevant, robust data.
The BRD is based on historical data, in part from the peer reviewed literature, including
previous validation studies.
3

Goal of the study:

The BRD and VMG papers were prepared with a view to considering the scientific validity
and readiness for regulatory consideration of the test method protocols as part of a testing
strategy to minimise animal use to establish the eye irritancy potential of test materials.
4

Test definition:

In the view of the PRP the test system has been adequately described.
The mechanistic basis and its relevance to eye irritancy are explained. However in the viewof the PRP it is not clear precisely what class of interaction (e.g. lysis, saponification,
coagulation, or reactive chemistry) is modelled in the test system.
5

Data quality:

There is published and unpublished data, not obviously produced to GLP, although someoriginal data have been audited.
The Draize eye-test reference data were not obtained using the standard in vivo protocol:
topical anaesthesia was used to obtain the CTFA data set, and the LVET was used for thecompany data. The significance of this is not discussed in detail in the BRD or VMG report.
In the view of the PRP the HO/EC material is particularly weak in a number of places, and in
the view of the PRP there may a case for setting it aside if a further evaluation of validation
status is undertaken.

23EUROPEAN COMMISSION
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European Centre for the Validation of Alternative Methods (ECVAM)

6

Test materials:

INVITTOX 102 data relate to 53 water-soluble test materials – 21 chemicals and 32 multicomponent mixtures, 29 of which were non-surfactants. These are clearly described in thematerial made available to the PRP.
7

Intra-laboratory variability:

The intra-laboratory variability is considered acceptable by the PRP.
The low CVs tend to confirm low degree of temporal and operator variability.
8

Transferability:

The method requires specialist equipment and consumables which are no longer currently
manufactured.
The performance described seems adequate to the PRP, although there is no agreed
Performance Standard, with respect to laboratories with established cell culture expertise.
9Inter-laboratory variability:

Although there are no agreed Performance Standards in the opinion of the PRP the interlaboratory variability seems to have been adequately demonstrated with generally goodcorrelations.
10 Predictive capacity:
The prediction model was developed and refined during the evaluation, however the final
recommended protocol must more clearly state how it is applied.
For a Top-Down approach, the material contained in the BRD and reviewed by the VMG
shows that for the evaluation of water-soluble surfactants and multi-component water-soluble
surfactant containing mixtures the false positive rate was 3-10%, and false negative rate 222%. It is not known if the mixtures contained other constituents expected to be biologically
active.
With water-soluble non-surfactants the false positive rate was 0-6%, and the false negative
rate 43-55%.
11 Applicability domain:
Other than the HO/EC data, evaluation was primarily based on water-soluble surfactants,
multi-component water-soluble surfactant containing mixtures, and water-soluble
chemicals/materials – generally expected to be of low irritancy potential. It is not known if the
mixtures contained other constituents expected to be biologically active.
In the opinion of the PRP this test is not considered suitable, on the basis of the available
data, for consideration for regulatory use for the evaluation of non-water soluble or non-24

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European Centre for the Validation of Alternative Methods (ECVAM)

aqueous materials, mixtures containing other ingredients likely to be biologically active,
viscous materials, suspensions or solids.
12 Minimum Performance Standards:
No performance standards were provided to the PRP.
The PRP has assumed for a Top-Down approach, with negatives being further evaluatedbefore classification, that fewer than 10% of test materials should be over-classified.
13 Readiness for regulatory purposes:
On that basis, the CM INVITTOX Protocol 102 is considered by the PRP to be suitable forconsideration for regulatory use as a Top-Down screen to detect strong irritants (EPA
Category 1, GHS Category 1, and EU R41) as part of a tiered-testing strategy for theapplicability domain given.
The PRP wishes to emphasise the following:
1. This opinion is based upon the performance standards developed by the PRP itself,
and not yet validated or endorsed by an other party.
2. Any validation statement is relevant only to the hardware and software used togenerate the data presented in the BRD.
3. Any significant changes to the hardware or software should be subject to a catch-up
validation study conducted at a single centre with small number of well-characterised
and representative test materials.
4. All negative results must be further evaluated before test materials are classified in a
Top-Down testing strategy.
5. Further work is required to establish if the applicability domain can be broadened toadditional classes of test materials.
6. The test may not be suitable to evaluate test materials expected to have a direct
effect on cell respiration or glycolysis.
7. The VMG has recommended changes to the exposure conditions, but the detail andrationale are not clear. The VMG should be asked for clarification.
8. When interpreting results with water-soluble surfactants users should take account of
whether the test materials were evaluated at concentrations above or below thecritical micelle concentration (CMC).

25

EUROPEAN COMMISSION
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Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

CM ASSAY – INVITTOX PROTOCOL 102 – BOTTOM-UP APPROACH
1. General Comments:
INVITTOX Protocol 97 is considered redundant, and is not considered further.
INVITTOX Protocol 102 was previously used as in-house screen by test-developer.
Equipment and consumables are no longer manufactured, and the original software is now
obsolete.
2. Data collection:
Reasonable efforts have been made to obtain relevant, robust data.
The BRD is based on historical data, in part from the peer reviewed literature, including
previous validation studies.
3. Goal of the study:
The BRD and VMG papers were prepared with a view to considering the scientific validity
and readiness for regulatory consideration of the test method protocols as part of a testing
strategy to minimise animal use to establish the eye irritancy potential of test materials.
4. Test definition:
The test definition is, in the view of the PRP, adequately described.
The mechanistic basis and its relevance to eye irritancy are explained. However in the viewof the PRP it is not clear precisely what class of interaction (e.g. lysis, saponification,
coagulation, or reactive chemistry) the test system models.
5. Data quality:
The BRD included both published and unpublished data, not obviously produced to GLP,
although some have been audited.
The in vivo reference data were not obtained using the standard rabbit Draize eye-test
protocol: topical anaesthesia was used to obtain the CTFA data set, and the LVET was used
for the company data. Although the significance of this is not discussed in the BRD or VMG
report, in the view of the PRP the data must still be considered for the purposes of validation.
In the view of the PRP the HO/EC material is particularly weak in places, and in the view of
the PRP there may be a case for setting it aside of any further validation assessment isconducted.
6. Test materials:
INVITTOX 102 data relates to 53 water-soluble test materials – 21 chemicals and 32 multicomponent mixtures, 29 of which were non-surfactants.

26

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7. Intra-laboratory variability:
Intra-laboratory variability is considered acceptable by the PRP. Low CVs tended to confirm
low degree of temporal and operator variability.
8. Transferability:
Transferability requires essential specialist equipment and consumables which are no longer
manufactured.
The performance described seems adequate to the PRP, although there is no agreed
Performance Standard, with respect to laboratories with established call culture expertise.
9. Inter-laboratory variability:
Although there are no agreed Performance Standards, inter-laboratory variability seems tohave been adequately demonstrated in the view of the PRP with generally good correlations.
10.

Predictive capacity:

The prediction model was developed and refined during the evaluation, the final
recommended protocol must in the view of the PRP more clearly state how it is applied.
For a Bottom-Up approach the material contained in the BRD and reviewed by the VMG
shows that for the evaluation of water-soluble surfactants and multi-component water-soluble
surfactant containing mixtures the false negative rate was 0-2% - with the materials
misclassified shown to be more than mild irritants when evaluated in the DET. It is not known
if the mixtures contained other constituents expected to be biologically active.
In contrast the false negative rate with non-surfactants was 24-38%, in the view of the PRP
this is too high for regulatory use in a bottom-up approach.
11.

Applicability domain:

Other than HO/EC data, the evaluation was primarily based on data generated with watersoluble surfactants and multi-component water-soluble surfactant containing mixtures – thatwere expected to be of low irritancy potential. It is not known if the mixtures contained otherconstituents expected to be biologically active.
Performance was poor with non-surfactants.
It is not suitable in the view of the PRP, on the basis of the available data, for the evaluationof non-aqueous or non-water soluble materials, mixtures containing other ingredients likely to
be biologically active, viscous materials, suspensions or solids.
12.

Minimum Performance Standards:

Minimum Performance Standards were not available.

27

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The PRP has assumed for a bottom-up approach, with positives being further evaluatedbefore classification, that there should be a negligible false negative rate, with no false
negative proving to be more than a mild irritant.
13.

Readiness for regulatory purposes:

On that basis CM INVITTOX Protocol 102 is considered by the PRP to be suitable forconsideration for regulatory use as a Bottom-Up screen to detect non-irritants (insert
classifications) as part of a tiered testing strategy only for water-soluble surfactants, andmulti-component mixtures containing water-soluble surfactants.
In the view of the PRP it is not suitable, on the basis of the available data, for the evaluationof non-aqueous or non-water soluble materials, mixtures containing other ingredients likely to
be biologically active, viscous materials, suspensions or solids.
The PRP emphasises that it considers:
1. This opinion is based upon the performance standards developed by the PRP itself,
and not yet validated or endorsed by an other party.
2. Any validation statement is relevant only to the hardware and software used togenerate the data presented in the BRD.
3. Any significant changes to the hardware of software should be subject to a catch-up
validation study conducted at a single centre with small number of well-characterised
and representative test materials.
4. All positive results obtained in a Bottom-UP approach must be further evaluatedbefore test materials are classified.
5. It is not recommended, on the basis of the available evidence, that the test be used toevaluate non-surfactants.
6. Further work is required to establish if the applicability domain can be broadened toadditional classes of test materials.
7. The test may not be suitable to evaluate test materials expected have a direct effecton cell respiration or glycolysis.
8. The VMG has recommended changes to the exposure conditions, but the detail andrationale are not clear. Clarification should be sought from the VMG.
9. When interpreting results with water-soluble surfactants users should take account of
whether the test materials were evaluated at concentrations above or below thecritical micelle concentration (CMC).

28