Name: eye irritation test (eit) for hazard identification of eye irritating chemicals using reconstructed human cornea-like epithelial (rhce) tissue model
Uploaded: 2015-08-23T14:00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model at JoVE.com

The authors would like to thank Dr. John Harbell for his scientific support and time dedicated to the EIT project. The authors also would like to thank Beiersdorf AG (Germany), IIVS (USA), Mary Kay Inc. (USA), Avon Products Inc. (USA), Procter &#38; Gamble/Cosmital (Switzerland), Laboratoire Pierre Fabre (France), Harlan Laboratories (United Kingdom), and LVMH Parfume (France) for participation in Multi-center International Pre-Validation and Validation Studies of the Eye Irritation Test15.

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	<li>National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Request+for+Ocular+Irritancy+Test+Data+From+Human,+Rabbit,+and+In+Vitro+Studies+Using+Standardized+Testing+Methods.">Request for Ocular Irritancy Test Data From Human, Rabbit, and In Vitro Studies Using Standardized Testing Methods.</a> Federal Register. 72 (109), 31582-31583 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+(EC)+No+1907/2006+of+the+European+Parliament+and+of+the+Council+of+18+December+2006+concerning+the+Registration,+Evaluation,+Authorisation+and+Restriction+of+Chemicals+(REACH),+establishing+a+European+Chemicals+Agency,+amending+Directive+1999/45/EC+and+repealing+Council+Regulation+(EEC)+No+793/93+and+Commission+Regulation+(EC)+No+1488/94+as+well+as+Council+Directive+76/769/EEC+and+Commission+Directives+91/155/EEC,+93/67/EEC,+93/105/EC+and+2000/21/EC.">Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC.</a> OJ. 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A., Bruner, L. H., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EC/HO+international+validation+study+on+alternatives+to+the+Draize+eye+irritation+test.">The EC/HO international validation study on alternatives to the Draize eye irritation test.</a> Toxicol in Vitro. 9, 871-929 (1995).</li><li>Curren, R. D., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+safety:+a+silent+(in+vitro)+success+story.">Ocular safety: a silent (in vitro) success story.</a> Altern Lab Anim. 30, 69-74 (2002).</li><li>Wilhelmus, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Draize+Eye+Test.">The Draize Eye Test.</a> Survey of Ophthalmology. 45, 493-515 (2001).</li><li>Curren, R. D., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+alternatives+for+ocular+irritation.">In vitro alternatives for ocular irritation.</a> Environ health persp. 106, 485-492 (1998).</li><li>McCain, N. E., Binetti, R. R., Gettings, S. D., Jones, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+ocular+irritation+ranges+of+market-leading+cosmetic+and+personal-care+products+using+an+in+vitro+tissue+equivalent.">Assessment of ocular irritation ranges of market-leading cosmetic and personal-care products using an in vitro tissue equivalent.</a> Toxicologist. 66, 243 (2002).</li><li>Niranjan, P., Dang, A. H., January, B. G., Gomez, C., Harbell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+EpiOcular+assay+for+preclinical+qualification+of+formulas+for+human+clinical+studies.">Use of the EpiOcular assay for preclinical qualification of formulas for human clinical studies.</a> Toxicologist. 96, 249 (2007).</li><li>Yin, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+ocular+irritation+potential+of+surfactants-based+formulations+at+different+concentrations+using+the+EpiOcular+model.">Prediction of ocular irritation potential of surfactants-based formulations at different concentrations using the EpiOcular model.</a> Toxicologist. 108, 378 (2009).</li><li>Harbell, J., Curren, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+for+the+prediction+of+ocular+and+dermal+toxicity.">In vitro methods for the prediction of ocular and dermal toxicity.</a> Handbook of Toxicology. , (2001).</li><li>Kaluzhny, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+EpiOcular(TM)+eye+irritation+test+for+hazard+identification+and+labelling+of+eye+irritating+chemicals+in+response+to+the+requirements+of+the+EU+cosmetics+directive+and+REACH+legislation.">Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation.</a> Altern Lab Anim. 39, 339-364 (2011).</li><li>Pfannenbecker, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cosmetics+Europe+Multi-Laboratory+Pre-Validation+of+the+EpiOcular+Reconstituted+Human+Tissue+Test+Method+for+the+Prediction+of+Eye+Irritation.">Cosmetics Europe Multi-Laboratory Pre-Validation of the EpiOcular Reconstituted Human Tissue Test Method for the Prediction of Eye Irritation.</a> Toxicol in vitro. , (2013).</li><li>MatTek Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals for use with Reconstructed Human EpiOcular Model (OCL-200-EIT). Protocol#MK-24-007-0055. , (2014).</li><li>Kaluzhny, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpiOcular+Eye+Irritation+Test+(EIT)+for+Hazard+Identification+and+Labeling+of+Eye+Irritating+Chemicals:+Protocol+Optimization+for+Solid+Materials+and+Extended+Shipment+Times.">EpiOcular Eye Irritation Test (EIT) for Hazard Identification and Labeling of Eye Irritating Chemicals: Protocol Optimization for Solid Materials and Extended Shipment Times.</a> Altern Lab Anim. 43 (2), 101-127 (2015).</li><li>Kolle, S. N., Kandarova, H., Wareing, B., van Ravenzwaay, B., Landsiedel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-house+validation+of+the+EpiOcular(TM)+eye+irritation+test+and+its+combination+with+the+bovine+corneal+opacity+and+permeability+test+for+the+assessment+of+ocular+irritation.">In-house validation of the EpiOcular(TM) eye irritation test and its combination with the bovine corneal opacity and permeability test for the assessment of ocular irritation.</a> Altern Lab Anim. 39, 365-387 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructed+Human+Cornea-like+Epithelium+(RhCE)+Test+Method+for+Identifying+Chemicals+Not+Requiring+Classification+and+Labelling+for+Eye+Irritation+or+Serious+Eye+Damage.+Draft+proposal+for+a+new+test+guideline..">Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage. Draft proposal for a new test guideline..</a> OECD Guideline for the Testing of Chemicals. , (2014).</li><li>Blazka, M. E., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+ocular+irritation+potential+of+54+test+articles+using+the+EpiOcular+Human+tissue+Construct+Model.">Evaluating the ocular irritation potential of 54 test articles using the EpiOcular Human tissue Construct Model.</a> Toxicol. In Vitro. , (2003).</li><li>Kandarova, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EpiDerm+Test+Protocol+for+the+Upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests+&#8212;+An+Assessment+of+the+Performance+of+the+Optimised+Test.">The EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests &#8212; An Assessment of the Performance of the Optimised Test.</a> Altern Lab Anim. 33, 351-367 (2005).</li></ol>

Publication fees for this article were paid by MatTek Corporation. The authors are employees of MatTek Corporation.

We have presented the Eye Irritation Test (Figure 1) that was developed for the EpiOcular tissue model. The EIT is able to separate ocular irritants and corrosives (GHS Categories 1 and 2 combined) from materials that do not require labeling (GHS No Category) with high degree of sensitivity and specificity17. The EIT as presented herein does not discriminate between GHS Category 1 from Category 2 chemicals. The EIT was validated for classification and labeling of ocular irritation potential of a wide range of chemicals, including cosmetic and pharmaceutical ingredients. In conjunction with other in vitro tests, the EIT will serve as a replacement for the in vivo rabbit eye irritation test.
The EIT uses two similar but distinct protocols for liquid and solid materials, which vary in the length of exposure and post-exposure incubation periods (Figure 1). The endpoint used in the EIT is tissue viability, determined by the MTT assay, which has been previously used in validated human epithelial tissue models20,21. To perform this assay, no special equipment besides standard cell culture equipment is needed. Due to the high level of tissue-to-tissue reproducibility, n = 2 tissues instead of the usually recommended n = 3 are used. The ability to use n= 2 tissues is a critical aspect of the protocol, since it allows an experienced operator to process two tissues simultaneously, thereby minimizing the variability of the assay that can arise due to the different handling of individual tissues14. Also, by using n = 2 tissues per test article, the irritancy of 10 test substances of the same physical state (liquid or solid), along with the positive and negative controls, can be evaluated using one kit (24 tissues).
Other key points that ensure reliable classification of materials are specifications for the positive control substance (tissue viability &#8804;50.0%), reproducibility between duplicate tissues (difference &#60;20.0%), and negative control OD readings (&#62;0.8 and &#60;2.5).
When performing the EIT test, it is important to adhere to the validated protocol and to the suggested dosing and rinsing schedules (Tables 3 and 4), since deviation from the protocol or changes in the incubation periods may result in altered outcome. Likewise, deviations from the 3 hr time for MTT incubation will result in different MTT readings and may affect assay result.
Occasionally, a test chemical may have optical or other properties which may interfere with MTT tissue viability assay or cause reduction of MTT. For instance, a test chemical may directly reduce MTT into blue-purple reaction product, or may be a colored substance that absorbs light in the same range as MTT formazan (~570 nm). However, these test chemicals will present a problem only, if at the time of the MTT assay, a sufficient amount of the material is still present on (or absorbed by) the tissue. To avoid this interference, extensive rinsing procedures are incorporated into the EIT protocol. If rinsing does not remove the TA and the TA interferes with MTT reduction, additional controls must be used to detect and correct for it. Briefly, if direct MTT reduction of the test chemical is suspected, 50 &#181;l (or 50 mg for solids) of the chemical in question is incubated for 3 hr with working MTT solution at SCC (NC, 50 &#181;l of sterile deionized water, should be run concurrently). If the MTT solution turns blue-purple, the test article is presumed to have reduced the MTT. In this case a functional check using freeze-killed tissue controls should be performed to evaluate whether the test material is binding to the tissue and leading to a false MTT reduction signal. If there is appreciable MTT reduction in the TA-exposed, killed tissue control (relative to the amount in the untreated viable tissue), the mean tissue viability of the test article must be corrected by subtracting the mean viability of the killed control.
The EIT errs on the side of safety, as demonstrated by the low incidence of false negative classifications14,15,18. Importantly, none of the GHS Category 1 chemicals, which are corrosive to the eye and which represent the most serious ocular hazard, were classified as non-irritating in this assay14,15,18,19. Finally, one of the major advantages of the RhCE in vitro test method is the possibility of testing neat liquid and solid materials (which is not possible with two-dimensional, submerged cell cultures).
The EIT will &#160;contribute significantly in determining the ocular irritation potential of a wide range of materials according to the UN GHS classification and labeling system. The replacement of animals to determine ocular toxicity has been a goal of toxicological research for many years. The EIT test method has completed a formal validation study supported by EURL ECVAM in 2014 and the EpiOcular EIT was implemented into the OECD test Guidelines as OECD TG 492&#160;in 2015.

Consumer products such as cosmetics, detergents, and household cleaners include a variety of chemicals that may induce serious damage if they contact the eyes. Therefore, testing of these agents for eye irritation is required by the US and EU regulatory agencies to ensure consumer safety1. An assessment of the eye irritation potential of mixtures and formulations is also a requirement for complying with REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) legislation for the labeling of cosmetic ingredients under the EU Cosmetics Directive for transport of chemicals, and for the labeling of pesticides and household products2. Currently, regulatory agencies require ocular hazard assessment using the Globally Harmonized System of Classification and Labeling of Chemicals (GHS)3. GHS is mainly based on the Draize eye irritation test, the most widely used eye irritation assay in which foreign substances and mixtures are introduced directly into the conjunctival sac of the rabbit eye4. According to GHS classification, GHS Category 1 (ocular corrosives) refers to test chemicals that cause severe initial injury to the eye tissues or serious damage to the eye and vision which is not fully reversible within 21 days following exposure3,5. GHS Category 2 refers to test chemicals that produce significant changes in the eye that are fully reversible within 21 days of exposure. Test chemicals that are not corrosives or irritants are referred to as GHS No Category.
For more than 40 years, the Draize rabbit eye test has been criticized for its lack of reproducibility, overestimation of human responses, and the use of live animals5-8. These concerns have encouraged many proposals for refinement, reduction, and replacement of the in&#160;vivo&#160;test9. The need for validated non-animal alternatives was further strengthened by the adoption of the Seventh Amendment to the Cosmetics Directive, which banned the use of animals in the safety evaluation of cosmetic products (in 2005) and ingredients (in 2009)2.
Since 1996, the reconstructed cornea-like tissue model has been widely used by the cosmetic industry to evaluate the irritation potential of raw materials, surfactant-based formulations, and compounded mixtures that are designed for use in, or in the vicinity of, the eye10-13. Use of the RhCE tissue model allows direct topical application of the test material onto the tissue surface in its native, undiluted form. In this way, non-water soluble formulations can be tested without diluting them with solvents. In response to EURL ECVAM&#8217;s (European Union Reference Laboratory European Centre for the Validation of Alternative Methods) request for a widely applicable, straightforward, and economic method the Eye Irritation Test (EIT) which utilizes a single exposure time and is able to separate ocular irritants and corrosives from materials that do not require labeling was developed (Figure 1)14. Based on the results for 83 chemicals (44 liquids and 39 solids), the EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A.
In 2007, a multi-laboratory pre-validation study sponsored by Cosmetics Europe (formerly Colipa) under the auspices of the EURL ECVAM assessed the relevance and reliability of the EIT with the goal of bringing it to formal validation15. In this study, 298 independent trials were performed in seven independent laboratories. Study results demonstrated 99.7% agreement in prediction with low coefficients of variation across all participating laboratories15. As a result, in 2010 the EIT protocol entered a formal EURL ECVAM validation program. The validation study utilized 104 coded test chemicals, including individual substances and chemical mixtures, for which in vivo reference data (Draize eye irritation data) were available. Based on the success of this work, an OECD draft test guideline was submitted in 2014. It is anticipated that the EIT will contribute significantly to classification of ocular irritation potential of a wide range of materials according to the UN GHS classification and labeling system.

eye irritation test (eit) for hazard identification of eye irritating chemicals using reconstructed human cornea-like epithelial (rhce) tissue model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Watch this Scientific Journal Video about Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model at JoVE.com

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

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