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1、 Series on Testing and Assessment No.400 Peer Review Report on Inclusion of OptiSafe Eye Irritation Test(EIT)in Test Guideline 496 on In Vitro Macromolecular Test Methods for Identifying the Eye Hazard Potential of Chemicals 2024 PUBE 2|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELI

2、NE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 OECD 2024 Attribution 4.0 International(CC BY 4.0)This work is made available under the Creative Commons Attribution 4.0 International licence.By using this work,you accept to be bound by t

3、he terms of this licence(https:/creativecommons.org/licenses/by/4.0/).Attribution you must cite the work.Translations you must cite the original work,identify changes to the original and add the following text:In the event of any discrepancy between the original work and the translation,only the tex

4、t of original work should be considered valid.Adaptations you must cite the original work and add the following text:This is an adaptation of an original work by the OECD.The opinions expressed and arguments employed in this adaptation should not be reported as representing the official views of the

5、 OECD or of its Member countries.Third-party material the licence does not apply to third-party material in the work.If using such material,you are responsible for obtaining permission from the third party and for any claims of infringement.You must not use the OECD logo,visual identity or cover ima

6、ge without express permission or suggest the OECD endorses your use of the work.Any dispute arising under this licence shall be settled by arbitration in accordance with the Permanent Court of Arbitration(PCA)Arbitration Rules 2012.The seat of arbitration shall be Paris(France).The number of arbitra

7、tors shall be one.Please cite this publication as:OECD(2024),Peer Review Report on Inclusion of OptiSafe EIT in Test Guideline 496 on In Vitro Macromolecular Test Methods for Identifying the Eye Hazard Potential of Chemicals,OECD Series on Testing and Assessment,No.400,OECD Publishing,|3 PEER REVIEW

8、 REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 About the OECD The Organisation for Economic Co-operation and Development(OECD)is an intergovernmental organisation in which representati

9、ves of 38 countries in North and South America,Europe and the Asia and Pacific region,as well as the European Union,meet to co-ordinate and harmonise policies,discuss issues of mutual concern,and work together to respond to international problems.Most of the OECDs work is carried out by more than 20

10、0 specialised committees and working groups composed of member country delegates.Observers from several Partner countries and from interested international organisations attend many of the OECDs workshops and other meetings.Committees and working groups are served by the OECD Secretariat,located in

11、Paris,France,which is organised into directorates and divisions.The Environment,Health and Safety Division publishes free-of-charge documents in twelve different series:Testing and Assessment;Good Laboratory Practice and Compliance Monitoring;Pesticides;Biocides;Risk Management;Harmonisation of Regu

12、latory Oversight in Biotechnology;Safety of Novel Foods and Feeds;Chemical Accidents;Pollutant Release and Transfer Registers;Emission Scenario Documents;Safety of Manufactured Nanomaterials;and Adverse Outcome Pathways.More information about the Environment,Health and Safety Programme and EHS publi

13、cations is available on the OECDs World Wide Web site(https:/www.oecd.org/en/topics/chemical-safety-and-biosafety.html).This publication was developed in the IOMC context.The contents do not necessarily reflect the views or stated policies of individual IOMC Participating Organizations.The Inter-Org

14、anisation Programme for the Sound Management of Chemicals(IOMC)was established in 1995 following recommendations made by the 1992 UN Conference on Environment and Development to strengthen co-operation and increase international co-ordination in the field of chemical safety.The Participating Organis

15、ations are FAO,ILO,UNDP,UNEP,UNIDO,UNITAR,WHO,World Bank,Basel,Rotterdam and Stockholm Conventions and OECD.The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by the Participating Organisations,jointly or separately,to achieve the sound management of chemicals

16、 in relation to human health and the environment.4|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 Foreword This document encloses the Peer Review Report(PRR)of the validatio

17、n study for OptiSafeTM Eye Irritation Test(EIT)for its inclusion in OECD Test Guideline 496 on In Vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage(UN GHS Cat.1)and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage(UN GHS No Cat).The

18、 OptiSafeTM EIT is validated for identifying UN GHS No Cat.The Peer Review was organized by the OECD Secretariat from November to December 2022 based on evaluation principles from OECD Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessme

19、nt.The peer review report was endorsed by the Working Party of the National Coordinators of the Test Guidelines Programme(WNT)at their 36th meeting in April 2024.This document is published under the responsibility of the Chemicals and Biotechnology Committee.|5 PEER REVIEW REPORT ON INCLUSION OF OPT

20、ISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 1.Summary In April 2022,WNT approved the Standard Project Submission Form(SPSF)submitted by the U.S on inclusion of OptiSafeTM as a me-too test method in TG 496:In

21、Vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not Requiring classification for Eye Irritation or Serious Eye Damage.The OptiSafeTM had been validated by the validated management team(VMT)comprised of experts within NICEATM,and the validation rep

22、ort was published in a peer-reviewed journal(1).The OptiSafeTM Test Method was presented and discussed at the teleconferences of OECD Expert Group on Skin and Eye Irritation on July 15th and September 26th,2022.The summary records from these teleconferences are available on the restricted community

23、site for the OECD Expert Groups(Link).The OECD Secretariat organized an independent Peer Review with a panel comprised of members of the OECD Expert Group on Skin and Eye Irritation during November December 2022.The results from the Peer Review were put together for a Peer Review Report(PRR)containe

24、d in this document.2.Background The OptiSafeTM Test Method uses a similar test platform as the Validated Reference Method(VRM)in TG 496,which contains a macromolecular reagent composed of a mixture of proteins glycoproteins,carbohydrates,lipids,and low molecular weight components,that when rehydrate

25、d forms a complex macromolecular matrix which mimics the highly ordered structure of the transparent cornea(2).The OptiSafeTM Test Method exhibits a few distinctions when compared with the VRM in TG 496.These deviations include a few differences in the test method components and protocols,and the ha

26、zard categories that the test method is intended for predictions.For the intended Peer Review Report on inclusion of OptiSafeTM EIT in Test Guideline 496 on In Vitro Macromolecular Test Methods for Identifying the Eye Hazard Potential of Chemicals 6|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN

27、 TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 hazard categories,the VRM is recommended for identifying chemicals that induce serious eye damage(i.e.,UN GHS Category 1)and for chemicals that do not require classification(i.

28、e.,UN GHS No Category),while the OptiSafeTM is recommended for chemicals that are not UN GHS Category 1(i.e.,chemicals that do not cause serious eye damage)and for chemicals that do not require classification.Finally,the prediction model for the OptiSafeTM is presented for three hazard categories(i.

29、e.,UN GHS Category 1,Category 2 and No Category).3.The peer review process The secretariat solicited participations from the OECD Expert Group on Skin and Eye Irritation to serve as a member of Peer Review Panel for the OptiSafeTM Test Method in October 2022.The Peer Review Panel(PRP)was confirmed o

30、n 21 October 2022.The selected members of the Panel are listed in Annex I.The secretariat drafted 8 Peer Review charge questions based on the validation principles outlined in OECD Guidance Document 34(3).The intention of these questions was to evaluate i)the study objective and test method purpose,

31、ii)the need and benefits of the test method in comparison to existing test methods,iii)the comparison of the test method with the essential test method components and performances as described in the Performance Standards for TG 496(5),iv)the biological and mechanistic relevance,v)the test method pr

32、otocol,vi)the appropriateness of the validation study management and conduct,and vii)the availability of all data supporting the assessment of the validity of the analysis.The final and eighth charge question asked for the panels final remarks and additional comments on the proposal.The charge quest

33、ions are listed in Annex II.The developer of the OptiSafeTM test method had given two presentations at the OECD Expert Group teleconferences.As all members of the PRP were from the OECD Expert Group who attended the teleconferences,no additional presentation was given to the PRP prior to the review

34、process.Panel members were asked to perform their review based on following materials.All materials are available on the restricted community site for the OECD Expert Group(Link).a.The Performance Standards for TG 496(5)b.The draft Appendix 2 on OptiSafeTM to TG 496 c.Validation Report(1)d.Standard

35、Operating Protocol e.Raw data for reference chemicals from performance standards f.Raw data for all chemicals in the database The members of PRP reviewed the supporting materials for OptiSafeTM,from November 7th to November 28th,2022 and provided their feedback.The secretariat compiled the feedback

36、and prepared this report.|7 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 4.Evaluation Principle 1.Study objective and test method purpose Charge Question 1:Were the object

37、ive and the purpose of the test method adequately described?2.Overall,the PRP does not consider the objective and the purpose of the test method to be adequately described.The draft Appendix 2 on OptiSafeTM for TG 496 proposes that the test method is recommended to identify chemicals that do not ind

38、uce serious eye damage(i.e.,Not UN GHS Category I)and for chemicals that do not require classification(UN GHS No Category).Thus,the purpose and Prediction Model differ notably from both the purpose proposed in the SPSF and the VRM presented in TG 496.This then suggests that,in addition to identifyin

39、g chemicals that do not require classification,OptiSafeTM can also identify chemicals causing eye irritation as GHS Category 2.The validation(1)was focused on validating the method for identifying chemicals that do not require classification,as a bottom-up test method.3.With the very high over predi

40、ction rate of GHS category 2 materials predicted as GHS Category 1,it is clear that this test method is not particularly suited for discriminating between GHS category 1 and category 2 materials.Nonetheless,there is considerable confidence in a category 2 prediction,given the very low under predicti

41、on of materials as non-classified.4.It is not clear from the available documentation whether criteria for accuracy,sensitivity and specificity for predicting category 2 materials have been prospectively defined.In the Table 2 of the draft Appendix 2,the performance of the OptiSafeTM test(presented w

42、ith a 3 x 3 matrix)shows that for the 21 chemicals identified as category 2,four of the chemicals were category 1 materials that were under predicted,resulting in approximately 19%of the Cat.1 chemicals being identified as Cat.2,which in the absence of clear criteria for acceptance could readily be

43、considered unacceptable from a regulatory safety perspective.5.For the reasons described above and considering the absence of previously agreed and established acceptance criteria for evaluating a methods capacity to predict all three UNGHS eye hazard categories,the PRP suggests removing the methods

44、 protocol and performance regarding Category 2 prediction and update the draft Appendix 2 accordingly.6.The PRP also made a few comments on the methods usage within a weight of the evidence(WoE)approach and on the applicability domain.It would be useful to include details and references on the mecha

45、nistic background of the method to help assessors to better understand how to use the results within a WoE approach,when to expect false-positive and false-negative results as well as high variability(i.e.,usefulness and limitations as a result in a WoE assessment).7.It would also be useful to add c

46、larity on the chemicals resulting in“CNM”and being excluded from the applicability domain(i.e.,which substances,chemical groups,pH,and physico-chemical properties are outside of the applicability domain of the method).8.The PRP recommends deleting the following sections to avoid confusion:o Improvem

47、ents made to the OptiSafeTM method for when validation was conducted(4.,page 1)8|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 o Prediction models that go beyond the UN GHS

48、 categories,as these are not in scope for validation(5.,page 2 and 8.,page 3 and table 10 and 11,page 6)5.Evaluation Principle 2.Need and benefits in comparison to existing test methods Charge Question 2:Were the need and benefits in comparison to existing test methods appropriately addressed in ter

49、ms of(regulatory)purpose,IP rights,geographical availability,animal welfare,cost,analysis time,sample amount and other relevant aspects?9.Overall,the PRP considers that there are needs and benefits of OptiSafeTM in comparison to existing test methods.10.The OptiSafeTM test method shares its founding

50、 lineage with the validated reference method(VRM)and as such has a very similar biochemical basis for evaluation of eye irritation as the VRM.However,improvements to the OptiSafeTM test method provide an expansion on the applicability domain,and address some of the limitations of the VRM.Otherwise t

51、he test methods presumably would be available in similar markets globally,and provide similar benefits in terms of animal welfare considerations,costs,expertise and equipment required in a laboratory.As both test methods are based on similar biochemical modelling in a macromolecular matrix,both have

52、 limited mechanistic relevance to ocular injury in comparison to cell-based or ex vivo ocular irritation test methods.11.VRM for TG 496 can be used to identify UN GHS Cat.1 and GHS No Cat as a stand-alone method,while the OptiSafeTM can be used to identify UN GHS No Cat as a stand-alone method.Moreo

53、ver,the OptiSafeTM shows an improved prediction performance for UN GHS Cat.2 and Cat.1 chemicals,which can help to increase robustness of WoE assessments when using positive results from the OptiSafeTM in addition to other information and/or testing to substantiate UN GHS Cat.1 or Cat.2 classificati

54、ons.Additionally,the OptiSafeTM is an improved version of VRM in terms of biological and human relevance(ascorbic acid as antioxidant to mimic tear fluid).12.In terms of IP rights,it is not clear whether a patent has been filed for reagents and/or kit required for the OptiSafeTM.If OptiSafeTM can be

55、 used only with the kit(as it is currently written in the SOP),then the method becomes obsolete,once the kit is not commercially available anymore.There was also no information on availability,costs,analysis time,sample amount,etc.|9 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE

56、496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 6.Evaluation Principle 3.Comparison of the test method with the essential test method components and performances as described in the Performance Standards for TG 496 Charge Question 3:Are the

57、 essential test method components,the performance,and the accuracy and reliability values of the OptiSafeTM relevant to the Performance Standards for TG 496?In case of deviations from the Performance Standards,please describe your assessment of such deviations in considering OptiSafe as a me-too met

58、hod(i.e.,whether such deviations affect the merits of OptiSafe as a me-too method for TG 496).13.Overall,the PRP considers the OptiSafeTM test method relevant as a me-too method for TG 496.14.The proposed application of the OptiSafeTM test method differs from the VRM,which is validated and approved

59、to identify UN GHS Cat.1 and No Cat substances.Thus it is not clear that the OptiSafeTM test method fully falls within a me-too method characterizations.Accordingly,evaluations to determine whether a me-too method performs the same as or performs better than the VRM may not be fully possible,in part

60、icular in discriminating between UN GHS Cat.1 and Cat.2 substances.15.The surfactants(Sodium lauryl sulfate(3%)and Sodium lauryl glucose carboxylate(and)lauryl glucoside)as well as the alkylating agent Sodium chloroacetate seem to be overpredicted in the OptiSafeTM.However,data is very limited to co

61、nclude on chemical groups that may be outside of the applicability domain of the method.16.Nonetheless,the OptiSafeTM test method would indeed fit well within TG 496,particularly given that this test method is mechanistically the same as the VRM,but provides additional latitude in the applicability

62、domain and addressing limitations.The community should always expect that technological improvements and innovations from otherwise similar test methods come about which meet the same regulatory requirements but perhaps with higher performance characteristics.17.Based on the findings reported for th

63、e accuracy,sensitivity and specificity of the OptiSafeTM test method,as well as the inter-and intra-laboratory reproducibility,the OptiSafeTM test method would certainly be considered to perform as well as the VRM for identifying UN GHS Cat.1 and No Cat substances,and thus appear to meet or exceed t

64、he criteria established for the me-too method.10|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 7.Evaluation Principle 4.Biological and mechanistic relevance Charge Question

65、 4:Are the toxicological mechanism and the relationship between the test method endpoint(s)with the biological effect as well as the toxicity of interest adequately addressed?18.Overall,the PRP considers that the biological and mechanistic relevance of OptiSafeTM test method has similar limitations

66、as the VRM in TG 496.19.The test method provides a biochemically analogous endpoint of increase in turbidity and light scattering by irritant test chemicals in a plant-based matrix of proteins,lipids,carbohydrates and“low molecular weight components”;presumably modelling the denaturation of corneal

67、proteins and solubilization of membrane lipids,etc.The test method validation publication(1)and the Performance Standards document for TG 496(3)provide a reasonable description of the test system and the biological and mechanistic relevance of in vitro macromolecular test method.The draft Appendix 2

68、 for OptiSafeTM does not provide any further information and does not appear to be warranted.20.Whereas these biochemical events are reasonably modelled,much as a molecular initiating event might be envisioned for ocular and conjunctival injuries,it is not at all clear how this uniform matrix can mo

69、del the complexity of the corneal epithelium,basal lamina,and corneal stroma in its ability to discriminate between reversible injuries typically limited to the corneal epithelium and upper stroma,and those that result in irreversible damage throughout the cornea and into the deep stroma(according t

70、o the Depth of Injury concept by Maurer and Jester(4).Whereas the test method presumably responds to a continuum of toxic potencies by irritant chemicals,the cellular events which define the depth and degree of injuries associated with ocular injuries and discriminate between moderate reversible inj

71、uries and those more aggressive injuries resulting in irreversible corneal damage are simply not modelled.The specific barrier properties of the corneal epithelial layers,the epithelial basal lamina,and the stroma all play key roles and how different chemicals permeate into the cornea,and this is si

72、mply not modelled in the homogenous macromolecular matrix.In addition,the key cellular events associated with corneal damage,particularly where relevant to moderate and severe ocular injuries,are cell lysis,cell stress signalling and cell death,none of which are modelled by any of the macromolecular

73、 test methods.21.As an added note,a more detailed mechanistic background of the method,especially with regards to ascorbic acid as antioxidant to mimic tear fluid,could be described in the draft Appendix 2 to help assessors to better understand how to use the results within a WoE approach to establi

74、sh a final UN GHS classification and to ensure that the applicability domain is well defined for OptiSafeTM.|11 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 8.Evaluation P

75、rinciple 5.Test Method Protocol Charge Question 5:Is a detailed protocol for the test method(Standard Operating Procedure,or SOP)available?22.Overall,the PRP considers that the detailed protocol for OptiSafeTM is available.23.The test method developer has provided a highly detailed protocol(Link)for

76、 review by the PRP,and this test method protocol appears sufficiently detailed to be able to accurately and precisely carry out the test method,presumably to a level that would support Good Laboratory Practice(GLP)compliance.The test method protocol provides details on the principle of the test,spec

77、ial handling procedures for specific chemicals,definition of the applicability of domain and considerations for limitations,a detailed list of components,reagents and equipment required,specific details to execute the work,and the data collection,data analyses,prediction models and reporting procedu

78、res.In summary the test method protocol requirement appears adequate.24.However,the SOP is not sufficiently summarized in the draft Appendix 2 and the SOP(same as the draft Appendix 2)requires some more clarity with regards to data interpretation and reporting.For example,the draft Appendix 2 should

79、 contain the criteria for PCHP mentioned in the SOP and the terminology for the 3 pre-tests should be aligned throughout the SOP and the draft Appendix 2.The SOP should be referenced throughout the draft Appendix 2 where needed.25.Additionally,the section in the SOP“Materials provided with the OptiS

80、afe Kit”are not descriptive enough to obtain the materials without purchasing the kit.If the kit is not commercially available anymore,then the method becomes obsolete.26.Finally,acceptance criteria are not very clear(different criteria for different stages)as well as the criteria to define the 5 di

81、lutions of the test chemicals and when are dilutions based on neat vs.diluted test chemicals(e.g.,Cetylpyridinium bromide,glycerol)12|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OE

82、CD 2024 9.Evaluation Principle 6.Appropriateness of the validation study management and conduct Charge Question 6:Was the validation study conducted according to the principles and criteria documented in pages 20-23 of the OECD Guidance Document 34?27.Overall,the PRP considers that the validation st

83、udy for OptiSafeTM was conducted according to the principles and criteria detailed in OECD Guidance Document 34.28.The prospective validation of the OptiSafeTM test method,as presented in the validation study report(1),overall follows the basic principles and criteria for test method validation,as d

84、escribed in OECD guidance document 34(3).Specifically the validation management team(VMT)was comprised of validation experts within NICEATM,who were charged with reviewing and approving the study design,work plan,test method protocol,study timeline,and deliverables.The VMT selected the chemicals for

85、 the validation study and coordinated the coding and distribution of the reference chemicals to the participating laboratories.The validation management team ensured that an initial training effort was executed and allowed for discussions among participating laboratory staff to occur only within the

86、 training phase.Thereafter,all communications by participant laboratories were required only to be directed to the VMT.During the testing phase,participant laboratories submitted data on a weekly basis to the VMT.At the conclusion of the study the VMT was responsible for data analyses and reporting

87、of the validation outcome.29.The VMT presented on the following aspects of the validation:a.the rationale for evaluating and ocular test method using a macromolecular matrix as a test system was presented,b.the relationship between the changes in the test system and relevance to the biochemical chan

88、ges in ocular irritation was presented,c.a brief overview of the test method protocol using validation was presented.The validation report indicates that the detailed protocol can be obtained from the lead laboratory,d.the intra-and inter-laboratory reproducibility of the test method on 30 chemicals

89、 from Phase 2 was reported,e.test method performance was evaluated predominantly on reference chemicals appropriately within the applicability domain,as well as a few known to be outside of the applicability domain,thus evaluating the pre-screen methodology,f.the reference chemicals included in vivo

90、 classifications in both UN GHS and EPA categorization schemes,g.although no claims of full GLP compliance were made,the VMT report stated that the testing was conducted in accordance with the principles of OECD GLP.Note that the quality assurance personnel from the lead laboratory conducted the dat

91、a audit and analyses from all of the participating laboratories.Although it is customary for data auditing to be conducted by independent personnel from the participating laboratories,often under contract by the VMT,according to the principles of OECD GLPs,the quality assurance personnel may be cons

92、idered sufficiently independent in their abilities to execute their duties,|13 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 h.the VMT has provided Excel sheets with the re

93、presentative data from the validation.30.The VMT report included a narrative on the progress of the validation study through the phases,with commentary on modifications made to the protocol procedures as a result of discrepancies identified after training,as well as a result of Phase I test results.

94、The VMT reported on the accuracy,sensitivity,specificity,false-negative and false-positive rates from the validation relative to the in vivo EPA and UN GHS categorization schemes.31.One short coming was that not all validation studies were performed in compliance with GLP Principles and intra-and in

95、ter-lab as well as intra-and inter-study variability should be more specific for the actual measurement(OD400 values)versus final classification outcome.This way,acceptance criteria can be defined with more clarity.In addition,the applicability domain needs to be defined in more detail(chemical resu

96、lting in“CNM”should be clearly defined as outside of the applicability domain).14|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 10.Evaluation Principle 7.All data supportin

97、g the assessment of the validity of the analysis should be available for expert review Charge Question 7:Do you consider that all the data supporting the assessment of the validity of analysis are easily available for expert review?32.Overall,the PRP considers that the data supporting the assessment

98、 of the validity needs clarifications.33.Data for control chemicals(QC1 and QC2)could not be found and data is not easily available for review.One data table with all raw data(versus data per sheet)would be preferred to review raw data,calculations,variability,and final classification results.The dr

99、aft Appendix 2 and SOP should explain in more detail the relevance of and acceptance criteria for“a True Negative Checks”and“Depth of Migration(DM)Results”.34.One reviewer posed the following question:a.In the EXCEL sheet OptiSafeTM Data Log_Performance_10.18.22.xlsx(Link),Linda Nguyen is often iden

100、tified as“QA”and for a few chemical analyses also identified as“Technician”(see PS No.3,Run2).Are these roles for Linda Nguyen to be understood as operating Technician and as independent Quality Assurance auditor?If so,it does not appear that the lead lab quality assurance personnel were truly indep

101、endent from the data generation and collection duties for work conducted in the lead lab.b.The test method submitter responded with an acceptable explanation that Nguyen did not perform as both Technician and QA on the same set of data and added that clarification to the data sheet will be made.35.A

102、dditionally,one of the Peer Review Panellists was not able to follow all the data through to the analyses and did not conduct any audits of the data and the analyses in the Excel data sheets.It is not clear how to relate the data from individual chemicals in the Excel sheets with the analysiss table

103、s in the validation report.For example,in Table 2,chemical no.3(2-Ethyl-1-hexanol)(CAS 104-76-7)is presented as having been tested in the inter-laboratory study,but that chemical does not seem to appear in the Excel sheets.|15 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON

104、IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 11.Additional Remarks Charge Question 8:Do you have any suggestions or remarks to share?36.Establishing acceptance criteria for test method performance at either extreme of the eye irritation continu

105、um(i.e.,in for either UN GHS Cat.1 or No Cat)has historically been achievable.However,establishing the acceptance criteria in 3 x 3 matrix to discriminate both between GHS Cat.1 and Cat.2,as well as between Cat.2 and No Cat has proven difficult,if for no other reasons than due to the variability of

106、the animal data historically used as reference for performance standards and the data interpretation procedures used to assign GHS categories.The OptiSafeTM test method statistically appears to do well by being highly sensitive in predicting UN GHS Cat.1 materials,as well as high accuracy in identif

107、ying No Cat,not unlike the VRM.As has historically been observed in other alternative test methods the accuracy in the middle ground(predicting UN GHS Cat.2 with high confidence)is notably reduced,as both considerable over-and under prediction rates occur frequently.In the opinion of one of the PRP

108、panellists,the OptiSafeTM test method is not likely to perform better than most other test methods given our assertion that the reference animal data are inherently variable.More importantly,the OptiSafeTM test method does not appear to adequately discriminate between UN GHS Cat.1 and Cat.2 material

109、s as evidenced by the high over-prediction rate of the UN GHS Cat.2 materials and considerable under-prediction of UN GHS Cat.1 materials,and in the absence of any biological relevance to the cellular based mechanisms involved in discriminating between reversible and irreversible ocular injuries in

110、vivo,the PRP would not consider this test method adequate as a stand-alone to identify UN GHS Cat.2 materials.37.One of the PRP panellists provided additional comments and inquiries for clarifications,as described below in paragraphs 47 53,38.As this is a relatively simple test method,the draft Appe

111、ndix 2 as well as the SOP should describe in detail to allow labs to establish this method without the need to contact the test method developer for details.39.Equations and illustrations would be helpful in the draft Appendix 2,if they deviate from the VRM(i.e.,Annex 2b and paragraph 18,respectivel

112、y)and the flow diagram from SOP would be helpful in the draft Appendix 2.40.Some further background why OD400 needs to be measured vs.OD405(VRM)would be helpful for labs when establishing the OptiSafeTM test method.Does this wavelength reflect the max absorbance of a specific ingredient in the OPtiS

113、afeTM reagent?41.When a neat chemical tests positive,but the 5 dilutions do not show any dose-response,should the chemical be considered as outside of the applicability domain?42.Would it be possible to predict different UN GHS classifications based on the 5 different dilutions applied per test chem

114、ical?Cetylpyridinium bromide is tested at different concentrations(6%,1%,0.1%)in accordance with the Proficiency Chemical data set.However,the OptiSafeTM requires 5 dilutions per test material and thus,the dilutions for Cetylpyridinium bromide 6%,1%,0.1%seem to overlap at least partly.Would it be po

115、ssible to define different UN GHS classifications based on the 5 dilutions?43.Some sections in the draft Appendix 2 and in the SOP(e.g.,the test report section about results(page 7)should be in line with the according section in the TG for the VRM,which is more detailed.16|PEER REVIEW REPORT ON INCL

116、USION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 12.Conclusions and recommendations 44.Overall,the PRP considers that the OptiSafeTM meets the standards performance for TG 496 to be considered as a me-

117、too method to the VRM,but suggests to revise the draft Annex 2 based on the recommendations made throughout the Peer Review Report contained in this document.45.The PRP does not consider that the objective and purpose of the test method is adequately described.The PRP does not consider that informat

118、ion on the OptiSafeTMs performance for predicting UN GHS Cat.2 substances is adequate,and recommends removing information on the model and performance metrics intended for predicting three UN GHS eye hazard categories 46.The PRP considers that there are needs and benefits of OptiSafeTM in comparison

119、 to existing test method.The PRP considers that OptiSafeTM provides additional latitude in the applicability domain and address limitations of the VRM.47.The PRP considers that the biological and mechanistic relevance of OptiSafeTM test method has similar limitations as the VRM in TG 496.48.The PRP

120、considers that the detailed protocol(SOP)for OptiSafeTM is available,with a suggestion to provide more detail on the sections for data reporting and interpretation,materials provided with an OptiSafeTM Kit,and for acceptance criteria.49.The PRP considers that the validation study for OptiSafeTM was

121、conducted according to the principles and criteria detailed in OECD Guidance Document 34,but whether the process was fully GLP compliant is not clear.50.The PRP considers the data supporting and assessment of the validity of the method adequate in general.However,the PRP would have preferred to have

122、 a data table with all raw data,calculations,variability,and final classification results in order to evaluate the linkage between the raw data and the final classifications.13.Acknowledgements The OECD Secretariat thanks the Peer Review Panel for their review and valuable discussions and comments.|

123、17 PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 14.Literature(1)Choksi N.,Lebrun S.,Nguyen M.,Daniel A.,DeGeorge G.,Willoughby J.,Layton A.,Lowther D.,Merrill J.,Matheson

124、J.,Barroso K.,Yozzo K.,Casey W.,Allen D.(2020).“Validation of the OptiSafe eye irritation test.”Cutan Ocul Toxicol.39(3):180192.doi:10.1080/15569527.2020.1787431 (2)OECD.(2019).“Test No.496:In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Re

125、quiring Classification for Eye Irritation or Serious Eye Damage.”OECD Guidelines for the Testing of Chemicals,Section 4,OECD Publishing,Paris.Available at:https:/doi.org/10.1787/970e5cd9-en.(3)OECD.(2005).“OECD Series on Testing and Assessment,Number 34.Guidance Document on the Validation and Intern

126、ational Acceptance of New or Updated Test Methods for Hazard Assessment.”Available at:https:/ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd-gd34.pdf.(4)Jester J.V.,Li H.F.,Petroll W.M.,Parker R.D.,Cavanaugh H.D.,Carr G.J.,Smith B.,Maurer J.K.(1998).Area and depth of surfactant-induced corneal i

127、njury correlates with cell death.Investigative Ophthalmology&Visual Science 39,922-936.(5)OECD.(2019).“OECD Series on Testing and Assessment No.312 Performance Standards for the assessment of proposed similar or modified in vitro macromolecular test method for identifying eye hazard potential a desc

128、ribed in Test Guideline 496.”Available at:https:/www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2019)32&docLanguage=en 18|PEER REVIEW REPORT ON INCLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL

129、OF CHEMICALS OECD 2024 Annex 1.Peer Review Panel Composition Hans Raabe Institute for In Vitro Sciences,Inc.U.S.Nora Krutz NV Procter&Gamble Services Company SA Belgium David Lehmann Environmental Protection Agency(EPA)U.S.Peer Review Manager Eugene Choi(OECD Secretariat)|19 PEER REVIEW REPORT ON IN

130、CLUSION OF OPTISAFE EIT IN TEST GUIDELINE 496 ON IN VITRO MACROMOLECULAR TEST METHODS FOR IDENTIFYING THE EYE HAZARD POTENTIAL OF CHEMICALS OECD 2024 Annex 2.Charge Questions for the Peer Review of OptiSafeTM Test Method as a me-too assay for TG 496 Charge Question 1:Were the study objective and the

131、 purpose of the test method adequately described?Charge Question 2:Were the need and benefits in comparison to existing test methods appropriately addressed in terms of(regulatory)purpose,IP rights,geographical availability,animal welfare,cost,analysis time,sample amount and other relevant aspects?C

132、harge Question 3:Are the essential test method components,the performance,and the accuracy and reliability values of the OPtiSafeTM relevant to the Performance Standards for TG 496?In case of deviations from the Performance Standards,please describe your assessment of such deviations in considering

133、OptiSafeTM as a me-too method.(i.e.,whether such deviations affect the merits of OptiSafeTM as a me-too method for TG496)Charge Question 4:Are the toxicological mechanism and the relationship between the test method endpoint(s)with the biological effect as well as the toxicity of interest adequately

134、 addressed?Charge Question 5:Is a detailed protocol for the test method available?Charge Question 6:Was the validation study conducted according to the principles and criteria documented in pages 20-23 of the OECD Guidance Document 34?Charge Question 7:Do you consider that all the data supporting the assessment of the validity of analysis are easily available for expert review?Charge Question 8:Do you have any suggestions or remarks to share?