ICCVAM Evaluation of the Murine Local Lymph Node Assay: I. The ICCVAM Review Process

New test methods are being developed to improve the prediction of human and environmental risks and to benefit animal welfare by reducing, refining, and replacing animal use. Regulatory adoption of new test methods is often a complex and protracted process, requiring test method validation, regulatory acceptance, and implementation. Assessments of new test methods have not always been uniform within or among regulatory agencies. Thus, there have been increased pressures for a harmonized approach to test method evaluation and acceptance. In 1997, in response to these pressures and to U.S. Public Law 103-43, the National Institute of Environmental Health Sciences (NIEHS) established the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to coordinate interagency consideration of new and revised test methods. This article describes the validation and acceptance criteria and process used for the first test method evaluated by ICCVAM, the murine local lymph node assay (LLNA). Based on ICCVAM's conclusions and recommendations, the LLNA has been accepted by U.S. regulatory agencies as a stand-alone assay for allergic contact dermatitis. Two related articles in this series of three present the results of the independent peer review evaluation of the LLNA and summarize the performance characteristics of the database substantiating the validity of the LLNA.     

Local lymph node assay - validation, conduct and use in practice.

The validation of alternative methods is a relatively new activity in toxicology. The local lymph node assay (LLNA), a novel method for the identification of chemicals that have the potential to cause skin sensitization, was the first test to pass through the formal regulatory validation process established in the USA under the auspices of ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods. ICCVAM approved the LLNA as an alternative to guinea pig tests for the identification of skin sensitisation hazards. In this report, we explore the nine recommendations made by ICCVAM and discuss their interpretation in relation to the new OECD Guideline 429, which describes the LLNA. In particular, the value and limitations of the use of statistical evaluation of data and of the inclusion of routine positive controls is examined. It is concluded that the OECD Guideline as currently written embodies the necessary flexibility to permit conduct of the LLNA in a manner necessary to meet the varying needs of regulatory agencies and toxicologists around the world.     

ICCVAM evaluation of the murine local lymph node assay. Conclusions and recommendations of an independent scientific peer review panel.

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose-response information and statistical analyses. A standardized LLNA protocol is provided.     

ICCVAM Evaluation of the Murine Local Lymph Node Assay: II. Conclusions and Recommendations of an Independent Scientific Peer Review Panel

The validation status of the murine local lymph node assay (LLNA), a method for assessing the allergic contact dermatitis potential of chemicals, was evaluated by an independent peer review panel (Panel) convened by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). The LLNA measures lymphocyte proliferation using incorporation of radioactive thymidine or iododeoxyuridine into cells of the draining lymph nodes of mice topically exposed to a test article. The Panel concluded that the assay performed as well as currently accepted guinea pig methods [guinea pig maximization test (GPMT)/Buehler assay (BA)] for the hazard identification of strong to moderate chemical sensitizing agents, but that it might not correctly identify all weak sensitizers or metals (potential false negative response) or all strong irritants (potential false positive response). The Panel concluded also that the LLNA involves less pain and distress than conventional guinea pig methods. The Panel unanimously recommended the LLNA as a stand-alone alternative for contact sensitization hazard assessment, provided that certain protocol modifications were made. These included collection of individual, rather than pooled, animal response data; the inclusion of a concurrent positive control; and consideration of dose–response information and statistical analyses. A standardized LLNA protocol is provided.     

Performance standards and alternative assays: Practical insights from skin sensitization

To encourage the development and validation of alternative toxicity test methods, the effort required for validation of test methods proposed for regulatory purposes should be minimized. Performance standards (PS) facilitate efficient validation by requiring limited testing. Based on the validated method, PS define accuracy and reliability values that must be met by the new similar test method. The OECD adopted internationally harmonized PS for evaluating new endpoint versions of the local lymph node assay (LLNA). However, in the process of evaluating a lymph node cell count alternative (LNCC), simultaneous conduct of the regulatory LLNA showed that this standard test may not always perform in perfect accord with its own PS. The LNCC results were similar to the concurrent LLNA. Discrepancies between PS, LLNA and LNCC were largely associated with “borderline” substances and the variability of both endpoints. Two key lessons were learned: firstly, the understandable focus on substances close to the hazard classification borderline are more likely to emphasise issues of biological variability, which should be taken into account during the evaluation of results; secondly, variability in the results for the standard assay should be considered when selecting reference chemicals for PS.     

Prediction of skin sensitization potency using machine learning approaches

The replacement of animal use in testing for regulatory classification of skin sensitizers is a priority for US federal agencies that use data from such testing. Machine learning models that classify substances as sensitizers or non‐sensitizers without using animal data have been developed and evaluated. Because some regulatory agencies require that sensitizers be further classified into potency categories, we developed statistical models to predict skin sensitization potency for murine local lymph node assay (LLNA) and human outcomes. Input variables for our models included six physicochemical properties and data from three non‐animal test methods: direct peptide reactivity assay; human cell line activation test; and KeratinoSens™ assay. Models were built to predict three potency categories using four machine learning approaches and were validated using external test sets and leave‐one‐out cross‐validation. A one‐tiered strategy modeled all three categories of response together while a two‐tiered strategy modeled sensitizer/non‐sensitizer responses and then classified the sensitizers as strong or weak sensitizers. The two‐tiered model using the support vector machine with all assay and physicochemical data inputs provided the best performance, yielding accuracy of 88% for prediction of LLNA outcomes (120 substances) and 81% for prediction of human test outcomes (87 substances). The best one‐tiered model predicted LLNA outcomes with 78% accuracy and human outcomes with 75% accuracy. By comparison, the LLNA predicts human potency categories with 69% accuracy (60 of 87 substances correctly categorized). These results suggest that computational models using non‐animal methods may provide valuable information for assessing skin sensitization potency. Copyright © 2017 John Wiley & Sons, Ltd.     

Integrated decision strategies for skin sensitization hazard

One of the top priorities of the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) is the identification and evaluation of non‐animal alternatives for skin sensitization testing. Although skin sensitization is a complex process, the key biological events of the process have been well characterized in an adverse outcome pathway (AOP) proposed by the Organisation for Economic Co‐operation and Development (OECD). Accordingly, ICCVAM is working to develop integrated decision strategies based on the AOP using in vitro, in chemico and in silico information. Data were compiled for 120 substances tested in the murine local lymph node assay (LLNA), direct peptide reactivity assay (DPRA), human cell line activation test (h‐CLAT) and KeratinoSens assay. Data for six physicochemical properties, which may affect skin penetration, were also collected, and skin sensitization read‐across predictions were performed using OECD QSAR Toolbox. All data were combined into a variety of potential integrated decision strategies to predict LLNA outcomes using a training set of 94 substances and an external test set of 26 substances. Fifty‐four models were built using multiple combinations of machine learning approaches and predictor variables. The seven models with the highest accuracy (89–96% for the test set and 96–99% for the training set) for predicting LLNA outcomes used a support vector machine (SVM) approach with different combinations of predictor variables. The performance statistics of the SVM models were higher than any of the non‐animal tests alone and higher than simple test battery approaches using these methods. These data suggest that computational approaches are promising tools to effectively integrate data sources to identify potential skin sensitizers without animal testing. Published 2016. This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

Report of an ISRTP workshop: progress and barriers to incorporating alternative toxicological methods in the U.S

The workshop objectives were to explore progress in implementing new, revised and alternative toxicological test methods across regulatory evaluation frameworks and decision-making programs in the United States, to identify barriers and to develop recommendations to further promote adoption of approaches that reduce, refine, or replace the use of animal methods. The workshop included sessions on: (1) current research, development, and validation of alternative methods within the U.S. federal government; (2) emerging alternative methodologies with potential applications to a broad spectrum of toxicity evaluation strategies; (3) tiered evaluation ("intelligent testing") strategies; and (4) identification of, and recommendations to address, critical barriers that affect adoption and use of new, revised alternative toxicological test methods by U.S. regulatory agencies. Through facilitated discussion, a list of barriers and recommendations were developed and grouped into categories of economic/financial, scientific/technical, and regulatory/policy. Overall, participants from all sectors collectively supported catalyzing actions to promote more meaningful and rapid progress for research to develop alternative methods focused for use in regulatory programs, accelerated lab investigations to validate such alternative methods and adoption of regulatory frameworks which embrace and incorporate these validated alternatives.     

ICCVAM evaluation of the murine local lymph node assay. Data analyses completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods.

To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.     

ICCVAM Evaluation of the Murine Local Lymph Node Assay: III. Data Analyses Completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods

To evaluate the reliability of the murine local lymph node assay (LLNA), a test for allergic contact dermatitis activity, the inter- and intralaboratory consistency statistics (h and k, respectively) were calculated for validation studies testing multiple chemicals. The analysis indicated the absence of excessive variability in the dose calculated to induce a threefold or greater increase in the stimulation index (SI). To assess the appropriateness of using an SI of 3 as the decision criteria for identifying a sensitizing compound, LLNA results based on SI values of 2.0, 2.5, 3.0, 3.5, and 4.0 were compared with guinea pig or human results. The results supported the use of an SI of 3 as the decision criteria. Assay performance was determined by comparing LLNA results to results obtained for guinea pigs or humans. The accuracy of the LLNA was 89% when compared with results from the guinea pig maximization test (GPMT)/Buehler assay (BA). The performance of the LLNA and the GPMT/BA was similar when each was compared to human maximization test results plus substances included as human patch test allergens. The LLNA offered advantages over the GPMT in respect to both the time required to conduct the test and the assay cost.     

LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.     

Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants

The Local Lymph Node Assay (LLNA) is the preferred test for the identification of skin-sensitizing potentials of chemicals in Europe and is also the first choice method within REACH. In the formal validation, only a very few surfactant chemicals were evaluated and SDS was identified as a false positive. In this study, 10 nonionic sugar lipid surfactants were tested in an LLNA, guinea pig maximization test (GPMT) and human repeated insult patch test. Of the 10 surfactants tested in the LLNA, 5 showed stimulation indices above 3.0. Three of five positive reactions were concomitant with signs of skin irritation indicated by an increase in ear thickness. In the GPMT, all test products were classified as nonsensitizers. In human volunteers, no skin reactions suggestive of sensitization were reported. In conclusion, these results are indicative of the LLNA overestimating sensitization potentials for this category of chemicals. This may in part be due to irritant effects generated by these surfactants. Until suitable nonanimal alternative tests obtain regulatory acceptance, use of other tests, e.g. GPMTs, may in cases be justified. Results such as these need be taken into account when developing nonanimal alternative methods to ensure reliable data sets for method validation purposes.     

LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.     

United States regulatory requirements for skin and eye irritation testing

Purpose: Eye and skin irritation test data are required or considered by chemical regulation authorities in the United States to develop product hazard labelling and/or to assess risks for exposure to skin- and eye-irritating chemicals. The combination of animal welfare concerns and interest in implementing methods with greater human relevance has led to the development of non-animal skin- and eye-irritation test methods. To identify opportunities for regulatory uses of non-animal replacements for skin and eye irritation tests, the needs and uses for these types of test data at U.S. regulatory and research agencies must first be clarified.

Methods: We surveyed regulatory and non-regulatory testing needs of U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) agencies for skin and eye irritation testing data. Information reviewed includes the type of skin and eye irritation data required by each agency and the associated decision context: hazard classification, potency classification, or risk assessment; the preferred tests; and whether alternative or non-animal tests are acceptable. Information on the specific information needed from non-animal test methods also was collected.

Results: A common theme across U.S. agencies is the willingness to consider non-animal or alternative test methods. Sponsors are encouraged to consult with the relevant agency in designing their testing program to discuss the use and acceptance of alternative methods for local skin and eye irritation testing.

Conclusions: To advance the implementation of alternative testing methods, a dialog on the confidence of these methods to protect public health and the environment must be undertaken at all levels.     

The local lymph node assay in practice: a current regulatory perspective

Following the formal acceptance of the local lymph node assay (LLNA) as an Organization for Economic Cooperation and Development (OECD) guideline in April 2002, the UK Health and Safety Executive (HSE) informed notifiers that this was now the method of choice for the assessment of skin sensitization potential under the EU notification scheme for new industrial chemicals (NONS). This paper summarizes the experience of the HSE for the 2-year period immediately following the issuing of this statement, during which 48 LLNA study reports were assessed for notification purposes. The issues discussed here include adherence to the OECD guideline, interpretation of results, and classification outcomes. Generally, notifying laboratories followed the OECD guideline successfully, with regard to the sex/ strain/numbers of mice used, the precise process used for measurement of cell proliferation, and the use of recommended vehicles and positive controls. Initially, use of the individual animal approach (measuring the cell proliferation in each animal rather than for a pooled dose group) highlighted problems caused by technical inexperience, but these were overcome by practice. Toxicity or irritation were found to be minor factors in dose selection; more important was the choice of vehicle to correctly maximize the test substance concentration, while maintaining appropriate application properties. Contrary to concerns that the LLNA would prove to be less sensitive or more sensitive than the traditionally used Guinea Pig Maximization Test (GPMT), the proportion of new substances classified as skin sensitizers was within the range observed in previous years. Although the sample size is relatively small, the experience of the HSE indicates that the LLNA is satisfactory for routine regulatory use.     

Strategies for identifying false positive responses in predictive skin sensitization tests

It is important that predictive toxicological test methods are selective for their intended endpoint and that their limitations are understood and acknowledged. The local lymph node assay (LLNA) is a relatively new predictive test for skin sensitization potential that can replace traditional guinea pig tests and offers significant scientific and animal welfare advantages. However, there has been some concern that certain irritant materials may yield false positive results, although it must be emphasized that false positives also occur in guinea pig methods. Consequently, we have examined the performance in the LLNA of a range of skin irritants, from varying chemical classes and covering a range of irritation potency. The results presented here demonstrate clearly that the majority of skin irritants are negative in the LLNA. These results are reviewed in the context of the occurrence of false positive reactions in the guinea pig maximization test and the strategies for dealing with such results are discussed. The need for careful scientific evaluation of the results in all predictive tests for sensitization is thus emphasized. In terms of specificity, the LLNA has been more fully evaluated than other predictive test methods and is at least as accurate. In terms of animal welfare, objectivity, reproducibility and reliability it is superior to other methods. In summary, all predictive skin sensitization test results should be evaluated in a scientifically rigorous manner and the additional data provided herein further support the adoption of the LLNA as a complete replacement for the traditional guinea pig methods.     

Lessons learned from validation of in vitro toxicity test: from failure to acceptance into regulatory practice.

As no scientific approach or regulatory guidelines existed for the experimental validation of in vitro toxicity tests, in 1990 a US/European validation workshop agreed in Amden (Switzerland) on a simple definition of the validation process. Several international validation studies failed, although they were conducted according to these recommendations. Taking into account the lessons learned from this experience, a second validation workshop was held by ECVAM in Amden in 1994 to develop a more precisely defined validation concept. Prevalidation and the development of biostatistically defined prediction models were added as essential elements to the validation process. In 1995/1996 the ECVAM validation procedure was officially accepted by EU member countries and at the international level by the US regulatory agencies and the OECD. The improved validation concept was immediately introduced into ongoing validation studies. In 1996 the ECVAM/COLIPA validation study of the in vitro phototoxicity test, which was conducted according to the ECVAM/OECD validation concept, was finished successfully and in 1998 a supporting study on UV-filter chemicals was undertaken. In 1998 the 3T3 NRU PT in vitro phototoxicity test was the first experimentally validated in vitro toxicity test that was recommended for regulatory purposes by ESAC, the ECVAM Scientific Advisory Committee, and by the DG ENV of the EU Commission. Meanwhile, two in vitro skin corrosivity tests have successfully been validated by ECVAM. Finally, in June 2000 the three experimentally validated tests were accepted by EU member states for regulatory purposes as the first in vitro toxicity tests. In addition, ECVAM has funded a successful validation study of three in vitro embryotoxicity tests, which was conducted in 12 European laboratories and finished in July 2000. The three tests validated in this study were the whole embryo culture (WEC) test applied to rat embryos, the micromass (MM) test employing primary cultures of dissociated mouse limb bud cells and the mouse embryonic stem cell test (EST). Examples will be given of successful validation studies during the past decade with particular reference to in vitro toxicity tests that were evaluated for regulatory purposes either by the US validation centre ICCVAM or ECVAM in the fields of sensitisation, phototoxicity and embryotoxicity     

Non-animal methods to predict skin sensitization (I): the Cosmetics Europe database*

Cosmetics Europe, the European Trade Association for the cosmetics and personal care industry, is conducting a multi-phase program to develop regulatory accepted, animal-free testing strategies enabling the cosmetics industry to conduct safety assessments. Based on a systematic evaluation of test methods for skin sensitization, five non-animal test methods (DPRA (Direct Peptide Reactivity Assay), KeratinoSens^TM, h-CLAT (human cell line activation test), U-SENS^TM, SENS-IS) were selected for inclusion in a comprehensive database of 128 substances. Existing data were compiled and completed with newly generated data, the latter amounting to one-third of all data. The database was complemented with human and local lymph node assay (LLNA) reference data, physicochemical properties and use categories, and thoroughly curated. Focused on the availability of human data, the substance selection resulted nevertheless resulted in a high diversity of chemistries in terms of physico-chemical property ranges and use categories. Predictivities of skin sensitization potential and potency, where applicable, were calculated for the LLNA as compared to human data and for the individual test methods compared to both human and LLNA reference data. In addition, various aspects of applicability of the test methods were analyzed. Due to its high level of curation, comprehensiveness, and completeness, we propose our database as a point of reference for the evaluation and development of testing strategies, as done for example in the associated work of Kleinstreuer et al. We encourage the community to use it to meet the challenge of conducting skin sensitization safety assessment without generating new animal data.     

Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine (BrdU) incorporation

Allergic contact dermatitis is a serious health problem. Over the last decade, the murine local lymph node assay (LLNA) has been developed to detect chemical allergens, and international validation studies have been conducted. We have tried to establish an alternative non-radioisotopic endpoint for the LLNA by using 5-bromo-2'-deoxyuridine (BrdU) incorporation in place of radioisotopes, such as [3H]thymidine, employed in the standard method. BrdU was given as a single administration at 5 mg/animal 2 days following three consecutive daily applications of a test chemical. BrdU incorporation into draining lymph node cells was measured using an enzyme immunosorbent assay technique. In this study, p-benzoquinone(PBQ), trimellitic anhydride (TMA), citral(CT) and dextran (DEX) were used as pilot chemicals. PBQ, TMA and CT, which are classified as moderate to strong sensitizers in the guinea pig maximization test and were positive in the original LLNA, were also found to elicit positive responses in the alternative LLNA using BrdU incorporation. In contrast, DEX tested negative in the modified assay consistent with previous guinea pig and LLNA data. Consequently, the modified LLNA endpoint using BrdU incorporation may represent a useful alternative to the standard assay in situations, where there is a need to avoid the use of radioisotopes.