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.     

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.     

Assessment of the skin sensitization potency of eugenol and its dimers using a non-radioisotopic modification of the local lymph node assay

Allergic contact dermatitis is a serious health problem. There is a need to identify and characterize skin sensitization hazards, particularly with respect to relative potency, so that accurate risk assessments can be developed. For these purposes the murine local lymph node assay (LLNA) was developed. Here, we have investigated further a modi fi cation of this assay, non-radioisotopic LLNA, which in place of tritiated thymidine to measure lymph node cell proliferation employs incorporation of 5-bromo-2'-deoxyuridine. Using this method we have examined the skin sensitizing activity of eugenol, a known human contact allergen, and its dimers 2,2'-dihydroxyl-3,3'-dimethoxy-5,5'-diallyl-biphenyl (DHEA) and 4,5'-diallyl-2'-hydroxy-2,3'-dimethoxy phenyl ether (DHEB). Activity in the guinea pig maximization test (GPMT) also measured. On the basis of GPMT assays, eugenol was classified as a mild skin sensitizer, DHEA as a weak skin sensitizer and DHEB as an extreme skin sensitizer. In the non-radioisotopic LLNA all chemicals were found to give positive responses insofar as each was able to provoke a stimulation index (SI) of >or=3 at one or more test concentrations. The relative skin sensitizing potency of these chemicals was evaluated in the non-radioisotopic LLNA by derivation of an ec(3) value (the concentration of chemical required to provoke an SI of 3). The ec(3) values calculated were 25.1% for eugenol, >30% for DHEA and 2.3% for DHEB. Collectively these data suggest that assessments of relative potency deriving from non-radioisotopic LLNA responses correlate well with evaluations based on GPMT results. These investigations provide support for the proposal that the non-radioisotopic LLNA may serve as an effective alternative to the GPMT where there is a need to avoid the use of radioisotopes.     

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.     

Quantitative comparison of the results obtained by the multiple-dose guinea pig maximization test and the non-radioactive murine local lymph-node assay for various biocides

We compared the results of the multiple-dose guinea pig maximization test (GPMT) and the non-radioactive murine local lymph-node assay (LLNA) for various biocides. Thirteen out of 17 positive biocides in the GPMT gave positive results in the LLNA. In the GPMT, the minimum first induction doses ranged over four orders (0.00005-0.5%), while elicitation-threshold doses, which were evaluated using an optimally sensitized group of animals in the multiple-dose studies, ranged over five orders (0.00006-2.8%). In the LLNA, minimum induction doses ranged over more than three orders (0.01-30%). With respect to 13 biocides that were positive in both the GPMT and the LLNA, results were quantitatively compared. When compared after conversion to corresponding area doses (microg/cm), the minimum doses required to elicit skin reaction in guinea pigs were always lower than that for induction in mice with all biocides. Correlation between minimum induction doses from the GPMT and the LLNA seemed poor (r=0.57), while that between minimum induction doses in the LLNA and elicitation-threshold doses in the GPMT was relatively good (r=0.73). The results suggest the possibility to estimate human elicitation-threshold doses, which are definitely lacking in the process of risk assessment for skin-sensitizers, from the data of the LLNA.     

The local lymph node assay: results of a final inter-laboratory validation under field conditions.

The local lymph node assay (LLNA) assesses the sensitizing activity of chemicals by measurement of primary lymphocyte proliferation in lymph nodes draining the site of application. In this final inter-laboratory study the consistency of LLNA results between laboratories and with guinea pig maximization test (GPMT) data was examined under 'field' conditions. Nine chemicals were evaluated independently by each laboratory according to guidelines for test concentration and vehicle selection developed during previous validation studies to ensure assay optimization. Equivalent predictions of sensitization potential were obtained by all laboratories for eight chemicals. Five of seven chemicals identified as sensitizers in the GPMT were correctly identified in the LLNA--four by all laboratories and 1 (4-chloroaniline) by one laboratory only--although in this latter case, two other laboratories obtained clear dose responses, suggestive of sensitization. The LLNA identified correctly those chemicals predicted to be extreme or strong sensitizers in the GPMT. The remaining two chemicals were non-sensitizers in the guinea pig and failed to elicit positive proliferative responses in the LLNA. These data demonstrate that sensitivity and reliability of the LLNA is retained when chemicals are evaluated independently, and that it provides a reliable pre-screen for the identification of chemicals with significant sensitization potential.     

Inter‐laboratory validation of the modified murine local lymph node assay based on 5‐bromo‐2′‐deoxyuridine incorporation

The murine local lymph node assay (LLNA) is a well‐established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. ( 2001 ) has developed a modified LLNA based on the 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). The LLNA:BrdU‐ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical‐treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut‐off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU‐ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens.

The guinea-pig maximization test (GMPT) has been in use as a method for the prediction of skin sensitization potential for over 20 years, and is widely accepted by regulatory authorities because of its reliable detection of a wide variety of potential human contact allergens. Nevertheless, the method has some limitations and drawbacks, including the use of an adjuvant, the injection of the test substance at induction thus bypassing the normal skin barrier and metabolic function, a subjective endpoint, interference by irritant and/or coloured chemicals, and a relatively long and complex protocol. To address these points, an alternative technique, the local lymph node assay (LLNA), has been proposed and has become the focus of much attention. Recent data from interlaboratory trials have shown a good level of agreement between test facilities and with existing guinea-pig data. The present work investigated the correlation between LLNA results and those derived from the GPMT for 40 chemicals covering a range of chemical types and levels of skin sensitization potential. The LLNA assay was capable of detecting chemicals that exhibit a strong sensitization potential in the GPMT. For chemicals classified as moderate sensitizers in the GPMT, the LLNA was usually positive or provided an indication of sensitizing activity (that was not sufficient to satisfy the current criteria for regarding the result as positive). Weaker sensitizers in the GPMT were usually not detected by the LLNA. With the single exception of copper chloride, non-sensitizers were not positive in the LLNA. The results support the view that the LLNA can provide a rapid and objective screening test for strong sensitizers.     

An assessment of the skin sensitisation hazard of a group of polyfunctional silicones using a weight of evidence approach

Discordant results were observed when testing five prototype polyfunctional silicone materials for skin sensitization potential in the murine local lymph node assay (LLNA) and in the guinea pig maximization test (GPMT). While all five silicone materials were consistently negative in the GPMT, the testing in the LLNA revealed weak to moderate skin sensitisation potential for four of the five test materials. Neither study quality nor other known chemical factors could explain these findings. Further analysis did not provide sufficient evidence for a link between the LLNA responses and the irritancy of the test substances. Only in the case of one of the test materials, the occurrence of an excessive level of irritation could be linked to the positive LLNA result. Considering all existing information including physico–chemical and structure activity and animal data as well as existing human experience from silicone exposures at the workplace or their use in cosmetic products, the weight of evidence suggests that none of the examined silicone materials represents a significant skin sensitization hazard to humans. The suitability of the LLNA appears questionable for this class of materials. In case of any additional data needs for other or new silicone materials, the skin sensitization testing strategy will require careful evaluation and will need to be set up on a case by case basis.     

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.     

Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 3. Apparent discrepancies between LLNA and GPMT sensitisation potential: False positives or differences in sensitivity?

The Local Lymph Node Assay (LLNA) is the gold standard regulatory toxicology test for skin sensitisation along with the guinea pig maximisation test (GPMT). Compared with the GPMT, LLNA uses fewer animals, it is quantitative, and it gives a numerical prediction of potency. However several concerns have been raised with this assay, mainly related to false positives and false negatives. Over the years, many authors, including the developers of the assay, have presented cases where there have been discrepancies between the GMPT and LLNA results. Several theories have been put forward for these discrepancies, the main one being the “over-sensitivity” of the GPMT. This paper analyses the data from a systematic study, published in three papers from 2008 to 2011, covering several classes of chemicals, in particular unsaturated fatty acids, sugar surfactants and ethoxylated alcohols, with many cases of chemicals testing positive in the LLNA being negative in the GPMT. Based on consideration of reaction chemistry and structural alerts, it is concluded that these discrepancies are not LLNA false positives, but can be rationalised in terms of the different protocols of the assays.     

Allergenicity evaluation of p-chloro-m-cresol and p-chloro-m-xylenol by non-radioactive murine local lymph-node assay and multiple-dose guinea pig maximization test

p-Chloro-m-cresol (PCMC) and p-chloro-m-xylenol (PCMX) are known to cause allergic contact dermatitis. For risk assessment of skin sensitizers, information on dose–response profiles in the induction and elicitation phases and cross-reactivity with analogous chemicals are important. In the non-radioactive local lymph-node assay (LLNA) using 5-bromo-2′-deoxyuridine instead of ³H-methyl thymidine, significant effect on lymph node cell proliferation was detected at 10% PCMC and 25% PCMX, while in the multiple-dose guinea pig maximization test (GPMT) at least one animal tested in the group was sensitized at a 5 ppm induction dose of either chemical. When mean skin reaction score in an animal group maximally sensitized with each allergen with the GPMT was plotted against log challenge concentration, linear regression lines with high correlations were obtained in both cases. The calculated elicitation threshold was lower for PCMC than PCMX. The area under the linear regression line between the threshold point and 1% of the elicitation concentration, another index of relative elicitation potency, was also greater for PCMC. Bidirectional cross-reactivity between PCMX and PCMC was detected in the GPMT. PCMC was thus identified in both LLNA and GPMT as a stronger sensitizer than PCMX in both the induction and elicitation phases. These results suggest that the non-radioactive LLNA is a simple and useful method for evaluating allergenicity in the induction phase, while the GPMT using a maximally sensitized animal group is more suitable for assessing the dose–response profile and cross-reactivity in the elicitation phase.     

Skin sensitization testing in potency and risk assessment.

The purpose of this article is to review, and make recommendations for, the use of relevant skin sensitization test methods, for the purposes of determination of relative potency and the threshold dose necessary for the induction of skin sensitization, and for risk assessment. In addressing the first area, the utility of three guinea pig tests (the guinea pig maximization test, the occluded patch test, and the open epicutaneous test) of the local lymph node assay (LLNA) and of human volunteer testing for the assessment of relative potency and identification of thresholds for sensitization were considered. The following conclusions were drawn. (1) Although attempts have been made to modify the guinea pig maximization test for the purposes of deriving dose-response relationships, this method is usually unsuitable for determination of relative sensitizing potency. (2) Guinea pig methods that do not require the use of adjuvant and which employ a relevant route of exposure (the occluded patch test and the open epicutaneous test) are more appropriate for the assessment of relative skin-sensitizing potency. (3) The LLNA is suitable for the determination of relative skin sensitizing potency, and the adaptation of this method for derivation of comparative criteria such as EC3 values (the estimated concentration of test chemical required to induce a stimulation index of 3 in the LLNA) provides an effective and quantitative basis for such measurements. (4) For all the methods identified above, potency is assessed relative to other chemical allergens of known skin sensitizing potential. The estimation of likely threshold concentrations is dependent upon the availability of suitable benchmark chemicals of known potency for human sensitization. (5) Human testing (and specifically, the Human Repeat Insult Patch Test) can provide information of value in confirming the absence of skin sensitizing activity of formulations and products under specific conditions of use and exposure. Based on the above, the following recommendations are made. (1) If results are already available from suitable guinea pig tests, then judicious interpretation of the data may provide information of value in assessing relative skin sensitizing potency. This option should be explored before other analyses are conducted. (2) The LLNA is the recommended method for new assessments of relative potency, and/or for the investigation of the influence of vehicle or formulation on skin sensitizing potency. (3) Whenever available, human skin sensitization data should be incorporated into an assessment of relative potency. With respect to risk assessment, the conclusion drawn is that all the available data on skin-sensitizing activity in animals and man should be integrated into the risk-assessment process. Appropriate interpretation of existing data from suitable guinea pig studies can provide valuable information with respect to potency, as the first step in the development of a risk assessment. However, for de novo investigations, the LLNA is the method favored for providing quantitative estimations of skin-sensitizing potency that are best suited to the risk assessment process. Finally, human testing is of value in the risk assessment process, but is performed only for the purposes of confirming product safety.     

Skin sensitization,false positives and false negatives: experience with guinea pig assays

The advent of the local lymph node assay (LLNA), and efforts to develop in vitro alternatives for the identification of skin sensitizing chemicals has focused attention on the issue of false positive and false negative results. In essence, the question becomes ‘what is the gold standard?’ In this context, attention has focused primarily on the LLNA as this is now the preferred assay for skin sensitization testing. However, for many years prior to introduction of the LLNA, the guinea pig maximization test and the occluded patch test of Buehler were the methods of choice. In order to encourage a more informed dialogue about the relative performance, accuracy and applicability of the LLNA and guinea pig tests, we have here considered the extent to which guinea pig methods were themselves subject to false positives and negative results. We describe and discuss here well‐characterized examples of instances where both false negatives (including abietic acid and eugenol) or false positives (including vanillin and sulfanilic acid) have been recorded in guinea pig tests. These and other examples are discussed with particular reference to the fabrication of a gold standard dataset that is required for the validation of in vitro alternatives. Copyright © 2010 John Wiley & Sons, Ltd.     

On the skin sensitisation potential of rosin and oxidised rosin

In the EU rosin is classified as a skin sensitiser, apparently on the basis of its oxidation to sensitising agents. Rosin (gum, tall oil or wood) is not a skin sensitiser when examined in the guinea pig maximisation test (GPMT). Oxidised rosins are sensitisers in the GPMT. Oxidised gum rosin was further tested in the mouse local lymph node assay (LLNA) and the Buehler test, but is not a sensitiser in either of these tests. Further, the outcome of the LLNA can be used to assess the potency of oxidised rosin as an inducing agent in humans, and oxidised rosin is, at most, a weak sensitiser in this test. Thus, oxidised rosin is not a potent inducing agent for skin sensitisation unless the dermal barrier is bypassed and/or there is deliberate use of Freund’s Complete Adjuvant to induce greater susceptibility.The material used for human patch testing (‘colophony’) is in oxidised form. A re-examination of epidemiological studies suggests that patients in dermatological clinics show higher response rates than do the general population or those occupationally exposed to presumably oxidised rosin. Thus, the differences seen in susceptibility in the regulatory tests may be reflected in the human population.These results are discussed in terms of possible testing and classification strategies for dealing with existing chemicals, with particular reference to the new European Union legislation.     

Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test

Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, beta-O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, beta-O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, beta-O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization.     

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.     

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT)

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon–carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)  3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.     

Interlaboratory Evaluation of the Local Lymph Node Assay with 25 Chemicals and Comparison with Guinea Pig Test Data

Abstract

Summary: The murine local lymph node assay (LLNA) has been developed as an alternative method for the identification of skin sensitizing chemicals. Measurement is made of the proliferation of lymphocytes within lymph nodes draining the site of exposure to the test chemical. This report describes a collaborative study in which 25 test chemicals were evaluated in each of four participating laboratories and the results compared with existing data from guinea pig predictive tests. Nineteen chemicals were predicted to be sensitizers in the guinea pig. Of these, 14 were correctly identified in the LLNA (9 by all laboratories and 5 by two or three laboratories). Five chemicals predicted to be contact allergens by guinea pig tests failed to elicit positive LLNA responses. With adoption of a 5 day rather than a 4 day exposure period to the test chemical and administration of maximum soluble test concentrations, positive reactions could be obtained with each of the chemicals initially negative in the LLNA. The LLNA and guinea pig tests were in agreement for all three chemicals predicted to be nonsensitizers in the guinea pig. Positive LLNA responses were obtained with four chemicals (including a re-evaluation of one initially negative in the LLNA) for which guinea pig results were equivocal in three cases and negative in another. These results suggest that the LLNA may provide a rapid and reliable elective prescreen for the identification of contact allergens.