An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8--10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A--C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.     

The murine local lymph node assay: results of an inter-laboratory trial

The local lymph node assay is a novel predictive test for the identification of contact allergens. The collaborative study reported here was performed to evaluate the reliability of the method when performed in independent laboratories. Eight chemicals were examined in each of 4 participating laboratories and results compared with predictions of skin-sensitizing activity made from concurrent Magnusson and Kligman guinea-pig maximization tests performed in a single laboratory. The local lymph node assay has as its theoretical basis the fact that contact allergens induce T-lymphocyte proliferative responses. In practice, predictions of contact-sensitizing potential are made following measurement of proliferation in lymph nodes draining the site of exposure to chemical, and derivation of a stimulation index using control values as the comparator. Although in the present study there was some variation between laboratories with respect to the absolute stimulation indices recorded, it was found that with all chemicals each laboratory made the same predictions of sensitizing activity. Six chemicals (2,4-dinitrochlorobenzene, formalin, eugenol, isoeugenol, p-phenylenediamine and potassium dichromate) yielded positive responses, and two (methyl salicylate and benzocaine) were negative, in each laboratory. Furthermore, with 7 of the 8 chemicals tested there was no significant difference between laboratories in terms of the characteristics of the dose-response relationships recorded. With the exception of one chemical (benzocaine), predictions made with the local lymph node assay were in accord with those derived from guinea-pig maximization tests. These inter-laboratory comparisons demonstrate that the local lymph node assay is a robust and reliable method for the identification of at least moderate and strong contact allergens.     

Local lymph node assay with non-radioisotope alternative endpoints

The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.     

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.     

Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.     

Draining lymph node cell activation in guinea pigs: comparisons with the murine local lymph node assay

The local lymph node assay in the mouse is a novel predictive test for the identification of contact sensitizing chemicals. The purpose of the studies described was to determine whether a similar local lymph node assay could be performed successfully in guinea pigs; currently the species of choice for assessment of sensitizing potential for regulatory purposes. Ten sensitizing chemicals (oxazolone, picryl chloride, 2,4-dinitrofluorobenzene, benzocaine, cinnamic aldehyde, 2,4,-dinitrothiocyanobenzene, p-nitrosodimethylaniline, formaldehyde, p-phenylenediamine and cyanuric chloride) and equal concentrations of sodium lauryl sulphate were examined in a guinea pig local lymph node assay. Animals received three consecutive daily applications of various concentrations of the test chemical on the dorsum of both ears. Control animals were untreated. Five days following the initiation of exposure, draining auricular lymph nodes were excised and weighed. Suspensions of lymph node cells (LNC) were prepared and cultured for 24 or 48 h and proliferation measured by incorporation of [3H]thymidine. Exposure to at least one concentration of all sensitizing chemicals, other than benzocaine, induced proliferation by draining LNC. Responses were higher at 24 h rather than 48 h. Evidence is presented that guinea pig LNC proliferation may be enhanced or maintained by addition to culture of an exogenous source of the T cell growth factor interleukin 2 (IL-2). Draining lymph node weight was increased following exposure to some sensitizing chemicals but, compared with LNC proliferation, provided a less sensitive correlate of lymph node activation. Exposure to sodium lauryl sulphate failed to induce changes in either lymph node weight of LNC proliferation. Data are compared with three-day murine local lymph node assays performed concurrently. The available information indicates that the local lymph node assay may be performed in guinea pigs.     

Cytokine endpoints for the local lymph node assay: consideration of interferon-gamma and interleukin 12.

The murine local lymph node assay (LLNA) is a method for the prospective identification of contact allergens. Skin sensitization potential is assessed as a function of induced proliferative responses in lymph nodes draining the site of topical exposure measured in situ by incorporation of radiolabelled thymidine ([3H]thymidine). The results of previous investigations have demonstrated that the analysis of [3H]thymidine incorporation represents a robust and reliable endpoint for the LLNA for the assessment of skin sensitizing activity for strong and moderate allergens and, in addition, many weaker sensitizers. The aim of the current experiments was to explore the utility of the production of the cytokines interferon-gamma (IFN-gamma) and interleukin 12 (IL-12) by draining lymph node cells (LNC) as alternative readouts for the LLNA. Animals were exposed to a range of skin sensitizers at two application concentrations. The first of these was chosen on the basis of results from previous investigations to stimulate a strong proliferative response (tenfold or greater increase in proliferation compared with concurrent vehicle controls). The second concentration of test material in each case was the amount of chemical estimated to be necessary mathematically for elicitation of a stimulation index of 3 (EC3 value); the induction of a threefold or greater increase in proliferation is the current criterion for a positive response in the LLNA. In addition, analyses were conducted with para-aminobenzoic acid (PABA), a non-sensitizing chemical shown previously not to induce LLNA responses. Secretion of IFN-gamma and the p40 subunit of IL-12 by draining LNC was measured by cytokine-specific enzyme-linked immunosorbent assay. In parallel experiments, LNC activity was assessed as a function of [3H]thymidine incorporation in situ. All the chemical allergens tested provoked robust proliferative responses, with the stimulation indices recorded at both test concentrations reflecting only small changes in activity compared with previously recorded data. Exposure to vehicle (4:1 acetone:olive oil, AOO) alone resulted in detectable, although variable, expression of both IFN-gamma and IL-12. Treatment with chemical allergen in each case caused a marked increase in IFN-gamma secretion, with particularly vigorous production of cytokine being stimulated following exposure to oxazolone or hexyl cinnamic aldehyde. In contrast, application of chemical allergens was not generally associated with elevated IL-12 p40 secretion. Exposure of mice to PABA did not result in increased IFN-gamma or IL-12 production compared with vehicle-treated controls. In general, however, cytokine secretion did not correlate closely with the induction of LNC proliferation. These data indicate that expression by allergen-activated LNC of IFN-gamma or IL-12 does not provide a reliable or sufficiently sensitive endpoint for the LLNA compared with [3H]thymidine incorporation in situ.     

A murine local lymph node assay for the identification of contact allergens

The development of an alternative predictive test for the identification of contact sensitizing chemicals is described. The method is based upon the fact that, following epicutaneous application, sensitizing chemicals initiate a primary immunological response in the draining lymph node(s) which is characterized by lymphocyte proliferation. Experimental conditions for the measurement in vitro of the induced lymph node cell proliferative response have been optimized. On the basis of the data presented a local lymph node assay was developed in which CBA/Ca strain mice were exposed daily, for 3 consecutive days, to various concentrations of the test chemical, or to vehicle alone, on the dorsum of the ear. Lymph node activation was measured subsequently as a function of increased node weight, the frequency of large pyroninophilic cells and lymphocyte proliferation in the presence or absence of an exogenous source of interleukin 2 (IL-2). The results of a validation study are reported in which 22 well-characterized sensitizing chemicals of varying potency were examined. With the exception of three chemicals where water was used as the application vehicle, positive responses, defined as a substantial increase in lymphocyte proliferative activity, were recorded with all these test materials. Under the conditions employed non-sensitizing chemicals, including non-sensitizing irritant chemicals, failed to influence the immunological status of the draining lymph node. Taken together, the data suggest that the local lymph node assay provides the basis for a rapid and cost-effective alternative to the currently available guinea pig predictive test methods. The local lymph node assay may be of particular value for the evaluation of coloured or irritant chemicals.     

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025–0.1% and 5–20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated.     

Anamnestic responses to contact allergens: application in the murine local lymph node assay

The murine local lymph node assay, an alternative predictive test for the identification of contact sensitizing chemicals, is based upon the fact that skin allergens induce proliferation in lymph nodes draining the site of application. In the present study we have examined whether pre-exposure to the test chemical at a distant site enhances subsequent draining lymph node cell proliferation and, thereby, the sensitivity of the assay. Experiments were performed using both in vitro and in situ measurement of induced lymph node cell proliferation. It was found that, with the exception of potent skin sensitizers such as picryl chloride and oxazolone, which impair subsequent proliferative activity as a consequence of induced immunoregulatory processes, pre-treatment with the test allergen resulted in enhanced proliferation. Evidence is presented that the local lymph node assay response to a variety of skin allergens (including eugenol, isoeugenol, dihydrocoumarin, 4-vinylpyridine, cinnamic aldehyde and 2,4,5-trichlorophenol) was augmented when mice received a single exposure to the same chemical 5 days earlier. It is concluded that the use of a modified protocol, incorporating pre-exposure to the test material, can enhance local lymph node assay responses to all but the most potent skin allergens, and may be of particular value when increased sensitivity is required.     

The murine local lymph node assay: regulatory and potency considerations under REACH

From June 2007, new chemicals legislation on the registration, evaluation, authorization and restriction of chemicals (REACH) will come into force across the European Union. This will require the submission of data on human health effects of chemicals, including chemical safety assessments which will require measurements of potency. For skin sensitization hazard identification, REACH states that the first-choice in vivo assay is the local lymph node assay (LLNA). This test has also been the UK competent authority's preferred test for skin sensitization since 2002, and has now replaced guinea pig tests in dossiers submitted to it under the Notification of New Substances Regulations. Advantages of the LLNA over guinea pig tests include improvements in animal welfare, a more scientific approach to hazard identification, and the inclusion of a dose-response element in the endpoint, which enables an estimation of potency. However, notifiers to the UK competent authority have sometimes been reluctant to use the assay because of concerns over false-positive reactions. Across Europe, these concerns have been heightened in the lead-up to the introduction of REACH, since the use of in vivo alternatives to the LLNA will require scientific justification. This review will address some of these concerns from a regulatory perspective.     

Evaluation of an ex vivo murine local lymph node assay: multiple endpoint comparison

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.     

Local lymph node assay responses to paraphenylenediamine: intra- and inter-laboratory evaluations.

The murine local lymph node assay (LLNA) is a method for the prospective identification of skin sensitizing chemicals. Proliferative responses induced in lymph nodes draining the site of topical application of the test chemical are measured and those chemicals that induce a stimulation index of three or more compared with concurrent vehicle-treated controls are considered to have the potential to cause skin sensitization. Dose-response data from the LLNA may be used to derive an estimate of relative skin sensitizing potency, based upon derivation of the concentration of chemical required to cause a stimulation index of 3 (EC3 value) as calculated by linear interpolation. The purpose of the present investigations was to examine the stability of LLNA responses and the consistency of derived EC3 values induced by the contact allergen paraphenylenediamine (PPD). Analyses were conducted once a month over a 4-month period in each of two independent laboratories. In all assays, and in both laboratories, PPD elicited a positive response. Although some minor differences in responses between and within laboratories were observed, the derived EC3 values were generally very consistent. In Laboratory 1, EC3 values varied between 0.06 and 0.09% PPD, whereas in Laboratory 2 the range was 0.09-0.20%. These EC3 values are consistent with clinical experience of this material insofar as it is a common and relatively potent cause of allergic contact dermatitis in humans. Taken together, these data confirm the stability of LLNA responses both with time and between laboratories and provide additional support for the use of derived EC3 values in the assessment of relative skin sensitizing potency.     

Detection of contact sensitivity of metal salts using the murine local lymph node assay.

The local lymph node assay (LLNA) is a predictive test for the detection of contact allergens. Nickel and chromium sensitization are common cases in man. However, in a previous study topical application of nickel sulfate and potassium dichromate in aqueous solution failed to induce activation in the draining lymph node. This study describes the application of LLNA to evaluate the contact sensitivity of metal salts. The metal salts were applied in dimethylsulfoxide or aqueous ethanol solution. In some experiments, the skin of the ears was gently abraded using a needle prior to application of metal salts. The ability of seven metal salts to induce lymph node cell (LNC) proliferation was compared. Nickel, cobalt, chromium and copper salts increased LNC proliferation, whereas zinc, manganese and iron salts failed to induce LNC proliferation in this assay.     

ICCVAM evaluation of the murine local lymph node assay. 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.     

Evaluation of skin sensitization potential of jet fuels by murine local lymph node assay

Jet A and JP-8 are the major jet fuels used in civilian and military (US Air Force) flights, respectively. JP-8+100 is a new jet fuel recently introduced by the US Air Force. Besides lung exposure, skin is the potential route of exposure to jet fuels. The purpose of the present study was to investigate the skin sensitization potential of jet fuels (Jet A, JP-8 and JP-8+100) using murine Local lymph node assay (LLNA). Female CBA/Ca mice (8-12-weeks-old) were used in the study. Dinitrochlorobenzene (DNCB, 0.25% w/v) and paraaminobenzoic acid (PABA, 2.5% w/v) were used as positive and negative control, respectively and acetone: olive oil (4:1, AOO) was used as the vehicle (control). All three jet fuels caused a proliferative activity significantly greater than the control (P<0.01). Our results demonstrate that JP-8 is a weak skin sensitizer [stimulation index (SI)=3.17]. The SI of Jet A and JP-8+100 were 2.44 and 2.38, respectively, hence are not considered as skin sensitizers. Interestingly, the SI of JP-8 with butylated hydroxytoluene (BHT) was consistently lower than JP-8, though the difference was not statistically significant (P>0.05). BHT, which is an antioxidant additive of JP-8+100, reduced the skin sensitization potential of JP-8. Furthermore, the lower SI of JP-8+100 could be partially attributed to the presence of BHT. The findings reported here suggest that care should be taken to minimize dermal exposure to jet fuels especially JP-8 to avoid skin sensitization.     

The murine local lymph node assay: a commentary on collaborative studies and new directions.

The murine local lymph node assay is a predictive test for the identification of contact allergens. This paper provides a historical background to the development of the assay and describes the performance of a recently completed interlaboratory trial designed to evaluate further the utility of the method as an alternative or adjunct to guinea-pig predictive tests. On the basis of these and supplementary investigations, a number of recommendations can be made regarding the use and interpretation of the local lymph node assay. Finally, a number of issues arising from recent studies are discussed, including comparisons of the local lymph node assay with guinea-pig methods.     

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method. This method involves a three‐step approach: (1) ultraviolet (UV) absorption analysis; (2) determination of no observed adverse effect level for skin phototoxicity based on LLNA; and (3) photoallergy evaluation based on LLNA. Photoallergic potential of chemicals was evaluated by comparing lymph node cell proliferation among groups treated with chemicals with minimal effect levels of skin sensitization and skin phototoxicity under UV irradiation (UV+) or non‐UV irradiation (UV?). A case showing significant difference (P < .05) in lymph node cell proliferation rates between UV? and UV+ groups was considered positive for photoallergic reaction. After testing 13 chemicals, seven human photoallergens tested positive and the other six, with no evidence of causing photoallergic dermatitis or UV absorption, tested negative. Among these chemicals, both doxycycline hydrochloride and minocycline hydrochloride were tetracycline antibiotics with different photoallergic properties, and the new method clearly distinguished between the photoallergic properties of these chemicals. These findings suggested high predictability of our method; therefore, it is promising and effective in predicting human photoallergens.     

The reduced local lymph node assay: the impact of group size

The local lymph node assay (LLNA) is a skin sensitization test that provides animal welfare benefits. To reduce animal usage further, a modified version (rLLNA) was proposed. Conducting the rLLNA as a screening test with a single high dose group and vehicle control differentiated accurately between skin sensitizers and non-sensitizers. This study examined whether a reduction in animal number/group is feasible. Historical data were utilized to examine the impact of conducting the rLLNA with two mice/group. To assess the effect on the stimulation index (SI) 41 datasets with individual animal data derived using five mice/group were analysed. SIs were calculated on all possible combinations of two control and two high dose group disintegrations per minute (dpm) values. For 25 of 33 sensitizer datasets, > 96% of possible dpm combinations resulted in a calculated SI > 3. The lowest percentages of positive SIs were observed with weak allergens when, in the standard LLNA, the mean SIs would have been nearer to the threshold value of 3. The results indicate that moderate, strong and extreme allergens are more likely than weak allergens to be identified as sensitizers when group sizes of two mice are used within the rLLNA. It is concluded that a rLLNA with two mice/group would display decreased sensitivity and is inappropriate for use in hazard identification.