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Local Lymph Node Assay

Table of Contents

  1. Test Submission
    1. Cover Letter
    2. Submission
  2. Appendices
    1. Local Lymph Node Assay Bibliography
    2. List of Chemicals
      1. Chemicals Tested in Local Lymph Node Assay
      2. Discordant Results Between Local Lymph Node Assay and Guinea Pig or Human Test Methods
      3. Disintegrations Per Minute Data and Stimulation Indices for Discordant Results
    3. Key Local Lymph Node Assay Papers
    4. Sample Local Lymph Node Assay Protocol
    5. ICCVAM Local Lymph Node Assay Test Submission Guidelines

INTRODUCTlON AND RATIONALE

Allergic contact dermatitis is a frequent occupational health problem, and in common with other forms of allergic disease, develops in two phases. The first or induction phase is initiated when a susceptible individual encounters on the skin sufficient amounts of an inducing allergen to stimulate a primary cutaneous immune response. This results in allergic sensitization. If the now sensitized individual is subsequently exposed, at the same or a different skin site, to the same allergen then an accelerated and more aggressive secondary immune response will be provoked at the site of contact. Allergen-responsive T lymphocytes are activated in the skin at the site of contact and release cytokines and other inflammatory mediators which cause the accumulation of mononuclear cells and the inflammatory reaction that is recognized clinically as allergic contact dermatitis.

For many years the species of choice for the identification of contact allergens was the guinea pig. A variety of guinea pig test methods has been described and while these vary in detail, the principles of the assays are in each case the same, sensitizing activity being measured as a function of challenge-induced erythematous and edematous reactions in previously sensitized animals. There is no doubt that some at least of these guinea pig methods have served toxicologists well. Nevertheless, it is clear that such assays are subject to some important limitations, including the fact that the endpoint is subjective and may be difficult to measure and interpret if colored or irritant chemicals are evaluated. Moreover, some of the more sensitive guinea pig methods demand the use of adjuvant. These limitations encouraged consideration of alternative approaches.

Some ten years ago the local lymph node assay (LLNA) was described (Kimber et al, 1986; Kimber et al, 1989; Kimber and Basketter, 1992; Kimber et al, 1994; Kimber, 1996). This method was founded on the belief that an increasingly sophisticated appreciation of the immune system would facilitate the design of alternative methods for the identification of chemical allergens that cause adverse effects through the stimulation of specific immune responses. LLNA employs mice, the experimental species where there is the most detailed information available about the induction and regulation of immunological responses. In contrast to guinea pig test methods, the LLNA identifies potential skin sensitizing chemicals as a function of events associated with the induction, rather than elicitation, phase of skin sensitization.

The induction phase of skin sensitization is characterized by the stimulation of an allergen-specific immune response in Iymph nodes draining the site of exposure. Epidermal Langerhans cells (LC) recognize, internalize, and process the chemical hapten associated with protein. LC are induced to migrate to draining Iymph nodes. While in transit they develop into immunostimulatory dendritic cells which in the lymph nodes are able to interact with and present antigen to responsive T lymphocytes (Kimber and Cumberbatch, 1992; Kimber and Dearman, 1996). Immune activation in draining Iymph nodes is characterized by T lymphocyte division and differentiation, the production by activated cells of cytokines and other mediators and an increase in the size, weight, and cellularity of the lymph nodes. The division of activated T cells results in an increase in the number of allergen-reactive Iymphocytes: this clonal expansion being the cellular basis of immunological memory and allergic sensitization. The importance of clonal expansion is reflected by the fact that the vigor of proliferative responses induced by chemicals in draining Iymph nodes correlates closely with the extent to which sensitization develops (Kimber and Dearman, 1991; Kimber and Dearman, 1996).

In initial investigations, several parameters of draining Iymph node activation were measured following topical exposure of mice to contact allergens and to non-sensitizing chemicals. These composed changes in lymph node weight and cellularity and lymphocyte proliferation measured as a function of radiolabeled thymidine incorporation during culture of Iymph node cells (Kimber at al, 1986; Kimber and Weisenberger, 1989a; Kimber, 1989). The marker that proved to be the most sensitive and selective correlate of slain sensitizing activity was the induction of Iymph node cell proliferation and subsequent investigations focused upon this. Another change introduced following these preliminary experiments was to measure the proliferative activity in situ, by intravenous injection of tritiated thymidine, rather than following culture of isolated Iymph node cells (Kimber and Weisenberger, 1989b; Kimber et al, 1989). It is this version of the method that has been evaluated extensively in the context of national and international collaborative trials and which has been the subject of detailed comparisons with guinea pig tests and with human data. The results of these evaluations and comparisons will be discussed later.

A criterion of positivity was required to facilitate decisions regarding the sensitizing potential of chemicals based on activity in the LLNA. The decision was made, based on extensive experience gained with the method that a chemical should be classified as a skin sensitizer if, at one or more test concentrations, proliferative activity threefold or greater than that measured in concurrent vehicle treated controls was induced. The validity of due use of a stimulation index of 3 for the identification of contact allergens is discussed later in this submission.

In summary, the LLNA provides a novel approach to the identification of skin allergens where immunobiological events stimulated during the induction phase of skin sensitization are measured. Decisions are based upon assessment of draining lymph node cell proliferative responses--responses that are known to be essential for and to correlate with the induction of skin sensitization.

For practical purposes the following recommendations are made for use of the LLNA:

  • A chemical which, at one or more test concentrations, elicits a threefold or greater increase in proliferative activity compared with concurrent vehicle treated controls should be classified as being a contact allergen and handled and labeled accordingly.
  • Chemicals that fail even one test concentration to elicit positive response in the local lymph node should be classified as lacking significant skin sensitizing potential and should be handled and labeled accordingly. No further confirmation of negative results is required.

There is currently some interest in comparing and contrasting the nature of immune responses induced in mice by different types of chemical allergens. It is very important to emphasize, however, that the proposal is that the LLNA can be used to identify those chemicals that are able to cause skin sensitization. A case is not being made here for use of the LLNA in the identification of any other classes of mechanical allergen. Moreover, this submission is focused on the standard LLNA, for which the method is described in detail in Section B. Consequently, papers describing modified versions of the assay are not reviewed in this document.

The proposal is that the LLNA provides an alternative method for use in the identification of skin sensitizing chemicals and for confirming that chemicals lack a significant potential to cause skin sensitization. This does not necessarily imply that in all instances the LLNA should be used in place of guinea pig tests, but rather that the assay is of equal merit and may be employed as a full alternative in which positive and negative results require no further confirmation.

The LLNA is not an in vitro method and as a consequence will not eliminate the use of animals in the assessment of contact sensitizing activity. It will, however, permit a reduction in the number of animals required for this purpose. It has been estimated tool in practice, on average half the number of animals required for a standard guinea pig test is needed for conduct of a LLNA. Moreover, the LLNA does offer a substantial refinement of the way in which animals are used for contact sensitization testing. One important point is that, unlike some of the guinea pig methods, such as the guinea pig maximization test (GPMT), the LLNA does not require the use of adjuvant. Furthermore, the LLNA is based upon consideration of immunobiological events stimulated by chemicals during the induction phase of sensitization. Unlike guinea pig tests, the LLNA does not require that challenged-induced dermal hypersensitivity reactions are elicited.

Due to the fact that the LLNA requires far fewer animals than needed for standard guinea pig tests it can be conducted for approximately half the cost. The time taken for conduct of a LLNA is some eight times less than that needed for a standard guinea pig method.

It is estimated currently that in excess of 25 separate laboratories worldwide are conducting the LLNA.

TEST METHOD PROTOCOL

The contact allergenic potential of a test substance, the conditions of this protocol, is evaluated by its ability to cause proliferation of draining lymph node cells in mice treated topically compared to appropriate concurrent vehicle treated controls. Direct epicutaneous application of a test substance to the ears is an appropriate route of administration for assessing the contact allergic potential of a test substance. Incorporation of 3H-thymidine into DNA of lymphocytes results from the stimulation of S-phase prior to proliferation of the cells after receipt of antigenic stimulation. Measurement of 3H-thymidine uptake by the cells is an objective and quantifiable correlate of immune activation.

Protocol

The standard protocol described previously (Kimber and Basketter, 1992) utilizes young adult (6-16 week old) female CBA/Ca strain mice. In strain comparisons, CBA/Ca mice were found to exhibit a more marked response to contact allergens than did the other strains examined (Kimber and Weisenberger, 1989a). However, female CBA/J and CBA/JHsd strain mice are also acceptable for use in the assay as, in several interlaboratory validation studies, they display responses comparable with those of CBA/Ca strain mice (Kimber et al, 1995; Loveless et al, 1996). Mice are housed under standard conditions, individually or by treatment group, in plastic shoe box type cages for the duration of the study. Food and tap water are provided ad libitum. Control of bias is addressed by randomization of mice prior to initiation of the study.

Groups of mice (n = 4 or 5) are treated by topical application, on the dorsum of both ears, of 25 µl of one of several concentrations of test material or with an equal volume of the relevant vehicle alone. Treatments are performed daily for three consecutive days and the mice are then rested for two days prior to analysis. On the sixth day (five days after initiation of treatment), the mice are injected intravenously via the tail vein with 250 µl of sterile phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine (3H-TdR: sperms activity between 2 and 7 Ci/mmol). Five hours later, the mice are killed and the draining auricular Iymph nodes excised and pooled for each experimental group or for each individual animal. Single cell suspensions of lymph node cells (LNC) are prepared by gentle mechanical disaggregation through 200-mesh nylon or stainless steel gauze. LNC are washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C. Twelve to eighteen hours later the samples, pelleted by centrifugation, are resuspended in 1 ml 5% TCA and transferred to 10 ml of scintillation cocktail. Incorporation of 3H-TdR is measured by ?-scintillation counting and expressed as disintegrations per minute (dpm). The use of 125IUdR rather than 3H-TdR as the isotope has been shown to be comparably robust in the LLNA (Kimber et al, 1995; Loveless et al, 1996).

A sample protocol for the standard LLNA is provided in Appendix D.

Dose Selection

No additional animals are used for dose range finding. The current practice is to select at least three consecutive concentrations from the following range: 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1% (w/v). The selection is made to provide the biggest possible test concentration, limited by compatibility with the vehicle chosen (and the suitability of the resultant preparation for unoccluded dermal application), while avoiding dermal trauma or systemic toxicity. The test chemical is dissolved in an appropriate vehicle. Vehicle selection is important and a variety of organic solvents is suitable. The following are recommended, in order of preference: acetone-olive oil (4:1) (AOO), acetone, dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethylsulfoxide (Kimber and Basketter, 1992). While aqueous vehicles are not recommended, aqueous and aqueous-organic mixtures such as 3:1 acetone:water have been used successfully.

Control Materials

The current OECD positive control sensitizers hexyl cinnamic aldehyde (HCA), 2-mercaptobenzothiazole and benzocaine have each been evaluated in the local lymph node assay. Results with these positive controls in the LLNA met or exceeded the minimum acceptable standard set forth by the OECD (Basketter et al, 1993). The strong sensitizer 2,4-dinitrochlorobenzene (DNCB) may be used as a positive control as it has produced consistent responses in the LLNA, including when tested in two recent international interlaboratory trials (Kimber et al, 1995; Loveless et al, 1996). Currently, there are no recommended negative controls for the LLNA as is the case with the reference guinea pig methods. However, methyl salicylate, tested at 1, 2.5, 5, 10, and 20% (w/v) in acetone:olive oil (4:1) (Kimber et al, 1995; Kimber et al, 1998) and para-aminobenzoic acid tested at 0.5, 1, 2.5, 5, and 10% (w/v) in acetone:olive oil (Loveless et al, 1996) have been used successfully as negative control chemicals in interlaboratory validation studies. In common with other skin sensitization tests, a control substance for irritation has not been defined for the LLNA.

Data Collection and Analysis

In vivo 3H-thymidine incorporation into Iymph node cell DNA associated with proliferation induced by application of a contact sensitizer (measured by liquid scintillation counting) is an objective and quantifiable response. Data are collected as disintegrations per minute (dpm).

The data are expressed as mean dpm for each experimental group and the stimulation indices (SI) for each experimental group are determined as the increase in 3H-TdR incorporation relative to concurrent vehicle-treated controls (test/control ratio). A test material which at one or more concentrations causes a stimulation index of 3 or greater is considered to have skin sensitizing activity. Thus, whether the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal, the threefold or greater increase in proliferative activity compared with concurrent vehicle-treated control animals is the sole criterion for a classification of skin sensitizing activity.

In cases when individual mice are being used for determining the mean dpm value for an experimental group, statistical analysis may be performed. The value of statistical analysis, either alone or in conjunction with the threefold stimulation index, has not yet been established and is still the subject of investigations. Where isotope incorporation is determined for individual mice, a mean dpm value standard ever of the mean (SEM) is calculated for each experimental group. A stimulation index is derived for each experimental group by dividing the mean dpm of that group by the mean dpm of the vehicle control group.

One approach to the development of statistical methods that may prove of value in the LLNA is as follows. For statistical analyses, the mean dpm values for each treatment group and the vehicle control group are initially normalized by obtaining their log value. Bartlett's test (Bartlett, 1937) is then used to examine the data for homogeneity of the within-chemical treatment variance. If Bartlett's best for homogeneity of variance is not significant, comparisons with the control group (and other specific, pair-wise comparisons of groups) are based on the least significant difference criterion. If Bartlett's test is significant, these comparisons are based on Wilcoxon's rank sum test. Alternately, when the data follow a normal distribution, then parametric statistical methods such as Dunnett's t test (Dunnett, 1955) can be used to compare experimental groups with vehicle-treated controls. For data which does not follow a normal distribution, a non-parametric method such as the Kruskal-Wallis test (Kruskal and Wallis, 1952) may be used followed by Dunn's multiple comparison procedure (Dunn, 1964). Groups differing from vehicle-treated controls at the level of P>0.05 are considered significantly different.

In addition, an estimate of the test material concentration required to produce a stimulation index of 3 (EC3) can be calculated using fitted quadratic regression analyses. An advantage of the EC3 calculation is that data from the entire dose response curve are used to produce a single value of intrinsic potency (Loveless et al, 1996). The EC3 value can then be used to rank in order the skin sensitizing potential of chemicals. Stronger sensitizers such as DNCB and oxazolone have lower EC3 values than more moderate sensitizers such as HCA and eugenol (Loveless et al, 1996). Dose response analyses in the local lymph node assay, combined with the mathematical derivation of the lowest test concentration of a chemical required for a defined stimulation index, such as the EC3, provides a convenient, reliable, and realistic approach to evaluation of relative potency (Kimber and Basketter, 1997).

An examination of the application of statistical analyses to the LLNA is continuing. At present, it is not clear whether, or in what way, an evaluation of statistical significance would add value to the interpretation of the LLNA. This, together with consideration of EC3 values for measurement of relative potency are areas of investigation that may pay dividends in the future, but which are not currently part of the standard protocol.

Summary of Control Data

The recommended positive control material, HCA, was tested independently by five laboratories over a dose range of 2.5, 5.0, 10.0, 25.0, and 50% (w/v) in AOO (Loveless et al, 1996). All five correctly identified HCA as a contact allergen. Four of the five laboratories found the lowest concentration to produce an Sl of 3 or greater was 10%. The fifth laboratory reported an SI of 2.5 for this concentration. Calculations of the EC3 for HCA ranged from 7.0 to 8.4%. DNCB was tested in two separate trials by the same five laboratories at concentrations of 0.01, 0.025, 0.05, 0.1, and 0.25% (w/v) in AOO. EC3 calculations for DNCB from both trials ranged from 0.03 to 0.09%.

Recently the stability with time of responses induced in the LLNA by HCA has been evaluated in a single laboratory. Over a ten month period HCA elicited very simiiar EC3 values in the LLNA (Dearman et al, 1998). These issues are discussed further in Section D below.

CHARACTERIZATION OF MATERIALS TESTED

Two of the interlaboratory evaluations of the LLNA were cabled out under conditions where all details of the test materials and test conditions were not known to the participating laboratories. In the first of these studies, 20 substances were coded and supplied to each of four laboratories (Basketter et al, 1991). In a subsequent study, the chemical names were given, but no advice on dose/vehicle selection was provided (Scholes et al, 1992). The results from both of these investigations demonstrated a high degree of interlaboratory agreement. It is interesting to compare these results with those from non-blinded interlaboratory studies of the GPMT and the Buehler test (Robinson et al, 1990; Andersen et al, 1985). In these instances, relatively poor interlaboratory reproducibility was achieved, which is in sharp contrast to experience with LLNA.

ASSESSMENT OF RELIABILITY (REPEATABILITY AND REPRODUCIBlLITY)

There are considerable data on intralaboratory reproducibility of the LLNA, some of which has been published (Basketter et al, 1996; Kimber et al, 1998) and some of which is based on unpublished individual laboratory experience. Table 1 summarizes the information on this topic.


Table 1: Intralaboratory Reproducibility of the LLNA

ChemicalTest 1Test 2Test 3Test 4Test 5Test 6
DNCB+++NDNDND
Hexyl cinnamic aldehyde++++++
Isoeugenol++++NDND
Eugenol+++++ND
Methyl salicylate----NDND
Benzocaine--+/----

ND = No data


Although it is not the aim within the current validation to examine assessment of relative skin sensitizing potency, it is possible to derive such information from the LLNA (Basketter et al, 1996; Kimber and Basketter, 1997). For this, the estimated concentration of the test chemical which is sufficient to cause a threefold stimulation (EC3) is determined by interpolation of the dose response data. What precise value this may have for risk assessment is currently the subject of various pieces of work (e.g. Basketter et al, 1996; Kimble and Basketter, 1997; Basketter, 1998). However, the approach taken also allows better comparison of individual LLNA results. Examples of this type of data are contained in Table 2.


Table 2: Reproducibility of LLNA Quantitative Data

ChemicalTest 1Test 2Test 3Test 4Test 5Test 6
DNCB - Laboratory 10.0510.03ND2NDNDND
DNCB - Laboratory 20.060.05NDNDNDND
DNCB- Laboratory 30.040.06NDNDNDND
DNCB - Laboratory 40.060.09NDNDNDND
DNCB - Laboratory 50.030.06NDNDNDND
Isoeugenol0.30.40.40.40.6ND
Hexyl cinnamic aldehyde - Laboratory 17.96.99.68.74.09.2
Hexyl cinnamic aldehyde - Laboratory 27.67.28.89.510.011.9
Eugenol5.16.110.511.914.5ND
Methyl salicylateNS3NSNSNSNDND
BenzocaineNSNS?4NSNSNS

1% concentration required to give a stimulation index of 3
2ND = Not done
3NS = Not a sensitizer
4Not possible to determine an EC3 value from the dose response data


The first collaborative LLNA validation trial involved four independent laboratories in the UK which evaluated the same batch of eight chemicals, using the same protocol, vehicles, and test concentrations. Each laboratory identified 2,4-dinitrochlorobenzene (DNCB), formalin, eugenol, isoeugenol, paraphenylenediamine (p-PDA), and potassium dichromate as positive with benzocaine and methyl salicylate as negatives. With the exception of isoeugenol, no significant differences between the laboratories were found with respect to the characteristics of dose-response curves (Kimber et al, 1991).

The same four laboratories participated in a more extensive evaluation involving 25 chemicals (Basketter et al, 1991). Of the 25 chemicals, equivalent predictions of sensitizing potential were made for 18 chemicals by all laboratories. An additional five chemicals were identified as potential sensitizers in the LLNA by two or three laboratories. Three of these subsequently gave a positive response in laboratories which initially failed to detect them when retested under identical or altered conditions (e.g. higher concentration, different vehicle). It should be noted that these investigations were conducted prior to publication of the definitive LLNA protocol.

For the final phase of this national collaboration, nine chemicals were evaluated and each laboratory independently selected the test concentrations and vehicles (Scholes et al, 1992). One modification that all laboratories employed was applying chemicals topically for three consecutive days and then terminating the experiment five days after the initiation of exposure, rather than four days. Chemicals were evaluated at three concentrations which were chosen independently by each laboratory with regard to potential toxicity. The choice of vehicle was based upon solubility and viscosity. For eight chemicals, equivalent predictions were made by all laboratories and by three of the four laboratories for the remaining chemical. Identical vehicles and concentrations were selected independently by all laboratories for two chemicals and by three laboratories for six chemicals. In those cases where different concentrations or vehicles were chosen, equivalent predictions (positive or negative LLNA results) were still made. To determine what effect minor protocol modifications would have on the predictive value of the test, the LLNA was evaluated in an international study by five independent labs, two of which had participated in the UK national validation exercise. Modifications to the standard protocol included exposure of mice for four, rather than three, consecutive days, removal of auricular lymph nodes four, rather than five days, after study initiation, the use of an alternative isotope and analysis of lymph nodes from individual mice to allow for statistical evaluation (reviewed in Gerberick et al, 1992; Ladies et al, 1995).

In the first phase of this international validation, two skin sensitizers, DNCB and potassium dichromate and one non-sensitizer, methyl salicylate, were evaluated (Kimber et al, 1995). In the LLNA, the criteria for a positive result is a threefold or greater stimulation of proliferative activity relative to vehicle controls. In the laboratories analyzing nodes from individual mice, a positive result was also defined for the purpose of this investigation, as treatment groups differing from vehicle treated controls at a predetermined level of statistical significance (p<0.05 or p<0.01 depending upon the statistical method employed). By either criterion, and regardless of the protocol utilized, all five laboratories identified the two known sensitizers as being positive in the LLNA. Estimates of the test concentration required to yield a stimulation index of three (EC3) were very similar for all laboratories for both chemicals. Using the stimulation index criteria, all laboratories reported a negative finding for methyl salicylate at all concentrations tested. Two of the three laboratories evaluating nodes from individual mice did detect a statistically significant increase in radioisotope incorporation at the highest of the five concentrations tested (20%).

In the second phase of the international collaborative trial, the sensitivity and selectivity of the assay were examined further by analysis of six additional chemicals: hexylcinnamic aldehyde (HCA), oxazolone isoeugenol, eugenol, sodium lauryl sulphate (SLS), and para-aminobenzoic acid (pABA) (Loveless et al, 1996). The last two are considered to be non-sensitizing chemicals, while the remainder exhibit skin sensitizing potential to varying extents, with HCA being one of three chemicals recommended by the OECD for use as positive controls in skin sensitization studies (OECD 1993). All laboratories retested DNCB under the conditions employed in Phase I of the trial (Kimber et al, 1995) to provide information on the temporal stability of assay data. All five laboratories identified as positive the five moderate to strong sensitizers (DNCB, HCA, oxazolone, isoeugenol, and eugenol). SLS, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. PABA, a non-sensitizing chemical, was negative in each laboratory.

Oxazolone was clearly the most potent sensitizer evaluated in Phase II, with predicted EC3 values ranging from 0.0007 to 0.0026%. This chemical highlights the benefit of utilizing the entire dose response curve for predicting the concentration required for a SI of three, since four of the five laboratories recorded stimulation indices of above three at the lowest concentration tested. It also demonstrates that determination of an EC3 may be useful in assessing the relative sensitizing potency of a class of chemicals. Results with HCA, eugenol, isoeugenol, and pABA were similar to published LLNA results (Basketter et al, 1993; Basketter and Scholes, 1992; Basketter et al, 1994).

The results of Phase I and II provide strong support that the incorporation of minor procedural modifications did not affect the performance of the LLNA. In that regard, applying a test chemical for either three or four consecutive days, with removal of lymph nodes five or four days, respectively, after the initiation of treatment did not change the ability of the assay to detect skin allergens. Three consecutive daily exposures to a chemical is, therefore, considered sufficient for the purpose of the identification of potential skin sensitization hazard.

Concerning the choice of isotope utilized for detection of proliferation, there was no difference in the ability of 3HTdR or 125IUdR to identify correctly the chemicals evaluated in this study. Either isotope can be used in the LLNA (Ladies et al, 1995; Kimber et al, 1995; Loveless et al, 1996).

An important modification assessed during Phase I and II of this international validation study was the analysis of proliferation within lymph nodes of individual mice as opposed to lymph nodes pooled for each experimental group. In the majority of cases, the lowest concentration yielding a positive response was identical by either method of analysis.

One objective of Phase II was to examine interexperimental variability by evaluating DNCB twice. Three of the five laboratories obtained identical results to the first study (Kimber et al, 1995). Depending upon which of the criteria were used, the other two participating laboratories had either identical interexperimental results or were within one adjacent concentration level. Therefore, the intralaboratory interexperimental variability was very low.

The overall conclusion from this and the previous phase of the validation study (Kimber et al, 1995) is that five independent laboratories, despite the use of procedural modifications and different methods for data analysis, successfully and consistently employed the LLNA to reach identical conclusions on the sensitizing potential of nine chemicals.

The most recent interlaboratory validation study involved the same five laboratories working in collaboration with the U.S. FDA. In this study (Kimber et al, 1998), a small series of chemicals used in topical drug products was examined. Again there was very close agreement between laboratories with all five identifying correctly benzoyl peroxide, hydroquinone, penicillin G, and methyl salicylate. Streptomycin sulfate induced equivocal responses, insofar as this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride was uniformly negative. Collectively these data serve to confirm that the LLNA is sufficiently robust to yield equivalent results when performed independently in separate laboratories. The data indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

A total of seven laboratories have been involved in interlabatory validations of the LLNA. The results of the work have appeared in the several associated publications (Kimber et al, 1991; Basketter et al, 1991; Scholes et al, 1992; Kimber et al, 1995; Loveless et al, 1996; Kimber et al, 1998). This work has involved investigation of more than 40 different chemicals. An overview of the time frame for the development and validation of the LLNA is displayed in Figure 1 (adapted from Chamberlain and Basketter, 1996). Information on consistency/performance over time has been given earlier in this section.

REFERENCE DATA

A variety of guinea pig tests has been developed for evaluation of the skin sensitizing potential of chemicals. Among those most widely applied are the guinea pig maximization test (GPMT) (Magnusson and Kligman, 1969, 1970) and the occluded patch test of Buehler (Buehler, 1965, 1985; Robinson et al, 1990). These two assays are the preferred guinea pig sensitization tests outlined in the OECD 406 guideline for skin sensitization. The GPMT used for comparisons with LLNA results is based on and similar to that described by Magnusson and Kligman (1970) which uses Freud's adjuvant. Albino Dunkin-Hartley guinea pigs weighing approximately 350g at the start of each study, are used. Preliminary irritation tests are carried out to determine the concentrations of the test substances suitable for induction of sensitization and for challenge. Guinea pigs are then treated by a series of six intradermal injections in the shoulder region to induce sensitization. After six to eight days, sensitization is boosted by a 48 hours occluded patch placed over the injection site. Twelve to fourteen days later, the animals are challenged on one flank by a 24 hours occluded patch at the maximum nonirritant concentration. Challenge sites are scored for erythema (scale 0-3) and edema 24 and 48 hours after removal of the patches. The EC guidelines state that a material is positive if the incidence is >/=30% (European Communities, 1993).

The standard Buehler test (BT) protocol uses an occluded topical patch technique for the induction and elicitation of contact sensitization (Buehler, 1965, 1985; Robinson et al, 1990). The procedure calls for 20 animals in the test (sensitized) group, 10 naive (control) animals for challenge, and 10 separate naive control animals for rechallenge. For induction, a single dorsal site is used for three six hour induction patches (applied occluded once per week to the same preshaven induction site on the dorsal surface of the test animals). Following a two week rest period, the test and noninduced control animals receive 6 hour challenge patches at a naive skin site for the primary challenge. The same test animals and additional new control animals can be rechallenged by this procedure 7-15 days after primary challenge at any remaining naive skin sites. Reactions are graded for erythema 24 and 48 hours after patch removal, according to a five point grading scale. The grades "1","2", and "3" denote increasing severity of erythema with grades >/="1" considered positive. The EC guidelines state that a material is positive if the incidence is >/=15% (European Communities, 1993).

In addition to comparison of the LLNA with guinea pig sensitization test, the LLNA has also been compared with human data (Basketter et al, 1994; Basketter et al, 1996). Specifically, the LLNA has been compared with the human maximization test (HMT) (Kligman, 1966a,b,c). This method was specifically designed to provide a rigorous assessment of the skin sensitization potential of chemicals in humans. In principle, a group of 25 subjects is subjected to 48 hour occlusive patch treatments with as high a concentration of test chemical as possible. This treatment is repeated five times over a two week period. If the substance is not sufficiently irritating, the irritancy is enhanced by prior treatment of the site for 24 hours with sodium lauryl sulfate prior to each 48 hour patch. The extent of sensitization in the panel is assessed by 48 hour treatments on a slightly irritated skin site using the maximum nonirritant concentration of the test substance. The challenge sites are scored at 48 hours and 96 hours post-application. In essence, this procedure can provide a stringent assessment of intrinsic sensitization hazard and its relative potency.

To define the role of the LLNA in predictive testing, results from the assay have been compared with predictions from guinea pig and human tests. In some instances, the LLNA results and the reference results (guinea pig or human) are presented together. In other cases, LLNA studies have been conducted with chemicals whose sensitization potential, or lack thereof, are well known. Basketter and Scholes (1992) investigated the correlation between results in the LLNA and those derived from the GPMT for materials that covered a range of chemical types and levels of skin sensitization potency. Kimber et al (1990) reported comparative analyses in which 24 chemicals of previously unknown contact sensitizing potential were evaluated in both the LLNA and the occluded patch test of Buehler. The data reported demonstrate that the LLNA identified successfully those chemicals that were classified as moderate or strong skin sensitizers in the Buehler test. Basketter et al, (1991) evaluated the performance of the LLNA with 25 chemicals for which GPMT or Buehler occluded patch test data were available. The 25 chemicals included preservatives, perfume ingredients, surfactants, plastics/resin chemicals, and oil additives. A high level of agreement between the results of LLNA and guinea pig test data was found.

As stated above, an essential point of comparison for the LLNA is with human data. Basketter et al (1994 and 1996) compared human maximization tests results with those obtained with the LLNA for the same 38 chemicals. The former being a rigorous assessment of the sensitization potential of chemicals in humans. The authors reported that the LLNA identifies those chemicals that are significant human contact allergens and that the specificity of the assay is good. A comprehensive review of published and unpublished LLNA data is given in Appendix A.

TEST METHOD RESULTS AND PERFORMANCE ASSESSMENT

The predictive power of the LLNA in comparison to standard guinea pig methods is given in Appendix B-Table 1. This type of information has been reviewed in detail in a recent paper (Basketter et al, 1996). While it is clear that the LLNA is not quite as sensitive as the GPMT, it is of similar or greater sensitivity than the Buehler test. It is important to note that this comparison is only true where the guinea pig tests have been conducted to the very highest standard. In terms of predictive identification of important skin sensitizers, the LLNA is at least as sensitive as, and much more reliable than, current guinea pig tests. Of the 130 chemicals tested in one of the reference guinea pig tests, approximately 88% gave the same result in the LLNA and the guinea pig tests. An overview of this information is contained in the 2 X 2 contingency table (Table 3).


Table 3: Comparison of LLNA and Guinea Pig Classifications

Guinea Pig Classificationa
LLNA Classification
Guinea Pig PositiveGuinea Pig NegativeUnclearTotal
LLNA Positive
86
6
0
92
LLNA Negative
10
28
0
38
total
96
34
0
130

Table Statistics for the Shadowed 2 X 2 Table

Sensitivity:90%
Specificity:82%
Prevalence:2.82
Positive predictivity:93%
Negative predictivity:74%
Accuracy:88%
X2:88% (p<0.001)

aGuinea pig classifications are based on GPMT or Buehler results - some of the results are derived from nonstandard GPMT guinea pig tests.


The 2 x 2 contingency table is a means to compare the in vivo classifications of skin sensitization of the guinea pig test with the in vivo predictions obtained in the LLNA. This procedure is recommended as a standard way of assessing data from validation studies (Balls et al, 1990). However, it is critical to point out that not all the guinea pig results are based on data generated by a standard protocol. Moreover, the guinea pig classifications are derived from both GPMT and Buehler studies. With these limitations in mind, the accuracy of the prediction of the LLNA amounts to 88%, With a sensitivity of 90% and a specificity of 82%. The test is characterized by a high positive predictivity of 93% and by a negative predictivity of 74%. Obviously, the LLNA does an excellent job of correctly identifying chemicals that are classified as skin sensitizers in the guinea pig tests. The high x2 value confirms that the classification of test chemicals by the LLNA is significant (p<0.001). Overall, the results given in Appendix B-Table 1, and Table 3 above, reveal a high level of concordance between the LLNA and guinea pig data in the determination of skin sensitization potential of a wide range of chemicals.

Appendix B-Table 2 lists those chemicals for which there is discord in results between the LLNA and guinea pig or human test methods. It is important to emphasize, however, that comparisons between LLNA data and the results of guinea pig tests should be viewed with caution. Guinea pig test data cannot be regarded as representing the 'gold standard' in skin sensitization testing. Thus, for instance, it should not be concluded that the failure of the LLNA to identify as a contact allergen a chemical that is known to elicit a positive response in a guinea pig test necessarily suggests a false negative in the former method. A case in point is sulfanilic acid, a chemical that is positive in the GPMT but which fails to provoke a response in the LLNA. There is compelling evidence that sulfanilic acid fails to induce allergic contact dermatitis in humans despite extensive occupational exposure (Basketter et al, 1992). In contrast to the case of sulfanilic acid, ammonium thioglycolate, a well described and important occupational contact allergen, notably among hairdressers, was positive in the LLNA, but was found not to give a significant response in the GPMT. This particular chemical would be expected to test positive in a predictive assay. Thus, the LLNA result is the correct one. Ethylene glycol dimethacrylate (EGDMA) produced a positive LLNA response, but was negative in guinea pig testing. Acrylate allergy is a complex subject, with many acrylate derivatives being suspected of giving rise to at least some degree of clinical disease. In the case of EGDMA, the LLNA result may be the more accurate reflection of the true characteristics of this substance as a potential human contact allergen; however, the clinical evidence is lacking.

Guinea pig or mouse data may not always mirror precisely and quantitatively the extent of the hazard to humans. Benzocaine, a substance selected as an OECD positive control for skin sensitization (OECD, 1993), has proven notoriously difficult to obtain reliable/reproducible positive results in either the LLNA or the GPMT (Basketter et al, 1993). Although it is well known as a skin sensitizer, the most common presentation arises from its use in puritis ani. In this situation, it is the repeated semi-occlusive exposure to inflamed mucosal tissue that renders a rather weak allergen positive. At the opposite end of the spectrum from ammonium thioglycolate, is the preservative propyl paraben. It is negative in both the LLNA and GPMT (Basketter and Scholes, 1992). This is not altogether surprising, except for behaving as a medicament allergen, notably in stasis ulcers, it is a very rare skin sensitizer, despite extensive dermal exposure, e.g. from cosmetics. The consequence, is that it is unreasonable to expect a normal predictive skin sensitization test to identify this substance as an allergen. Neither nickel chloride nor nickel sulphate produced clear positive results in the standard LLNA. In contrast, and although nickel has been documented as a difficult allergen in predictive tests (Wahlberg, 1989), positive results can be obtained in the GPMT. While nickel is a common allergen, it is not a strong allergen, since it is the extensive and intimate exposure (e.g. pierced ears) which result in the high incidence of allergy. Thus, the conclusion is that the failure of the LLNA to identify nickel salts as allergens is as unsuprising as it is unimportant.

Comparison of skin sensitization data from predictive tests such as the GPMT and the LLNA with human clinical information is far from simple. Clinical data are complicated by the varying nature and extent of exposure to which individuals may have been subjected together with their individual sensitivities. Thus, it is easy to confuse a strong allergen with a common one (e.g. nickel) or to expect that the parabens esters or lanolin should be positive in predictive tests because clinicians often refer to these as allergens. In this latter case, skin allergies do arise, but most commonly in a special group of patients (stasis eczema/medicament allergy) which cause dermatologists particular problems. However, it is evident from the large list of chemicals in Appendix B-Table 1, that the LLNA is quite capable of detecting essentially all of the major human contact allergens. It is worth repeating here what has been said elsewhere about metals -- that the precise mechanisms of metal allergy are probably rather different than those for organic chemicals; since it is known which metals are allergens and which are not, and given that new metals are not being invented, the ability of the LLNA, or indeed any other predictive sensitization assay, to detect metal allergens is rather irrelevant to the main need - the identification of new organic chemical skin sensitizers.

The data for the discordant results are reported in Appendix B-Table 3. Specifically, the disintegrations per minute (dpm) and stimulation indices (SI) are given for each concentration of test material tested. For comparison a positive control (hexyl cinnamic aldehyde) and negative control (para-aminobenzoic acid) are listed to illustrate typical results obtained in the LLNA. For the allergen benzocaine, one can see that the SI increase with increasing concentrations tested, but the threefold level is not reached and the material is classified as negative in the LLNA. In contrast, the irritant, sodium lauryl sulphate, leads to SI above the threefold level leading to its positive classification in the LLNA.

In relation to the mouse ear swelling test (MEST) (Gad et al, 1986), the LLNA offers several important animal welfare advantages, not least that unlike the MEST it does not use adjuvant. In addition, the state of validation of the MEST is quite preliminary. The data which does exist suggests that results are not wholly reliable, but clearly a great deal more work would be required to establish in detail its merits as a full replacement for the current guinea pig methods.

It is not expected, from our current knowledge of the mechanism of skin sensitization to organic chemicals, and what is known of the immunology of guinea pigs, mice, and man, that the LLNA will face special problems. Little is known of the impact of interspecies differences in skin metabolism of prohaptens and its importance in predictive testing. What limited information exists has suggested that there may be species differences (Bertrand et al, 1997) but examination of the concordance in the identification of skin sensitizers implies that these may not be of major practical importance.

One question commonly asked about skin sensitization tests concerns their ability to discriminate allergens from irritants. This question has been posed for the LLNA (Montelius et al, 1995), as it has for the GPMT (Kligman and Basketter, 1995; Buehler, 1996). In practice, all guinea pig skin sensitization tests may have such difficulties, and strategies for dealing with them are available (Kligman and Basketter, 1995; Frankild et al, 1996). The LLNA deals well with irritancy, it is not a confounding factor for dose selection and the majority of irritants are negative in the assay. Strategies for dealing with potential false positives in the LLNA and other predictive skin sensitization tests have been reviewed recently (Basketter et al, 1998).

If the LLNA is determined to be an acceptable alternative, then it will enhance further what is already happening, that this assay begins to be used ever more widely as the first choice method when it is necessary to assess skin sensitization potential of an unknown chemical. The limitations of the assay are minor compared with its advantages. They comprise the inability to evaluate the elicitation response and to test for cross challenge reactions. This latter item is of some use in research, but rarely forms part of testing for regulatory purposes, which is the season for this assay validation.

DATA INTERPRETATION

In the LLNA, skin sensitizing activity is measured as a function of proliferative activity induced in draining lymph nodes by repeated topical exposure of mice to a test chemical. For the purposes of developing a criterion for identification of contact allergens a stimulation index of 3, relative to background cell turnover measured in concurrent vehicle treated controls, was proposed as an empirical arbiter. This value was chosen on the basis of previous experience with the local lymph node assay and an apparent high level of discrimination between contact allergens and non-sensitizing chemicals. Since that proposal was first adopted in 1990, a number of independent laboratories has gained considerably greater experience with the method and in excess of one hundred additional chemicals have been tested. The accumulated evidence reveals that the use of a stimulation index of 3 continues to provide an accurate and reliable criterion for the identification of skin sensitizing chemicals. However, as discussed in a review article published in 1992 (Kimber and Basketter, 1992), while the threefold stimulation index provides a very useful criterion for judging sensitizing activity, in practice a dose-related increase in proliferative activity that approaches but does not reach, a stimulation index of 3 might trigger a repeat analysis using higher concentrations and/or an alternative application vehicle (Robinson and Cruze, 1996). In this context the potential utility of a higher or lower stimulation index for the identification of sensitizing activity has been considered, but there is no evidence that this would enhance further the specificity or selectivity of the method.

Whether the draining auricular lymph nodes are excised and pooled for each experimental group or for each individual animal, a stimulation index of 3 is used as the sole criterion against which to judge skin sensitizing activity. The use of statistical analysis for classifying the skin sensitization potential of chemicals is still under investigation. This is also the case for using EC3 values for determining the potency of a sensitizing chemical. Further research will be required to determine the usefulness of these approaches in LLNA testing. In the meantime, the approach is the use of the three-fold stimulation index.

In the standard LLNA protocol test, chemicals are evaluated using three application concentrations. In the vast majority of assays, conventional dose responses are recorded with sensitizing chemicals such that increasing concentrations of the allergen provoke increasingly more vigorous proliferative responses. In some instances the dose response profile may be relatively flat which suggests either that saturation kinetics for absorption have been achieved or that maximal immune stimulation has been induced. In such instances where a repeat analysis is performed using lower concentrations of the test chemical then invariably a conventional dose response profile is achieved. Very rarely there may be some indication at the top concentration of an inversed dose response. In these cases the cause is either local or systemic toxicity. Again, repeat studies conducted with reduced application concentrations yield normal dose responses. The LLNA is not associated normally, and certainly no more frequently, than any other biological analytical system, with ambiguous dose responses.

In conclusion, the view is that the LLNA should be employed as a 'stand-alone' method for reaching decisions about the skin sensitizing potential of chemicals. There would be no added value in using instead a battery of methods that included, with the LLNA for instance, analyses of skin penetration or identification of structural alerts using structure-activity relationships. The LLNA provides a holistic mechanistically-based assessment of the ability of a test chemical to provoke the cutaneous immune response necessary for the induction of contact sensitization. If the chemical tested fails to gain access through the skin or is unable to interact with protein to form an immunogenic hapten-macromolecular complex, then immune activation will not be initiated and sensitization will fail to develop. The current status of the LLNA and its application in regulatory toxicology has been reviewed in detail elsewhere (Basketter et al, 1996).

DATA QUALITY

Much of the data used here to support this submission and much of the data contained within the publications cited in this document have been derived from audited Good Laboratory Practices (GLP) compliant studies. Where this is not the case all investigations have been conducted to the spirit of GLP or Good Research Practice in GLP compliant facilities. Data quality audits, when conducted, have been satisfactory.

It is worth emphasizing that in all collaborative studies, both national and international, all data from each of the participating units have been made available to, and have been scrutinized by all laboratories.

There is now a long history of the local lymph node assay being used successfully in many independent laboratories for conduct of GLP compliant studies.

SUPPORTING MATERIALS

The LLNA is already mentioned in detail in the main internationally accepted regulatory guideline describing test methods, namely, by the OECD (1993), where it is presented as a screening method. It is also similarly represented in EU guidelines (EC, 1996). If the result is positive, then the chemical can be defined as a contact allergen. On the basis of this OECD update to the skin sensitization test guideline, the European Commission adopted the LLNA as a screening method acceptable for the identification of skin sensitizers which in its view should be formally classified and labeled as such (European Communities, 1993). Chemicals classified would carry the R43 risk phrase 'May cause sensitization by skin contact'. However, both the OECD and EC tests state that when the result of the LLNA is negative, it is necessary to conduct a confirmatory guinea pig test according to the standard protocol. It is important to point out that these guidelines were crafted before most of the LLNA validation work had been completed. In fact, the references cited in the OECD 406 guidelines dated from 1989 and 1990.

Recently Dr. Peter Evans (UK-Health and Safety Executive) stated that the LLNA has been extensively and rigorously validated against both animal and human data and that the assay should be adopted by the OECD and accepted by the EU as a suitable method for classification purposes for skin sensitization (Evans, 1998). In light of advancing knowledge and experience, and given animal welfare considerations, it is our opinion that the LLNA is now fully validated as a methodology for the identification of significant skin sensitizers and, therefore, should be adopted formally as an alternative skin sensitization test and incorporated fully into OECD Guideline 406.

Since the initial publication on the LLNA in 1986 by Kimber and his associates, there have been numerous publications addressing the immunological mechanisms underlying the assay as well as its use in regulatory toxicology. In Appendix A, a bibliography of 61 relevant publications is provided. These papers are related directly to the development of the LLNA for its use in assessing the skin sensitization potential of chemicals. Copies of ten selected manuscripts are included in Appendix C to permit reference to specific information supporting the validation of this assay for regulatory toxicology.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Rebecca J. Dearnan, Linda J. Lea, and Cindy A. Ryan for their contributions to the preparation of this submission.

REFERENCES

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