Quantitative relationship between the local lymph node assay and human skin sensitization assays

The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to skin ("R43") in the European Union, by differentiated concentration limits derived from the limits for the four potency groups.     

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.     

Potency values from the local lymph node assay: Application to classification,labelling and risk assessment

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: ‘extreme’, ‘strong’, ‘moderate’ and ‘weak’. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.     

Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI = 3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI = 150 for CD86 (EC150), and for CD54 (EC200) with LLNA’s EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R = 0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.     

A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses.

Effective risk assessment and management of allergic contact dermatitis require three key factors: adequate hazard identification, measurement of the relative potency of identified hazards and an understanding of the nature, extent and duration of exposure. Suitable methods for hazard identification, such as the murine local lymph node assay (LLNA) and the guinea-pig maximization test, are well established and conditions of human exposure normally can be well anticipated. Thus, the need is for a robust and quantitative method for the estimation of relative skin sensitizing potency. One possible approach is via the analysis of LLNA dose-response data. In the LLNA, contact allergens are defined currently as those chemicals that cause a threefold or greater increase in lymph node cell proliferative activity compared with concurrent vehicle-treated controls. It is possible to estimate the concentration of a sensitizer required to generate a threefold stimulation of proliferation in draining lymph nodes; such a concentration is known as the EC3 value. Using a variety of statistical approaches to derive EC3 values from LLNA dose-response data for 10 chemicals, it has been demonstrated that simple linear interpolation between the values either side of the threefold stimulation index provides a robust assessment of the EC3 value without the need for recourse to more sophisticated statistical techniques. Provided that the appropriate concentrations of test chemical have been selected, EC3 values obtained in this way are reproducible both within and between laboratories and form the basis for examination of the utility of this approach for the estimation of relative skin sensitizing potency.     

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.     

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.     

Proposal for a risk assessment methodology for skin sensitization based on sensitization potency data

In this paper, we propose a quantitative risk assessment methodology for skin sensitization aiming at the derivation of 'safe' exposure levels for sensitizing chemicals, used e.g., as ingredients in consumer products. Given the limited number of sensitizers tested in human sensitization tests, such as the human repeat-insult patch test (HRIPT) or the human maximization test (HMT), we used EC3 values from the local lymph node assay (LLNA) in mice because they provide the best quantitative measure of the skin sensitizing potency of a chemical. A comparison of LLNA EC3 values with HRIPT and HMT LOEL, and NOEL values was carried out and revealed that the EC3, expressed as area dose, can be used as a surrogate value for the human NOEL in risk assessment. The uncertainty/extrapolation factor approach was used to derive (a) an 'acceptable non-sensitizing area dose' (ANSAD) to protect non-allergic individuals against skin sensitization and (b) an 'acceptable non-eliciting area dose' (ANEAD) to protect allergic individuals against elicitation of allergic contact dermatitis. For ANSAD derivation, interspecies, intraspecies and time extrapolation factors are applied to the LLNA EC3. For ANEAD derivation, additional application of a variable sensitization-elicitation extrapolation factor is proposed. Values for extrapolation factors are derived and discussed, the proposed methodology is applied to the sensitizers methylchloroisothiazolinone/methylisothiazolinone, cinnamic aldehyde and nickel and results are compared to published risk assessments.     

Introduction of a methoxymethyl side chain into p-phenylenediamine attenuates its sensitizing potency and reduces the risk of allergy induction

The strong sensitizing potencies of the most important primary intermediates of oxidative hair dyes, p-phenylenediamine (PPD) and p-toluylenediamine (PTD, i.e. 2-methyl-PPD) are well established. They are considered as the key sensitizers in hair dye allergic contact dermatitis. While modification of their molecular structure is expected to alter their sensitizing properties, it may also impair their color performance. With introduction of a methoxymethyl side chain we found the primary intermediate 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance but significantly reduced sensitizing properties compared to PPD and PTD: In vitro, ME-PPD showed an attenuated innate immune response when analyzed for its protein reactivity and dendritic cell activation potential. In vivo, the effective concentration of ME-PPD necessary to induce an immune response 3-fold above vehicle control (EC3 value) in the local lymph node assay (LLNA) was 4.3%, indicating a moderate skin sensitizing potency compared to values of 0.1 and 0.17% for PPD and PTD, respectively. Finally, assessing the skin sensitizing potency of ME-PPD under consumer hair dye usage conditions through a quantitative risk assessment (QRA) indicated an allergy induction risk negligible compared to PPD or PTD.     

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.     

Anchoring molecular mechanisms to the adverse outcome pathway for skin sensitization: Analysis of existing data

Allergic contact dermatitis (ACD) is a hypersensitivity immune response induced by small protein-reactive chemicals. Currently, the murine local lymph node assay (LLNA) provides hazard identification and quantitative estimation of sensitizing potency. Given the complexity of ACD, a single alternative method cannot replace the LLNA, but it is necessary to combine methods through an integrated testing strategy (ITS). In the development of an ITS, information regarding mechanisms and molecular processes involved in skin sensitization is crucial. The recently published adverse outcome pathway (AOP) for skin sensitization captures mechanistic knowledge into key events that lead to ACD. To understand the molecular processes in ACD, a systematic review of murine in vivo studies was performed and an ACD molecular map was constructed. In addition, comparing the molecular map to the limited human in vivo toxicogenomic data available suggests that certain processes are similarly triggered in mice and humans, but additional human data will be needed to confirm these findings and identify differences. To gain insight in the molecular mechanisms represented by various human in vitro systems, the map was compared to in vitro toxicogenomic data. This analysis allows for comparison of emerging in vitro methods on a molecular basis, in addition to mathematical predictive value. Finally, a survey of the current in silico, in chemico, and in vitro methods was used to indicate which AOP key event is modeled by each method. By anchoring emerging classification methods to the AOP and the ACD molecular map, complementing methods can be identified, which provides a cornerstone for the development of a testing strategy that accurately reflects the key events in skin sensitization.     

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM–H₂DCFDA), for 15 min, then incubated with test chemicals for 30 min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.     

Novel approach for classifying chemicals according to skin sensitizing potency by non-radioisotopic modification of the local lymph node assay

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone sensitization test for determining the sensitizing potential of chemicals, and it has the advantage of yielding a quantitative endpoint that can be used to predict the sensitization potency of chemicals. The EC3 has been proposed as a parameter for classifying chemicals according to the sensitization potency. We previously developed a non-radioisotopic endpoint for the LLNA based on 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA), and we are proposing a new procedure to predict the sensitization potency of chemicals based on comparisons with known human contact allergens. Nine chemicals (i.e. diphencyclopropenone, p-phenylenediamine, glutaraldehyde, cinnamicaldehyde, citral, eugenol, isopropyl myristate, propyleneglycol and hexane) categorized as human contact allergen classes 1-5 were tested by the non-RI LLNA with the following reference allergens: 2,4-dinitrochlorobenzene (DNCB) as a class 1 human contact allergen, isoeugenol as a class 2 human contact allergen and alpha-hexylcinnamic aldehyde (HCA) as a class 3 human contact allergen. Consequently, nine test chemicals were almost assigned to their correct allergen class. The results suggested that the new procedure for non-RI LLNA can provide correct sensitization potency data. Sensitization potency data are useful for evaluating the sensitization risk to humans of exposure to new chemical products. Accordingly, this approach would be an effective modification of LLNA with regard to its experimental design. Moreover, this procedure can be applied also to the standard LLNA with radioisotopes and to other modifications of the LLNA.     

Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency

The assessment of the potency of a skin sensitizing chemical is a key starting point for its subsequent risk assessment/management. The Local Lymph Node Assay can provide information on the relative skin sensitizing potency of contact allergens by interpolation from the dose response curve the concentration of a chemical required to elicit a threshold positive response (EC3 value). However, interpolation requires that the dose response curve have at least one stimulation index (SI) value above and one SI value below the threshold value of 3. For instances where all test concentrations result in SI values above 3, there was a need to develop a method that would permit estimation of EC3 values. This has been achieved by log-linear extrapolation using the two lowest test concentrations from the dose response curve. Before applying this approach, it is important that data quality is assessed. The dose response must include concentrations on the linear portion of the curve and, ideally, the SI induced by the lowest dose should approach 3. Judicious use of this approach for extrapolating EC3 values can provide information on a likely potency classification for use in risk assessment and may avoid the need for repeat animal testing.     

Effect of prolonged exposure to low antigen concentration for sensitization

The local lymph node assay (LLNA) is an assay in mice to identify potential allergens. Compounds that do not induce a stimulation index (SI)>or=3 are not considered sensitizers. Of the chemicals that do, the SI of 3 is used as a benchmark, and indicates the sensitizing potency of a chemical. Compared to the exposure duration of the LLNA (3 days), real life exposure often lasts for months or years. We therefore investigated whether prolonged exposure to sensitizers at concentrations that do not induce a SI>or=3 in the LLNA, were able to surpass this threshold. Mice were treated for 2 months at 7-day intervals with a range of concentrations of the known allergens ethyl-p-aminobenzoate (benzocaine, BENZ), 2,4-dinitrochlorobenzene (DNCB), and tetramethyl thiuram disulfide (TMTD). Both proliferative activity and cytokine production were established at day 60. Neither BENZ nor TMTD showed a significant increase in the proliferation rate compared to vehicle controls. Only DNCB at concentrations originally above the EC(3) a significant increase in proliferation was seen after prolonged exposure. No significant effect on IFN-gamma and IL-4 production was observed for all three compounds compared. These findings indicate that for classification of sensitizers the shorter exposure period employed in the standard LLNA is sufficient, and longer periods of exposure have no bearing on this classification.     

Examination of a vehicle for use with water soluble materials in the murine local lymph node assay.

The murine local lymph node assay (LLNA) is a validated method for identifying skin sensitization hazard. Vehicle choice can influence the sensitization potential of haptens in both the LLNA and in humans, therefore selection of an appropriate vehicle is important. Suggested vehicles for the LLNA include organic solvents and organic-aqueous mixtures. However, due to its high surface tension and poor wetting qualities, water is not recommended and therefore testing aqueous soluble materials can be problematic. The aims of this investigation were to identify a water-based vehicle that possesses better skin wetting properties than water alone, and to assess its performance relative to other solvents in the LLNA using aqueous soluble haptens. The selected wetting agent was the surfactant Pluronic(R) L92 (L92). Concentrations of L92 of up to 50% did not induce positive responses in the LLNA. 1% aqueous L92 was chosen for further examination. Dose-response analyses were performed with dinitrobenzene sulfonic acid (DNBS) and formaldehyde formulated either in water, 1% L92, dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). Potassium dichromate (PDC) and nickel sulfate were tested in 1% L92, DMSO or DMF. The highest concentration of potassium dichromate was retested in each vehicle and in water to assess the effect of the wetting agent. Estimates of the relative sensitizing potency in each vehicle were determined by calculation of EC3 values (the estimated concentration required to induce a threshold positive response). While DNBS and formaldehyde produced positive responses in all four vehicles, their relative potency varied among the vehicles. The rank ordering of potencies for both materials was, from highest to lowest, DMF > or = DMSO > 1% L92 > water. Compared with water, use of 1% L92 resulted in >2-fold increase in potency for DNBS and >3-fold increase for formaldehyde. PDC was positive in DMF, DMSO and 1% L92. The potency ranking was DMF > or = DMSO > 1% L92. Re-evaluation of 0.5% PDC confirmed that formulations of both DMSO and DMF induced strong proliferative responses, whereas somewhat less proliferation was recorded with the 1% L92 vehicle. PDC in water was without activity. The performance of 1% L92 as a vehicle for nickel sulfate was assessed relative to DMSO and DMF. In DMSO, nickel sulfate produced a stimulation index (SI) >3 at only the highest level. Testing in DMF induced low levels of proliferation, but failed to produce a SI of 3 at any concentration tested. When formulated in 1% L92, nickel sulfate caused a SI of 3 when tested at 2.5%. Based on the results of these experiments, for identification of sensitization hazard of aqueous soluble materials using the LLNA, DMF and DMSO are the preferred vehicles. However, if a test material is not soluble in DMF or DMSO, or if higher test concentrations can be achieved in an aqueous vehicle, then 1% L92 may provide a better alternative to water alone in terms of improved assay performance.     

Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation

For more than 15 years, the murine local lymph node assay (LLNA) has undergone development, evaluation and validation as an alternative approach to the predictive identification of skin sensitizing chemicals. The criteria by which sensitizing chemicals are distinguished from those without significant skin sensitising hazard were developed empirically and were based on experience rather than a mathematical formula or statistical method. The current practice is to classify, as skin sensitizers, those chemicals which at one or more test concentrations stimulate a threefold or greater increase in the proliferative activity in draining lymph node cells. Despite the apparent confirmation of the utility of this approach from the extensive data available, there has not previously been any attempt to substantiate the accuracy of this criterion. In this present investigations, data from 134 chemicals tested in the LLNA and in the guinea pig and/or for which there exists clear evidence relating to human skin sensitization potential, have been subjected to a rigorous statistical evaluation using Receiver Operating Characteristic (ROC) curves. Whether the analysis is based on a comparison with guinea pig or human data, the results indicate that the empirically derived threefold threshold is an acceptable practical value for hazard identification.     

Estimating skin sensitization potency from a single dose LLNA

Skin sensitization is an important aspect of safety assessment. The mouse local lymph node assay (LLNA) developed in the 1990s is an in vivo test used for skin sensitization hazard identification and characterization. More recently a reduced version of the LLNA (rLLNA) has been developed as a means of identifying, but not quantifying, sensitization hazard. The work presented here is aimed at enabling rLLNA data to be used to give quantitative potency information that can be used, inter alia, in modeling and read-across approaches to non-animal based potency estimation. A probit function has been derived enabling estimation of EC3 from a single dose. This has led to development of a modified version of the rLLNA, whereby as a general principle the SI value at 10%, or at a lower concentration if 10% is not testable, is used to calculate the EC3. This version of the rLLNA has been evaluated against a selection of chemicals for which full LLNA data are available, and has been shown to give EC3 values in good agreement with those derived from the full LLNA.