Biomedical subjects

E W Scholes

Publications and source records attributed to E W Scholes.

17 recordsLinked to original sources

PubMed1996

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

Animals↗

PubMed1996

Experimental assessment of the sensitizing properties of formaldehyde.

Formaldehyde causes upper respiratory tract irritation and has been reported in some investigations to be a cause of occupational allergic asthma. The data are equivocal, however, and it has proved difficult to confirm that exposure to formaldehyde induces respiratory sensitization or provokes the production of specific immunoglobulin E (IgE) antibody. In this study the sensitizing properties of formaldehyde were examined experimentally. This chemical elicited strong positive responses in three independent methods for the prospective identification of contact sensitizing chemicals-the guinea pig maximization test, the occluded patch test of Buehler and the murine local lymph node assay. In contrast, in a novel predictive test method for assessment of respiratory sensitization potential-the mouse IgE test-formaldehyde at the same test concentrations was negative. Furthermore, formaldehyde induced in mice a pattern of cytokine secretion by draining lymph node cells inconsistent with the stimulation of IgE antibody responses or respiratory sensitization. These data indicate that, although formaldehyde is a potent contact allergen, it lacks a significant potential to cause sensitization of the respiratory tract.

Administration, Topical↗

PubMed1996

Dichloronitrobenzene: a reappraisal of its skin sensitization potential.

Unlike the closely related chemical dinitrochlorobenzene (DNCB), which is a very strong contact allergen, dichloronitrobenzene (DCNB) has been widely regarded as a non-allergen and, as such, a useful control for its strongly sensitizing counterpart. Nevertheless, it is still an organic chemical species readily capable of penetrating skin and, rather than being regarded as completely inert, it has even been suggested to react with the immune system in such a way that it induces specific tolerance to its chemical structure. We investigated whether DCNB was in reality a non-allergen, or rather merely a weak contact sensitizer. In both a rigorously conducted guinea pig maximization test and in a modified murine local lymph node assay, DCNB was demonstrated to possess weak sensitizing activity. On this basis, DCNB cannot be regarded as inert with respect to contact allergic potential, and is therefore inappropriate as a negative control in studies of skin sensitization.

Animals↗

PubMed1995

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Analysis of Variance↗

PubMed1995

An alternative strategy to the use of guinea pigs for the identification of skin sensitization hazard.

For over half a century, guinea pig methods have dominated the field of toxicology concerned with the identification of skin sensitizers. Specific protocols, for example the guinea pig maximization test (GPMT), have been pre-eminent in the identification of skin sensitization hazard for regulatory purposes. However, there are increasingly several forces driving change, not least animal use/welfare considerations. In response to this and to address the need for a rapid screen for chemical allergens, an alternative strategy has been developed. In the first instance, a chemical is assessed by a computer-based expert system. This system is constructed from some 50 rules describing the key chemically reactive substructures of known skin sensitizers. The output from the expert system is also evaluated in the light of the understanding of the skin penetration characteristics of the chemical. In this way, and without use of animals, the likelihood that a chemical represents a skin sensitization hazard is assessed based on the two key characteristics of a skin sensitizer: (1) its direct or indirect ability to react with skin protein (i.e. does it contain a structural alert?); and (2) the ability of the chemical to partition into the appropriate epidermal compartment. When the chemical does possess a structural alert and has the capacity to penetrate skin sufficiently, then it may be regarded as a potential skin sensitizer. Subsequent to this screening phase, if necessary the chemical may be assessed in the murine local lymph node assay. This assay is quicker and cheaper than traditional guinea pig assays and importantly is less stressful to the fewer animals that it requires. The assay is well validated and produces objective results which are equivalent to the GPMT in terms of identifying significant skin sensitization hazard. In this paper, the above strategy is described in more detail, focusing on its relevance to hazard identification and its value in animal welfare terms. It is concluded that the strategy provides an important opportunity for both substantial reduction and refinement of animal use in a manner which will not compromise the existing standard of classification and labelling of skin sensitization hazard in the European Union.

Animal Testing Alternatives↗

PubMed1995

An evaluation of the suitability of benzocaine as a positive control skin sensitizer.

Although they are subject to some limitations, sensitization tests such as the guinea pig maximization test (GPMT) have for many years provided a valuable basis for the identification of skin sensitization potential. Thus they have been used widely by regulatory authorities, such as those in Europe, as a means to identify significant sensitization hazards associated with new chemicals. However, the standard of performance of guinea pig sensitization assays has been demonstrated to be widely variable. Consequently, the OECD sensitization test guideline (the de facto world standard) has been updated to incorporate recommendations for action whose aim is to achieve a minimum standard of test conduct. The principle is that a test laboratory should be able to demonstrate an acceptable level of response using a moderately sensitizing chemical. A list of 3 such chemicals is provided, hexyl cinnamic aldehyde, mercaptobenzothiazole and benzocaine. It is our experience that whilst good results can readily be obtained with the first 2 of these, benzocaine is much more difficult. Using both the GPMT and the local lymph node assay (LLNA), an OECD-recommended screening test, benzocaine has given highly variable results. A range of from 0% to 60% positive in the GPMT was found and, in most tests, benzocaine would not classify as a skin sensitizer according to EU criteria. In the LLNA, from a series of 12 tests conducted in 2 laboratories, only occasional positive results were obtained. Furthermore, these positive results were not reproducible. Reasons for this variability are discussed. However, the main conclusion must be that benzocaine does not represent a useful moderately sensitizing positive control.

Anesthetics, Local↗

PubMed1994

The local lymph node assay: developments and applications.

The murine local lymph node assay is a predictive test method for the identification of contact allergens in which sensitizing activity is measured as a function of induced proliferative responses in lymph nodes draining the site of application. In this article the development and validation of the assay are described and comparisons with guinea pig predictive test methods discussed. In addition we examine the advantages and limitations of the method and consider new opportunities and applications of the assay in the context of the toxicological evaluation of sensitizing potential.

Allergens↗

PubMed1994

The performance of the local lymph node assay with chemicals identified as contact allergens in the human maximization test.

For many years, tests in the guinea pig have been the favoured option for the identification of the skin sensitization potential of chemicals. However, the mouse has been used widely in immunology research and can represent a viable alternative. A variety of murine assays have been described, including several methods based on ear swelling as an endpoint. Another option is to assess induced lymph node cell proliferation and it is this which forms the basis of the murine local lymph node assay (LLNA). The LLNA has undergone several successful interlaboratory validations and compares well with standard guinea pig assays. In the present study, the performance of the LLNA was examined with chemicals tested previously in the human maximization test (HMT). 30 chemicals, 23 of which proved positive in the HMT and seven of which were negative, have been tested. All but four of the materials found positive in the HMT also tested positive in the LLNA. Of these four, sulfanilamide and paraben esters would not classify as skin sensitizers in the guinea pig maximization test and nickel has been found to yield variable results in a number of predictive animal tests. Of the seven substances which proved negative in the HMT, six were also negative in the LLNA, the exception being sodium dodecyl sulfate. These data demonstrate that the LLNA is able to identify accurately chemicals which have the potential to cause significant allergic contact dermatitis in humans.

Allergens↗

PubMed1993

Influence of sodium lauryl sulphate on 2,4-dinitrochlorobenzene-induced lymph node activation.

The influence of the anionic surfactant sodium lauryl sulphate (SLS) on the ability of the contact allergen 2,4-dinitrochlorobenzene (DNCB) to provoke draining lymph node cell proliferative responses, a correlate of skin sensitizing potential, has been examined in mice. Topical application of 10% SLS with 0.1% DNCB caused a more vigorous proliferative response than did exposure to 0.1% DNCB alone. Lower concentrations (0.1% or 1%) of SLS were ineffective and 10% SLS failed to influence proliferative responses to higher concentrations (0.5% or 1%) of DNCB. Using an in vitro model for measurement of percutaneous absorption 10% SLS was shown not to increase the skin penetration of 0.1% DNCB. We therefore examined the influence of SLS on the accumulation of dendritic cells (DC) in lymph nodes draining the site of exposure, an important early event during the induction phase of skin sensitization. The frequency of DC in draining nodes was measured following topical application of SLS, DNCB or a combination of both. Epicutaneous exposure to 0.1% DNCB caused only a modest increase in the number of lymph node DC. However, 10% SLS or a mixture of 10% SLS with 0.1% DNCB each resulted in a significant elevation of DC numbers. It is proposed that SLS augments the skin sensitizing potential of sub-irritant concentrations of DNCB via an increase in the number of immunostimulatory DC which reach the draining nodes.

Administration, Cutaneous↗

PubMed1993

Results with OECD recommended positive control sensitizers in the maximization, Buehler and local lymph node assays.

The guinea pig maximization test and the Buehler occluded patch test are used widely to identify the sensitization potential of new chemicals. This information enables toxicologists and/or regulatory authorities to determine whether a chemical should be classified formally as a skin sensitizer. Both to improve and to harmonize these assessments internationally, the OECD has recommended recently that moderate rather than strong contact sensitizers are used as positive control substances. The purpose is to ensure an adequate level of sensitivity in sensitization assays performed at specific testing establishments. Results from two laboratories reported here show that the minimum acceptable standard laid down by the OECD can be achieved and indeed commonly exceeded by a substantial margin. Furthermore, results with these positive controls in a new method, the local lymph node assay, also appear to satisfy similar criteria, suggesting results from this assay, including negative data, should be acceptable for classification purposes. However, a review of the way in which results with new chemicals will be interpreted for regulatory purposes, in the context of positive control data, reveals that considerable inadequacies still exist. It is recommended that ultimately, sensitization data can only be interpreted meaningfully (i.e. to protect humans from sensitization hazards) by considering the potency of the contact allergen in the context of the sensitivity of the assay performed at the particular testing institution.

Acrolein↗

PubMed1992

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.

Animals↗

PubMed1992

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

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

Allergens↗

PubMed1992

Sulphanilic acid: divergent results in the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has proven to be a valuable tool for the identification of the skin sensitization potential of chemicals. The method identifies a hazard which can lead in the EC to compulsory labelling of that chemical. In the present study, data on sulphanilic acid derived from the GPMT has been compared with results from a second guinea pig assay (the cumulative contact enhancement test) and the murine local lymph node assay, both of which require only topical application of chemical. Except for the GPMT, no test identified any sensitizing activity associated with exposure to sulphanilic acid. These latter results are consistent with the experience gained from substantial human exposure in an occupational setting and from which no cases of allergic contact dermatitis to sulphanilic acid have arisen over a 20-year period. In consequence, it is questioned which test protocol in practice has given the more accurate identification of sensitization hazard relevant to man.

Administration, Topical↗

PubMed1992

The value of the local lymph node assay in quantitative structure-activity investigations.

The development of quantitative correlations between the physicochemical properties of a compound and its ability to act as a skin sensitizer is complicated by the number of variables associated with the current sensitization test data, combined with the absence of a truly objective end point. Recently, however, a novel approach to the assessment of skin sensitization potential, the local lymph node assay (LLNA), has been described, which determines the skin sensitization by measuring lymphocyte proliferation in lymph nodes draining the site of chemical exposure. The assay offers several advantages over traditional methods in the context of quantitative structure-activity relationship studies. In the present work, a range of bromoalkanes has been employed which demonstrate the robustness and reproducibility of the LLNA. Sensitizing activity increased with chain length up to a maximum at C15/C16, whereafter the response declined. The data were modelled against hydrophobicity, expressed as Clog P and (ClogP)2 to fit the biphasic nature of the results. The results demonstrate the utility of LLNA data for interpretation in the context of quantitative structure-activity relationships, the limited number of variables, inter-test reproducibility and quantitative end point, lending themselves to mathematical interpretations.

Animals↗

PubMed1991

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.

Allergens↗

PubMed1991

The identification of photoallergic potential in the local lymph node assay.

Guinea pig test methods are the most commonly used and reliable of predictive models for contact photoallergenicity of chemicals. The murine local lymph node assay (LLNA) has been developed recently as an alternative method for the identification of skin-sensitizing chemicals. Sensitization potential is measured from an assessment of the proliferation of lymphocytes in lymph nodes draining the site of exposure to the test chemical. This work investigates the activity of 6 widely reported photoactive chemicals in a modified LLNA (a photo-LLNA). The photoallergens tetrachlorosalicylanilide and fentichlor elicited positive ultraviolet radiation (UV)-dependent proliferative responses that were greater than their positive UV-independent responses, suggesting that they are both contact and photoallergic in the mouse. The lack of a proliferative response to 6-methylcoumarin and the absence of a reproducible response to musk ambrette suggest that the assay is insufficiently sensitive to identify weak photoallergic potential. Positive UV-dependent responses to acridine and anthracene, both photoirritants, cast doubt on the specificity of the photo-LLNA. Positive LLNA responses to these chemicals may be due to skin protein modification, based on evidence from the in vitro photo-chemical protein-binding assay. These results demonstrate that the photo-LLNA is able to detect at least moderate photoallergic potential.

Acridines↗

PubMedDate not supplied

The local lymph node assay: an interlaboratory evaluation of interleukin 6 (IL-6) production by draining lymph node cells.

The murine local lymph node assay is a predictive method for the identification of skin-sensitizing chemicals in which activity is measured as a function of proliferative activity induced in lymph nodes draining the site of exposure. In the present study, the induction by topically applied chemicals of draining lymph node cell (LNC) production of the cytokine interleukin 6 (IL-6) has been evaluated as an alternative endpoint for the local lymph node assay. In addition, results derived from studies of IL-6 production by LNC performed independently in two separate collaborating laboratories have been compared. Of the nine skin sensitizing chemicals examined, six provoked detectable levels (> 150 pg ml-1) of IL-6 production by draining LNCs (as measured by enzyme-linked immunosorbent assay) following exposure of mice to at least one test concentration of the material in both of the laboratories. Three other sensitizing chemicals failed to induce measurable IL-6 production at any test concentration in either one or both of the participating laboratories. Both of the non-sensitizing chemicals evaluated (sodium lauryl sulphate and methyl salicylate) also failed to result in detectable IL-6 synthesis. There was a high level of agreement between the two laboratories. The rank order of chemicals with respect to IL-6 production by LNCs was identical in both cases, as was the dose-response relationship observed with each test material. These data reveal that, although inducible IL-6 production by draining LNCs provides a robust approach to the measurement of strong sensitizing activity, as performed here the method is of insufficient sensitivity for the routine identification of skin allergens.

Allergens↗