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G F Gerberick

Publications and source records attributed to G F Gerberick.

At least 37 records · Page 2Linked to original sources
PubMed2000

Use of the local lymph node assay for the estimation of relative contact allergenic potency.

The effective toxicological evaluation of skin sensitization demands that potential contact allergens are identified and that the likely risks of sensitization among exposed populations assessed. By definition, chemicals which possess the toxicological property of skin sensitization potentially are capable of causing allergic contact dermatitis (ACD) in humans. However, this hazard is not an all-or-none phenomenon; clear dose-response relationships can be discerned and thresholds identified for both the induction of sensitization and the elicitation of contact dermatitis. Commonly, these parameters are grouped under the heading of potency, determination of which is vital for risk assessment. In the present investigation, the local lymph node assay (LLNA) has been employed to determine the relative potency of a range of 20 chemicals. The parameter used is the estimated concentration required to produce a 3-fold increase in draining lymph-node cell proliferative activity, the EC3 value. These measurements have been compared with an assessment of the human sensitizing potency of the 20 selected chemicals, each being assigned to 1 of 5 classes based on their human sensitizing potency. The EC3 value, derived from LLNA work carried out in acetone/ olive oil vehicle, correlated well with the human classification, with the strongest sensitizers having low EC3 values (<O.1%), weaker sensitizers having EC3 values generally in the 1-10% range, and non-sensitizing chemicals having EC3 values in excess of 100%. In conclusion, the derivation of the EC3 for a chemical provides an objective and quantitative estimate of potency that is of considerable utility for skin sensitization risk assessment.

Allergens↗
PubMed2000

Activity of human contact allergens in the murine local lymph node assay.

The murine local lymph node assay (LLNA) is a predictive test for the identification of chemicals that have the potential to cause skin sensitization. Since its original development, the assay has been the subject of national and international evaluation studies and extensive comparisons with guinea pig tests and human data. On the basis of these investigations, the LLNA has recently been endorsed by ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) as a stand-alone method for skin sensitization hazard identification. At the same time, ICCVAM confirmed that, although the LLNA is not an in vitro method, it does represent a refinement in the way animals are used and can provide a means for reducing the number of animals used in sensitization hazard assessment. The investigations described here were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans. To that end we have measured, amongst 3 independent laboratories, LLNA responses induced by a total of 18 test chemicals, 11 of which are known to cause skin sensitization and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans. The LLNA correctly classified 16 of the 18 materials. The 11 chemicals tested which are associated with allergic contact dermatitis in humans were found to be positive in the LLNA. Of the 7 materials believed to be non-sensitizers, 5 were negative in the LLNA and 2 produced positive results. Collectively, these data provide additional evidence that the LLNA is able to discriminate skin sensitizers from those chemicals which do not possess a significant skin sensitization potential and thus provides a method for hazard identification that offers important animal welfare benefits.

Allergens↗
PubMed2000

A skin sensitization risk assessment approach for evaluation of new ingredients and products.

Skin sensitization risk assessment of new ingredients or products is critical before their introduction into the marketplace. The risk assessment process described in this article involves evaluation of skin sensitization hazard, consideration of all potential human exposures, comparative ingredient/product benchmarking, and, when appropriate, the management of the risk. In this article, a risk assessment process is reviewed along with a description of the risk assessment tools that are employed for evaluating a new ingredient or product. The basic process we use for evaluating the skin sensitization risk of a new product or ingredient is considered a no effect/safety factor approach. The tools used for conducting a risk assessment include structure activity relationship analysis, exposure assessment, preclinical testing (e.g., local lymph node assay [LNNA]) and clinical testing (e.g., human repeat insult patch testing [HRIPT]). The skin sensitization risk assessment process described in this paper has been used successfully for many years for the safe introduction of new products into the marketplace. This process is dynamic--it can be applied to a diversity of product categories (e.g., shampoo, transdermal drug). In summary, the skin sensitization risk assessment process described in this article allows one to carefully assess the skin sensitization potential of a new ingredient or product so that it can be safely introduced into the marketplace.

Allergens↗
PubMed2000

Local lymph node assay: validation assessment for regulatory purposes.

For the prediction of skin sensitization potential of substances, the murine local lymph node assay (LLNA) is an alternative to the widely used guinea pig tests. For more than 10 years, this method has undergone extensive development, evaluation, and validation. In this review, the validation status of the LLNA is considered, specifically with regard to its use for regulatory identification of skin sensitization hazards. The LLNA is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell proliferative responses stimulated by topical application of test chemicals. The LLNA has successfully passed all reasonable validation stages. It provides a reliable and relevant source of predictive skin sensitization data, which unlike results from guinea pig tests, are reproducible from laboratory to laboratory. In summary, the LLNA is now ready for acceptance as a viable and complete alternative to traditional methods, offering a substantial reduction in animal numbers and refinement opportunities without compromising the standards for the identification of important skin sensitizers.

Allergens↗
PubMed1999

Selective modulation of B-cell activation markers CD86 and I-Ak on murine draining lymph node cells following allergen or irritant treatment.

It is well known that T cells are key effector cells in the development of allergic contact dermatitis. However, we and others have shown that mice exposed to contact allergens show a preferential increase in B lymphocytes in the draining lymph nodes (DLN) as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. The purpose of the present investigation was to determine whether chemical allergens, in contrast to irritants, would modulate B-cell activation markers, CD86 and I-Ak, on B cells isolated from DLN of treated mice using the local lymph node assay (LLNA) protocol. Mice were treated on the ears for 3 consecutive days with concentrations of allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one, and trinitrochlorobenzene), or irritants (benzalkonium chloride and sodium lauryl sulfate), which caused an increase in the number of DLN cells. The DLN were excised 72 h following the final chemical treatment, and the cells were prepared for analysis by flow cytometry. In mice treated with allergens an increase in the median intensity of I-AK and CD86 on B220+ or IgG/IgM+ B cells was observed compared to mice treated with irritants or vehicles. Mice treated with allergens demonstrated an increase in the median intensity of CD86 on B220+ B cells that was dose dependent and peaked at 72 h following the final allergen treatment. The increase in the median intensity of I-AK also was dose dependent but peaked at 96 h. Finally, T and B cells isolated from both allergen- and irritant-treated mice demonstrated an increase in [3H]thymidine incorporation compared to vehicle-treated and naïve mice at 72 h following the final chemical treatment. The results suggest that B cells isolated from DLN of allergen-treated mice are activated and proliferating. Analysis of B-cell activation markers may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

Allergens↗
PubMed1999

Local lymph node assay: differentiating allergic and irritant responses using flow cytometry.

The murine local lymph node assay (LLNA) is a method for assessing the contact sensitization potential of chemicals. Based on events that occur during the induction phase of a contact sensitization response, the LLNA measures the in vivo proliferation of cells in the draining lymph nodes (DLNs) of mice following topical exposure to chemicals. In terms of predictive identification of important skin sensitizers, the LLNA has been shown to be at least as sensitive as, and much more reliable than, current guinea pig tests. However, proliferation has also been observed following treatment with some irritants. In an attempt to distinguish allergic from irritant-induced proliferation, flow cytometric techniques have been used to examine the phenotype of lymphocyte subsets in the DLNs as well as markers of T-lymphocyte activation and memory. Mice were treated on the ears for 3 consecutive days with allergens or irritants. The DLNs were harvested 72 h after the final treatment. Single-cell suspensions were prepared, counted, and stained for analysis of the percentages of T cells and B cells and T-cell expression of two adhesion molecules that have been associated with differentiating naïve and activated/memory T cells, CD62L (L-selectin) and CD44 (H-cam). Increases in lymph node cellularity were observed in both allergen- and irritant-treated mice relative to naïve and vehicle-treated animals. Mice treated with allergens showed a preferential increase in the percentage of B220(+) B cells compared with irritant-treated mice. Treatment with allergens, but not irritants, resulted in a selective increase in the percentages of CD4(+) and CD8(+) cells expressing the T-cell activation/memory phenotype CD62L(lo)CD44(hi). Taken together, flow cytometric analysis of cell phenotype and expression of T-cell activation/memory markers may provide important information for differentiating allergen- and irritant-induced proliferative responses in the DLNs of chemically treated mice.

Allergens↗
PubMed1999

Thresholds in contact sensitization: theoretical and practical considerations.

The existence of thresholds for both the induction and the elicitation of contact sensitization is an important issue for hazard assessment in this area of toxicology. In this paper, the evidence for such thresholds has been reviewed from both theoretical and practical perspectives. When the mechanisms of skin sensitization are considered, the existence of thresholds can be shown to occur at several stages. They are both quantitative, for example in terms of the degree of protein haptenation and in the sufficiency of the signals for Langerhans cell migration and maturation, as well as qualitative, in terms of the type of immune response that is engaged. Such considerations are evidenced by a substantial body of practical observation. In humans and in animal models of skin sensitization there is abundant evidence for the existence of thresholds for both the induction and the elicitation of reactions. In addition to, and in distinction from, the experimental situation, in the general human population there is extensive evidence for threshold effects. This evidence arises directly from the observation that only a proportion of those who are exposed become sensitized (i.e. are patch test positive), and of that latter group only a proportion develop allergic contact dermatitis.

Allergens↗
PubMed1999

Identification of metal allergens in the local lymph node assay.

BACKGROUND: The murine local lymph node assay (LLNA) has recently been endorsed as a validated alternative to guinea pig methods for the identification of skin sensitization hazard. Nevertheless, there has been some debate regarding the utility of this method for the detection of metal contact allergens. OBJECTIVE: In these investigations, we have used the LLNA to determine the skin sensitization potential of 13 metal salts, 8 of which were considered to possess a significant ability to sensitize man, whereas the remaining 5 were judged to lack such potential. RESULTS: The predictions from the LLNA were correct for 7 of the 8 (88%) sensitizing metals and for 4 of the 5 (80%) nonsensitizers when considered against the experience of these metals as human skin sensitizers. Thus, the overall predictive accuracy of the LLNA in relation to metals was 11/13 (85%), which is very similar to the accuracy of approximately 88% in relation to a much larger number of low-molecular-weight organic chemicals, as reported previously. CONCLUSION: These data provide support for the potential utility of the LLNA in hazard identification of metal contact allergens.

Allergens↗
PubMed1999

Cytokine mRNA expression in human epidermis after patch treatment with rhus and sodium lauryl sulfate.

BACKGROUND: Cytokines have been shown to play a pivotal role in the development and elicitation of contact hypersensitivity reactions. The sources of these cytokines in the skin include T cells, keratinocytes, and Langerhans cells. OBJECTIVE: In an effort to characterize the cytokines involved in the elicitation phase of a contact allergic response, we examined mRNA expression in human epidermis following patch testing with a known allergen and vehicle. METHODS: Allergic subjects were patch tested with poison ivy allergen (rhus), irritant (sodium lauryl sulfate [SLS]) and vehicle controls for 24 hours. Epidermal samples were obtained from the patch sites by a suction blister technique. Total RNA was isolated from the epidermis and the level of cytokine gene expression was determined using reverse transcriptase polymerase chain reaction (RT-PCR). PCR products for the various cytokines were confirmed and semiquantitated by liquid hybridization with (32)P-labeled product-specific probes. RESULTS: Results of liquid hybridization confirmed the presence of message for interleukin (IL)-2, IL-4 and IL-10 in rhus, SLS, and vehicle treated sites. Generally, in rhus treated sites, the steady state level of message for IL-2 was highest, followed by IL-4 and IL-10, in decreasing levels. In contrast, only minimal expression of mRNA for these cytokines was observed in irritant and vehicle treated sites. Interestingly, interferon (IFN)-gamma mRNA was not detected at 24 hours in rhus, SLS, or vehicle treated sites. CONCLUSION: These preliminary results indicate differences in the steady state levels of cytokine mRNA in allergen versus vehicle and irritant treated sites at 24 hours after treatment.

Cytokines↗
PubMed1998

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

Administration, Topical↗
PubMed1998

Strategies for identifying false positive responses in predictive skin sensitization tests.

It is important that predictive toxicological test methods are selective for their intended endpoint and that their limitations are understood and acknowledged. The local lymph node assay (LLNA) is a relatively new predictive test for skin sensitization potential that can replace traditional guinea pig tests and offers significant scientific and animal welfare advantages. However, there has been some concern that certain irritant materials may yield false positive results, although it must be emphasized that false positives also occur in guinea pig methods. Consequently, we have examined the performance in the LLNA of a range of skin irritants, from varying chemical classes and covering a range of irritation potency. The results presented here demonstrate clearly that the majority of skin irritants are negative in the LLNA. These results are reviewed in the context of the occurrence of false positive reactions in the guinea pig maximization test and the strategies for dealing with such results are discussed. The need for careful scientific evaluation of the results in all predictive tests for sensitization is thus emphasized. In terms of specificity, the LLNA has been more fully evaluated than other predictive test methods and is at least as accurate. In terms of animal welfare, objectivity, reproducibility and reliability it is superior to other methods. In summary, all predictive skin sensitization test results should be evaluated in a scientifically rigorous manner and the additional data provided herein further support the adoption of the LLNA as a complete replacement for the traditional guinea pig methods.

Animals↗
PubMed1998

In vitro and in vivo testing techniques for allergic contact dermatitis.

A variety of preclinical and clinical models have been developed for assessing the skin sensitization potential of chemicals. These models have been invaluable in identifying potential allergens and providing the information needed to conduct sound skin sensitization risk assessments. Of course, all models have inherent strengths and limitations. In the area of skin sensitization, significant advances have been made in understanding the underlying mechanisms of allergic contact dermatitis. This knowledge has been critical in the development of new in vitro and in vivo approaches for assessing the skin sensitization potential of chemicals. This article presents basic immunologic information that is being used to aid in the identification of cellular markers for differentiating irritant and allergic reactions in animals and humans. In addition, data are reviewed on the evaluation of skin equivalent cultures for the identification of skin allergens in vitro.

Animals↗
PubMed1997

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Allergens↗
PubMed1997

Selective modulation of T cell memory markers CD62L and CD44 on murine draining lymph node cells following allergen and irritant treatment.

Naive and activated T cells are known to express different adhesion molecules and are thought to exhibit different migratory patterns that result from their expression of discrete adhesion molecules. Two adhesion molecules that have been associated with differentiating naive and activated/memory T cells are CD62L (L-selectin) and CD44 (H-CAM). It has been demonstrated previously that naive T cells express a CD62LhiCD44lo phenotype, whereas memory T cells exhibit a CD62LloCD44hi phenotype. The purpose of the present investigation was to determine whether chemical allergens, in contrast to irritants, would induce a CD62LloCD44hi phenotype on CD4 and/or CD8 T cells isolated from draining lymph nodes (DLN) of treated mice. Mice were treated on the ears for 3 consecutive days with concentrations of allergens or irritants which caused an increase in the number of DLN cells. The DLN were excised 72 hr following the final chemical treatment and cells prepared for analysis by flow cytometry. In mice treated with the allergen trinitrochlorobenzene an increase in the percentage of CD4+ cells expressing CD62LloCD44(hi) was observed compared to cells isolated from mice treated with the irritant benzalkonium chloride or vehicle treated mice. Mice treated with dintrochlorobenzene had an increase in the percentage of CD4+ cells expressing CD62LloCD44(hi) that was dose dependent and peaked at 72 hr following the final allergen treatment. Concomitant with changes on CD4+ cells, increases in the percentage of CD8+ cells expressing CD62LloCD44hi were observed with allergens, but not with irritants. Increases in the percentage of CD4+ and CD8+ cells expressing CD62LloCD44(hi) were observed with other allergens including oxazolone and alpha-hexylcinnamaldehyde, but not the irritant sodium lauryl sulfate. These data demonstrate that allergens, but not irritants, cause a selective and reproducible increase in the percentage of CD4+ and CD8+ cells expressing the T cell activation/memory phenotype CD62LloCD44hi. Analysis of T cell activation/memory markers may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

Allergens↗
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

Phenotypic analysis of lymphocyte subpopulations in lymph nodes draining the ear following exposure to contact allergens and irritants.

The murine local lymph node assay (LLNA) measures in vivo proliferation in draining lymph nodes (DLN) following topical exposure to chemicals to assess contact sensitization potential. However, proliferation has also been observed with some irritants. To further characterize events in the DLN during the LLNA and distinguish allergens from irritants, phenotypic analysis of lymphocyte subsets was made following topical exposure. In preliminary studies, mice were treated on the ears for 3 consecutive days, and 48 hr following the final application, analysis of CD3, CD4, CD8, and B220 expression was evaluated by flow cytometry. The allergens oxazolone (OXAZ) and picryl chloride (TNCB) and the irritant benzalkonium chloride (BC) increased cell number compared to vehicle. The increase in lymph node cellularity for these materials was due to an increase in the total number of T and B lymphocytes. Interestingly, even though contact sensitization is a cell-mediated immune response (Th1), mice exposed to the contact allergens showed a preferential increase in B lymphocytes in the DLN as seen by an increase in the percentage of B220+ cells. The percentage of B220+ cells was 13.1 and 36.1% for OXA and TNCB, respectively, compared to percentages of 7.4 and 9.3% for irritant and vehicle, respectively. With some allergens, a concomitant decrease in the percentage of CD3+ cells was seen. Time course studies demonstrated the increase in the percentage of B220+ cells was seen in allergen treated mice by 24 hr after the final application of material, plateaued by 48 hr, and was still elevated by 96 hr. In allergen-treated mice, percentages of B220+ cells increased dose dependently. Further studies were performed to evaluate additional contact allergens and irritants and determine if evaluation of flow cytometric parameters could potentially identify contact allergens and differentiate them from irritants. Analysis of data from these studies, which examined a total of five contact allergens and six irritants, showed that the modifications to the LLNA improved the identification of irritants and allergens in individual experiments by including both phenotypic analysis of the DLN and cell number per node as endpoints rather than either endpoint alone.

Allergens↗
PubMed1996

The local lymph node assay: a viable alternative to currently accepted skin sensitization tests.

The prospective identification of skin sensitizing chemicals is a vital prerequisite for their proper risk management. Traditionally this has been achieved largely by the conduct of guinea pig assays such as the maximization and Buehler tests. These methods are recommended by the Organisation for Economic Cooperation and Development (OECD) and are required by the European Union (EU) for the evaluation of new substances. However, a novel mechanistically based method, the local lymph node assay (LLNA), has been the focus of substantial validation activity in recent years. This material is reviewed in this paper. It is shown that the LLNA has been validated successfully by five interlaboratory assessments as well as by comparisons with guinea pig tests and human data. The method also offers clear advantages to the user in terms of objectivity, time and cost, and delivers important animal welfare benefits. In consequence, it is recommended that the LLNA be formally adopted by the OECD in Guideline 406 and accepted by the EU and US EPA as a method suitable for the classification of the skin sensitizing potential of chemicals.

Allergens↗