Guinea-pig Test Research Articles

Local Lymph Node Assays

The murine local lymph node assay (LLNA) allows identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. In this test, contact allergens are identified as a function of events occurring during the induction phase of skin sensitization; specifically, stimulation of proliferative responses in draining lymph nodes measured as a function of radiolabeled thymidine incorporation in situ. Two methods are presented. In one, lymph nodes are pooled on an experimental group basis, while in the other, lymph nodes of individual animal are pooled, which enables statistical analyses. These methods have been developed for contact sensitization hazard assessment and provide a robust and reliable method as an alternative to guinea pig tests which measure skin sensitizing potential as a function of challenge-induced skin reactions in previously sensitized animals. It is also possible to use LLNA data for measurement of relative potency in terms of risk assessment.

A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence.

Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains. To determine whether these observations could be extended to Shigella, a dam mutant of Shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella dam mutant showed some unique characteristics; however, it retained virulence in vivo as well as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent, but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However, the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam needs to be re-evaluated.

PCS.01 Risk assessment: a brief history for ACD

Risk assessments of chemicals are key issues in our live.Prior to industrialisation, risk assessments based on individual experience, for the allergic contact dermatitis (ACD) for instance, after contact with allergic plants affecting the skin exposed. Today, the risk to get an ADC becomes more and more important, because of increasing contact to new chemicals because of frequent contact with consumer products. Therefore sufficient test systems are required, appropriate to estimate the sensitisation potential of individual chemicals. To be able to predict weak sensitisers, adjuvants were simultaneously injected to test animals to booster their allergic reactions. One of the most well known test method, is the maximisation test in guinea pigs according to Magnusson and Kligman. Regulatory authorities have required this method for chemical classification, as well as for estimating the risk to get sensitised when dermaly exposed.Beside the advantages of these guinea pig studies, the limitations became prominent not only in respect to ethical aspects, but also because of the lack to quantify the sensitisation potency of chemicals. To be able to perform accurate, realistic risk assessments, the knowledge of potency is becoming mandatory, especially when sensitive test methods are used. Based on the possibility to measure the biological response during induction of sensitisation, the local lymph node assay (LLNA) gets popular. Today, this test is officially accepted by regulators to give information on skin sensitisation potential. With the quantitative approach on potency, risk assessment of ACD will become much more reliable and useful for Men.

Iosimenol, a low-viscosity nonionic dimer: preclinical physicochemistry, pharmacology, and pharmacokinetics.

Newer radiologic techniques require fast bolus injections and thus low-viscosity, high-concentration, well-tolerated contrast media (CM), especially in vulnerable patients. To this end, we designed and developed iosimenol, a novel isotonic nonionic dimer, and have conducted tests to enable its clinical evaluation. Standard physicochemical methods were used. Effects on erythrocyte morphology and coagulation were investigated in human and rat blood. Neural tolerance was assessed by behavioral tests in rats after intracisternal injection. Immunosensitizing potential was evaluated by the skin sensitization test in guinea pigs and by the popliteal lymph node assay in rats. Pharmacokinetics and biotransformation were investigated in rats and dogs. Iosimenol is extremely hydrophilic, it is less viscous than any other isotonic CM, has little effect on erythrocytes and blood coagulation, and has good neural tolerance. No immunosensitizing effect was found in validated animal models. Pharmacokinetics are identical with other angio- and urographic CM. Iosimenol is the only CM which, although isotonic, affords, unlike current nonionic dimers, at the same iodine concentration the low viscosity of monomeric, nonionic agents, which are all hypertonic. Iosimenol's pharmacologic characteristics closely resemble those of iotrolan and iodixanol.

QSAR Study of Skin Sensitization Using Local Lymph Node Assay Data

Allergic Contact Dermatitis (ACD) is a common work-related skin disease that often develops as a result of repetitive skin exposures to a sensitizing chemical agent. A variety of experimental tests have been suggested to assess the skin sensitization potential. We applied a method of Quantitative Structure-Activity Relationship (QSAR) to relate measured and calculated physical-chemical properties of chemical compounds to their sensitization potential. Using statistical methods, each of these properties, called molecular descriptors, was tested for its propensity to predict the sensitization potential. A few of the most informative descriptors were subsequently selected to build a model of skin sensitization. In this work sensitization data for the murine Local Lymph Node Assay (LLNA) were used. In principle, LLNA provides a standardized continuous scale suitable for quantitative assessment of skin sensitization. However, at present many LLNA results are still reported on a dichotomous scale, which is consistent with the scale of guinea pig tests, which were widely used in past years. Therefore, in this study only a dichotomous version of the LLNA data was used. To the statistical end, we relied on the logistic regression approach. This approach provides a statistical tool for investigating and predicting skin sensitization that is expressed only in categorical terms of activity and nonactivity. Based on the data of compounds used in this study, our results suggest a QSAR model of ACD that is based on the following descriptors: nDB (number of double bonds), C-003 (number of CHR3 molecular subfragments), GATS6M (autocorrelation coefficient) and HATS6m (GETAWAY descriptor), although the relevance of the identified descriptors to the continuous ACD QSAR has yet to be shown. The proposed QSAR model gives a percentage of positively predicted responses of 83% on the training set of compounds, and in cross validation it correctly identifies 79% of responses.

Inhalation Toxicology of Fumed Dodecanediamine

Dodecanediamine (DDDA) is used in the production of specialty polymers. Exposure to this chemical was associated with dermal sensitization in pilot-plant workers, and the possibility that the chemical could produce dermal sensitization was confirmed in a guinea pig test. This property and its dermal irritative properties demonstrate the need to limit skin contact. The possibility of exposure via inhalation also exists. Although stable under ambient conditions, DDDA is processed at elevated temperatures where it may fume, forming a carbamate after reaction with atmospheric carbon dioxide. Some of the carbamate may be converted back to the diamine after hydrolysis in tissue. The rat was used to evaluate the effects of both acute and repeated exposure following inhalation. Fumed DDDA was found to be moderately toxic following a single 4-h exposure with lethality seen at concentrations of 680 mg/m3 or higher. Rats were then exposed to concentrations of either 0 (control), 11, 34, or 98 mg fumed DDDA/m3, 6 h/day, 5 days/wk for 2 wk. Mortality was seen at the highest concentrations, along with increased lung weights. In these rats, laryngeal and tracheal lesions consisting of acute necrosis and inflammation were seen, but surviving rats given a 14-day recovery period showed almost complete recovery. Tracheal and laryngeal lesions were not seen in rats exposed to either 11 or 34 mg/m3. Degenerative and necrotizing lesions were seen in the nasal regions, primarily the respiratory mucosa, of rats in all three treatment groups. The lesions were exposure related with regard to incidence and severity, but regeneration was seen following the recovery period. No evidence of systemic toxicity was seen. The dose-response characteristics of the nasal lesions and the sensitization potential suggest that workplace control levels of 0.1 mg/m3 should be sufficient to protect workers against the untoward effects of fumed DDDA.

Classification of contact allergens according to potency: proposals

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of potency and categorisation. Included here are detailed and specific recommendations for how best the results of the three test methods considered can be used for the categorisation of chemical allergens as a function of skin sensitisation potency.

Final Report on the Amended Safety Assessment of Sodium Polynaphthalenesulfonate and Sodium Naphthalenesulfonate

Sodium Polynaphthalenesulfonate (SPNS) and Sodium Naphthalenesulfonate (SNS) are sodium salts of naphthalene sulfonic acid. SPNS was used as an emulsion stabilizer, surfactant—hydrotrope, and/or surfactant—suspending agent at concentrations between 0.1% and 0.4%, in a wide range of products, including one lipstick. SNS is described as a surfactant—hydrotrope; no current uses were reported, but information was provided indicating that use concentrations would be typically below 2%. SNS is manufactured by reacting naphthalene with sulfuric acid to produce a sulfonic acid, which is then reacted with sodium hydroxide to produce the final product. The polymer form uses the sulfonic acid intermediate in a reaction with formaldehyde and water under conditions of heat and pressure to form the polymer sulfonic acid form, to which sodium hydroxide is added to make the final SPNS. The residue level of formaldehyde was 0.09%. Only around 1% of SNS in a 1-mg/ml solution applied to porcine skin penetrated the skin after 24 h, a similar amount was found noncovalently bound to the skin, and the concentration of material applied to the surface of the skin was largely unchanged. Both chemicals were not toxic in acute oral or dermal studies. In a subchronic oral toxicity study in rats, the effects noted were increases in urinary sugar in females and urine protein concentrations in males. Although undiluted SPNS was not a significant eye irritant in rabbits, undiluted SNS was a moderate eye irritant in rabbits. At 2%, SNS was a minimal eye irritant in rabbits. Undiluted SNS was at most a mild irritant in Guinea pigs, and was nonirritating at 20% and 2%. In a delayed contact hypersensitivity test in Guinea pigs, 30% SNS used in the induction phase and in the challenge phase produced no reactions. In a Guinea pig maximization test, 1% SNS used with Freund's complete adjuvant (FCA) injected in the initial sensitization, 50% SNS applied topically in the second sensitization, and up to 30% SNS applied topically in the challenge phase did not produce any irritation or sensitization. Both ingredients were negative in Ames mutagenesis assays. In clinical studies, SNS was neither an irritant (tested up to 2%), cumulative irritant (tested up to 1%), nor a sensitizer (tested up to 1%). The Panel considered the low penetration in concert with the low concentrations of use of these ingredients and the absence of significant overall toxicity and the limited negative genotoxicity findings sufficient to support a conclusion that SNS and SPNS are safe as used in cosmetic formulations intended to be applied to the skin. Use of SPNS in a lipstick formulation, was not considered to be different from application to the skin in that the barrier properties of the skin do not apply when these ingredients may contact mucous membranes or may be ingested. Accordingly, the Panel concluded that the available data are insufficient to support the safety of SNS and SPNS in cosmetic formulations that may contact mucous membranes or be ingested. The additional data needed to make a safety assessment for these uses include dermal reproductive and developmental toxicity data and one genotoxicity assay in a mammalian system, and if that study is positive, then a 2-year dermal carcinogenicity study using National Toxicology Program (NTP) methods may be needed.

Final Report on the Amended Safety Assessment of Sodium Polynaphthalenesulfonate and Sodium Naphthalenesulfonate

Sodium Polynaphthalenesulfonate (SPNS) and Sodium Naphthalenesulfonate (SNS) are sodium salts of naphthalene sulfonic acid. SPNS was used as an emulsion stabilizer, surfactant--hydrotrope, and/or surfactant--suspending agent at concentrations between 0.1% and 0.4%, in a wide range of products, including one lipstick. SNS is described as a surfactant--hydrotrope; no current uses were reported, but information was provided indicating that use concentrations would be typically below 2%. SNS is manufactured by reacting naphthalene with sulfuric acid to produce a sulfonic acid, which is then reacted with sodium hydroxide to produce the final product. The polymer form uses the sulfonic acid intermediate in a reaction with formaldehyde and water under conditions of heat and pressure to form the polymer sulfonic acid form, to which sodium hydroxide is added to make the final SPNS. The residue level of formaldehyde was 0.09%. Only around 1% of SNS in a 1-mg/ml solution applied to porcine skin penetrated the skin after 24 h, a similar amount was found noncovalently bound to the skin, and the concentration of material applied to the surface of the skin was largely unchanged. Both chemicals were not toxic in acute oral or dermal studies. In a subchronic oral toxicity study in rats, the effects noted were increases in urinary sugar in females and urine protein concentrations in males. Although undiluted SPNS was not a significant eye irritant in rabbits, undiluted SNS was a moderate eye irritant in rabbits. At 2%, SNS was a minimal eye irritant in rabbits. Undiluted SNS was at most a mild irritant in Guinea pigs, and was nonirritating at 20% and 2%. In a delayed contact hypersensitivity test in Guinea pigs, 30% SNS used in the induction phase and in the challenge phase produced no reactions. In a Guinea pig maximization test, 1% SNS used with Freund's complete adjuvant (FCA) injected in the initial sensitization, 50% SNS applied topically in the second sensitization, and up to 30% SNS applied topically in the challenge phase did not produce any irritation or sensitization. Both ingredients were negative in Ames mutagenesis assays. In clinical studies, SNS was neither an irritant (tested up to 2%), cumulative irritant (tested up to 1%), nor a sensitizer (tested up to 1%). The Panel considered the low penetration in concert with the low concentrations of use of these ingredients and the absence of significant overall toxicity and the limited negative genotoxicity findings sufficient to support a conclusion that SNS and SPNS are safe as used in cosmetic formulations intended to be applied to the skin. Use of SPNS in a lipstick formulation, was not considered to be different from application to the skin in that the barrier properties of the skin do not apply when these ingredients may contact mucous membranes or may be ingested. Accordingly, the Panel concluded that the available data are insufficient to support the safety of SNS and SPNS in cosmetic formulations that may contact mucous membranes or be ingested. The additional data needed to make a safety assessment for these uses include dermal reproductive and developmental toxicity data and one genotoxicity assay in a mammalian system, and if that study is positive, then a 2-year dermal carcinogenicity study using National Toxicology Program (NTP) methods may be needed.

Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan.

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.

Copper hypersensitivity: dermatologic aspects--an overview.

Reports of immune hypersensitivity reactions of both the immediate and the delayed type following cutaneous or systemic exposure to copper are reviewed here in an endeavor to draw a comprehensive profile of the immunogenic potential of that metal and its compounds. The immunotoxic potential of the metal is also briefly reviewed. In principle, as noted for other transition metals, the electropositive copper ion is potentially immunogenic because of its ability to diffuse through biological membranes, forming complexes when in contact with tissue protein. Based on the results of the predictive guinea pig test and the local lymph node assay (LLNA), copper has a low sensitization potential. Reports of immune reactions to copper include immunologic contact urticaria (ICU), allergic contact dermatitis (ACD), systemic allergic reactions (SAR) and contact stomatitis (STO), but considering the widespread use of copper intrauterine devices (IUDs) and the importance of copper in coinage, items of personal adornment and industry, unambiguous reports of sensitization to the metal are extremely rare, and even fewer are the cases that appear clinically relevant. Most reports of immune reactions to copper describe systemic exposure as a cause--predominantly to intrauterine devices and to prosthetic materials in dentistry--implicitly excluding the induction of hypersensitivity from contact with the skin as a risk factor.

Potency Control of Diphtheria Component in Adsorbed Vaccines by In Vitro Neutralization Tests

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the χ2 test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved .

Joint Positive Control Testing in Guinea Pig Skin Sensitization Tests: A Harmonized Approach

A scheme for the performance of positive control studies within a coordinated group of laboratories was proposed (joint positive control testing). The procedure has been described, as well as the first results of the validation phase of this joint positive control testing project. Adoption of this proposal within the participating six laboratories would lead to a reduction in the number of guinea pigs required for reliability and sensitivity checks from current approximate 12 studies per year down to 2 studies, i.e., 150–300 fewer animals per year. Another benefit would be the use of a harmonized, and therefore more comparable, method to perform guinea pig tests and interpret the data. In the validation phase of joint reading of the positive control studies, the congruency of reading could clearly be demonstrated. From the experience gained up to now, it was possible to draw the conclusion that a coordinated interlaboratory approach for positive control testing was fully acceptable and an improvement with regard to animal welfare.

Local lymph node assay — validation, conduct and use in practice

The validation of alternative methods is a relatively new activity in toxicology. The local lymph node assay (LLNA), a novel method for the identification of chemicals that have the potential to cause skin sensitization, was the first test to pass through the formal regulatory validation process established in the USA under the auspices of ICCVAM, the Interagency Coordinating Committee on the Validation of Alternative Methods. ICCVAM approved the LLNA as an alternative to guinea pig tests for the identification of skin sensitisation hazards. In this report, we explore the nine recommendations made by ICCVAM and discuss their interpretation in relation to the new OECD Guideline 429, which describes the LLNA. In particular, the value and limitations of the use of statistical evaluation of data and of the inclusion of routine positive controls is examined. It is concluded that the OECD Guideline as currently written embodies the necessary flexibility to permit conduct of the LLNA in a manner necessary to meet the varying needs of regulatory agencies and toxicologists around the world.

Predictive testing in contact allergy: facts and future.

Predictive testing in contact allergy: facts and future.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs.

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 +/- 6 and 2 +/- 1 in phases 1 and 2) and lickings (6 +/- 2 and 1 +/- 1 in phases 1 and 2) in the formalin test when compared with control (75 +/- 13 and 22 +/- 6 flinches in phases 1 and 2; 28 +/- 7 and 17 +/- 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 +/- 0.1 and 0.53 +/- 0.13, respectively) when compared with control (-0.10 +/- 0.08 at 30 min and -0.09 +/- 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses.

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.

Fewer guinea-pigs

A new skin sensitization test for experimental animals, the murine local lymph node assay (LLNA), has won the SmithKline Beecham Laboratory Animal Welfare Prize (2000) for its originators Ian Kimber (Syngenta), Frank Gerberick (Procter & Gamble) and David Basketter (Unilever). The LLNA test obviates the need for the injection of test materials and circumvents the elicitation of allergic reactions, which accompany standard guinea-pig testing. The Research Defence Society, a UK-based organization that awards the prize annually, believes that the introduction of LLNA testing will reduce the number of animals being used for skin allergy testing in the future. AS

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.

Characterization of Bacillus anthracis strains used for vaccination.

Three Bacillus anthracis strains, formerly used as anti-anthrax vaccine strains in Argentina, were characterized from genetic and pathogenic perspectives. Southern blotting and PCR with pXO1 and pXO2 probes and primers, as well as pathogenicity and protection tests in guinea pigs and mice, were performed. Two of the B. anthracis strains contained both pXO1 and pXO2 plasmids, as did the fully virulent strains, while the third was a Sterne-type strain (pXO1+, pXO2-). The three strains were, however, markedly less pathogenic than a wild-type virulent strain. The methodology applied here may be used to characterize other B. anthracis strains.