Decision making in next generation risk assessment for skin allergy: Using historical clinical experience to benchmark risk.

Our aim is to develop and apply next generation approaches to skin allergy risk assessment that do not require new animal test data and better quantify uncertainties. Quantitative risk assessment for skin sensitisation uses safety assessment factors to extrapolate from the point of departure to an acceptable human exposure level. It is currently unclear whether these safety assessment factors are appropriate when using non-animal test data to derive a point-of departure. Our skin allergy risk assessment model Defined Approach uses Bayesian statistics to infer a human-relevant metric of sensitiser potency with explicit quantification of uncertainty, using any combination of human repeat insult patch test, local lymph node assay, direct peptide reactivity assay, KeratinoSens™, h-CLAT or U-SENS™ data. Here we describe the incorporation of benchmark exposures pertaining to use of consumer products with clinical data supporting a high/low risk categorisation for skin sensitisation. Margins-of-exposure (potency estimate to consumer exposure level ratio) are regressed against the benchmark risk classifications, enabling derivation of a risk metric defined as the probability that an exposure is low risk. This approach circumvents the use of safety assessment factors and provides a simple and transparent mechanism whereby clinical experience can directly feed-back into risk assessment decisions.

Next generation risk assessment for skin allergy: Decision making using new approach methodologies.

Our aim is to develop and apply next generation approaches to skin allergy risk assessment (SARA) that do not require new animal test data and better quantify uncertainties. Significant progress has been made in the development of New Approach Methodologies (NAMs), non-animal test methods, for assessment of skin sensitisation and there is now focus on their application to derive potency information for use in Next Generation Risk Assessment (NGRA). The SARA model utilises a Bayesian statistical approach to infer a human-relevant metric of sensitiser potency and a measure of risk associated with a given consumer exposure based upon any combination of human repeat insult patch test, local lymph node, direct peptide reactivity assay, KeratinoSens™, h-CLAT or U-SENS™ data. Here we have applied the SARA model within our weight of evidence NGRA framework for skin allergy to three case study materials in four consumer products. Highlighting how to structure the risk assessment, apply NAMs to derive a point of departure and conclude on consumer safety risk. NGRA based upon NAMs were, for these exposures, at least as protective as the historical risk assessment approaches. Through such case studies we are building our confidence in using NAMs for skin allergy risk assessment.

A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

A comparison of TRECs and flow cytometry for naive T cell quantification.

Assessment of thymic output by measurement of naive T cells is carried out routinely in clinical diagnostic laboratories, predominantly using flow cytometry with a suitable panel of antibodies. Naive T cell measurements can also be made using molecular analyses to quantify T cell receptor excision circle (TRECs) levels in sorted cells from peripheral blood. In this study we have compared TRECs levels retrospectively with CD45RA+ CD27+ T cells and also with CD45RA+ CD31+ T cells in 134 patient samples at diagnosis or during follow-up. Both panels provide naive T cell measurements that have a strongly positive correlation with TRECs numbers and are suitable for use with enumerating naive T cell levels in a clinical laboratory.

Causality methods in cosmetovigilance: comparison of Colipa and PLM versus global introspection.

The European Cosmetics Regulation requires a post-marketing system for detection of undesirable effects on human health of cosmetic products. Colipa, the European Cosmetic, toiletry and perfumery association, provided guidelines for causality assessment of these effects. In addition another causality method originally designed for causality rating in Post Launch Monitoring (PLM) of novel foods has been employed to assess causality of cosmetic products. In this study these two causality schemes for consumer cosmetic products were validated against clinical assessment, using the method of global introspection (GI) in 100 reported cases. Causality assessments were performed by three experienced assessors in pharmacovigilance. In the event of discordance between the assessors, an adapted Delphi method was used. The overall Spearman correlation coefficient was 0.74 for comparison of Colipa versus GI, whereas this was 0.50 for PLM versus GI. According to current guidelines, the sensitivity was 0.95 for both the Colipa and PLM method, specificity was 0.84 for Colipa and 0.40 for PLM. From these results it can be concluded the performance of the Colipa causality method yielded better correlation to GI than PLM causality method. The factor identified from comparison of these two schemes as having greatest impact was the course of the reaction.

Refinement of the Dermal Sensitisation Threshold (DST) approach using a larger dataset and incorporating mechanistic chemistry domains.

An essential step in ensuring the toxicological safety of ingredients in consumer products is the evaluation of their skin sensitising potential. Where skin exposure is low, it is possible to conduct a risk assessment using the Dermal Sensitisation Threshold (DST), a process similar to that of the Threshold of Toxicological Concern. This paper describes work building on that previously published, whose aim was to produce a more robust tool for assessing the safety of consumer products. This consisted of expanding the Local Lymph Node Assay dataset used to define the original DST and classifying chemicals in the dataset according to their mechanistic chemistry domains. A DST of 900µg/cm(2) was derived for chemicals classified as non-reactive and non-proreactive. This value was benchmarked against human potency data for 58 fragrance allergens and was lower than the measured No Expected Sensitisation Levels for those classified as non-reactive. Use of this DST will help to eliminate the need for animal testing of non-reactive and non-proreactive chemicals where skin exposure is sufficiently low. For chemicals where a Quantitative Risk Assessment based on the DST does not give an adequate margin of safety, and those classified as reactive, a case-by-case risk assessment will be required.

A general population from Thailand: incidence of common allergens with emphasis on para-phenylenediamine.

BACKGROUND: Most studies on the prevalence of allergy to the permanent hair dye chemical para-phenylenediamine (PPD) are reported from populations of eczema patients attending patch-test clinics, and are assumed to be much higher than in the normal population. No data exist on incidence of senitization to PPD resulting from the use of commercial hair dye preparations over a defined time period. METHOD: A total of 2545 healthy adult volunteers (Bangkok) were screened for PPD allergy through standard patch testing. Volunteers not allergic to PPD were then recruited into two groups: one group applying a commercial hair dye brand as instructed on a monthly basis for 6 months (n=548) and the other group (controls) (n=516) was instructed not to dye their hair for 6 months. Sensitization to PPD resulting from the use of hair dye over this period was then detected by repeat patch testing. RESULTS: The prevalence of PPD allergy in a normal adult population was 2.7% (m=2.4%, f=3.2%). Projected to the adult Thai population, at least 1,000,000 Thai individuals could be allergic to PPD. The incidence of sensitization through the monthly application of standard commercial hair dye preparations over a 6-month period was 1.3%, substantially higher than in controls (0.4%), although numbers were small and not statistically significant. INTERPRETATION: There is a higher prevalence of hair dye allergy among the normal population than previously thought. The incidence of new cases of PPD allergy would indicate that current regulations and practice of hair dye exposure lead to PPD sensitization and allergy, which is a public health problem.

p-Phenylenediamine allergy: the role of Bandrowski's base.

p-Phenylenediamine (PPD) is a commonly used hair-dye and a potent skin allergen. The mechanism of sensitization is unknown, as PPD is protein unreactive. We studied Bandrowski's base (BB), a PPD trimer, as well as 1,4-benzoquinone (BQ), a PPD hapten. PPD patch-test positive patients were patch-tested to BB and BQ. All tests were negative to 0.01% BQ and 0.01% BB. Five of 14 (35.7%) tested had true positive reactions to 0.1% BQ. One percent BQ was found to be irritant. Seven of 43 tested (16%) were positive to either 0.1% or 1% BB. The positive reactions to BB were weak, even when PPD reactions were strong. Mice lymph node assay gave EC3 values of 0.14% for PPD compared with 0.03% for BB. Therefore, BB is approximately 10 times more potent than PPD, taking into account the molarity. We suggest that while PPD may act as a prohapten, there is probably a spectrum of antigenic determinants in vivo. BB may be bound or metabolized by keratinocytes before it reacts with Langerhans cells.

Utility of historical vehicle-control data in the interpretation of the local lymph node assay.

The accepted approach to the interpretation of local lymph node assay (LLNA) data requires comparison of responses in the test groups with background activity found in concurrent vehicle-treated controls. However, of established value in the interpretation of toxicity test data is the use of historical control values that provide one criterion against which to judge the integrity of individual experiments. Specifically, the availability of robust and relevant historical control data permits examination of whether, in any individual experiment, control values fall within the expected range. With the most commonly used vehicle employed in the LLNA, acetone/olive oil (4 : 1) (v/v), the mean values, standard deviations and normal ranges are increasingly well established for a given laboratory, although there is some variation between laboratories, particularly with regard to expected ranges. Against this background, it is possible to identify (and, if appropriate, eliminate) a concurrent vehicle-control value that falls well outside the expected range. To explore critically the potential merits of this approach, one specific example is examined in detail.

Vaccine containing iron-regulated proteins of Pasteurella haemolytica A2 enhances protection against experimental pasteurellosis in lambs.

A vaccine containing sodium salicylate extract (SSE) of Pasteurella haemolytica A2 cells grown in a medium chemically depleted of available iron by the addition of alpha alpha dipyridyl to induce iron-regulated proteins (IRPs) conferred protection to specific pathogen-free (SPF) lambs exposed to an aerosol of P. haemolytica A2. The disease score in these lambs was significantly lower (p less than 0.005) than those in unvaccinated lambs or in lambs immunized with SSE prepared from cells grown in iron-replete medium. Immunoblotting of sera from these SPF lambs against whole cell antigens of P. haemolytica A2 grown under iron-restricted conditions demonstrated that antibodies to IRPs were present only in the sera of animals immunized with SSE-IRP. The antibody profile of sera from the SSE-IRP group was similar to that obtained with serum from a lamb which had recovered from P. haemolytica A2 disease produced experimentally. Negligible levels of cytotoxin-neutralizing and bactericidal antibodies were detectable in the SSE-IRP group and therefore appear not to be involved in the protection observed in this experiment.

Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody.

Eight monoclonal antibodies (MAbs) were produced from mice immunised with whole cells of heat-killed Pasteurella multocida type A which had been cultured under iron-restricted conditions. The MAbs were selected by an enzyme-linked immunosorbent assay (ELISA) in which the antigen consisted of whole bacteria of the immunising strain. Their reactivity was investigated further by immunoblotting, indirect haemagglutination, a complement-mediated bactericidal assay and passive protection of mice. One of the eight MAbs was shown by immunoblotting to react with lipopolysaccharide (LPS), was bactericidal, and completely protected mice against homologous challenge with 10 LD50 of live bacteria. This MAb was selected for further study. Its reaction with LPS of 17 type-A strains and of single strains of types B, D and E was investigated by immunoblotting. Strains that reacted with the anti-LPS MAb in immunoblots were susceptible to its bactericidal activity and gave high ELISA absorbances. Those that did not react were not susceptible to its bactericidal activity and gave low ELISA readings. The relation between bactericidal activity and ELISA absorbance was highly significant (p less than 0.001). Five of the strongly reacting heterologous strains and one non-reacting strain were selected as challenge organisms in a passive protection experiment: only the mice receiving the reacting strains were protected.

Outer membrane proteins of bovine strains of Pasteurella multocida type A and their doubtful role as protective antigens.

Outer membranes were prepared by the Sarkosyl method from 30 strains of Pasteurella multocida and the closely related Taxon 13, which had been isolated from cattle. The patterns of the outer membrane proteins (OMPs) on SDS-PAGE were generally similar to one another, though the four major proteins (a-d) varied somewhat in molecular mass; these patterns allowed the strains to be arranged into 12 groups. Taxon 13 strains and typical P. multocida strains were indistinguishable, both types being found within the same group. Mice were vaccinated with heat-killed bacteria of three strains and challenged with 10 LD50 of homologous and heterologous live bacteria, representing groups based on OMP patterns; the best protection was afforded by strain W674, which protected against nine of the 17 challenge strains; but there was no correlation between protection and PAGE pattern. Pre-vaccination and pre-challenge sera were used in immunoblotting to probe OMPs from protective and non-protective strains. All three vaccines produced antibody to proteins a and d; these proteins appeared to be common to all strains, varying in molecular mass but not in overall antigenic expression. The antibody response to the other two major OMPs appeared to be PAGE-group specific. There was no correlation between protection and the antigen pattern seen by immunoblotting.

[Pasteurella haemolytica serotyping]. / Pasteurella haemolytica serotypering.

Forty-nine strains of Pasteurella haemolytica were serotyped in a pilot study. The majority of strains were isolated from sheep which had died from fibrinous pneumonia and small numbers from cases of sepsis, polyserositis, mastitis and meningitis. Serotype A2 was found to be the most prevalent: 69.4 per cent. In addition, serotype A7 with 18.4 per cent and serotypes A1 and A6, both 6.1 per cent, were identified. Biotype T strains were not detected in this study. Further research is required into the seasonal prevalence of P. haemolytica serotypes in sheep and cattle. This knowledge may help to improve herd health schemes for sheep and cattle in the Netherlands.

Use of long-acting oxytetracycline against pasteurellosis in lambs.

The efficacy of long-acting oxytetracycline in the control of pneumonic pasteurellosis in lambs was tested on seven Scottish farms. After laboratory confirmation of pasteurella-related deaths in lambs, half the lambs in each flock were given long-acting oxytetracycline (20 mg/kg intramuscularly) and half were left untreated. On three farms a single treatment was given and on four farms two doses were administered four days apart. Eighteen of the 878 control lambs died as a result of confirmed Pasteurella haemolytica pneumonia compared with one of the 878 treated lambs. In addition nine of the control lambs were diagnosed clinically to have pasteurellosis which responded to treatment with oxytetracycline. None of the treated lambs were seen to be ill during the trial.

A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation.

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.