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.

Ovine systemic pasteurellosis caused by Pasteurella haemolytica biotype T.

A detailed study was made of lambs aged 5--7 months naturally infected with Pasteurella haemolytica biotype T. In addition to the well known features of such infections, previously unreported necrotic lesions of the tonsil, oesophagus, pharynx and adjacent areas were consistently seen. Large numbers of P. haemolytica were present in the tonsil, oesophageal lesions, lung, liver and spleen, but few or none in other tissues. The evidence indicated that the disease was not a true septicaemia. It is postulated that P. haemolytica biotype T already present in the tonsils multiplies and invades the adjacent tissues of the upper alimentary tract; groups of organisms from this site enter the blood stream as emboli, most of which lodge in the capillary beds of the lung and liver; rapid multiplication of organisms in these tissues leads to death from the effects of endotoxin.

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.

Electronmicroscopy of the surface of Pasteurella haemolytica.

The surfaces of Pasteurella haemolytica, biotype A, serotypes 1, 2, 6, 7 and 9 and of P. haemolytica, biotype T serotypes 3, 4, 10 and 15 were examined by transmission electronmicroscopy with ruthenium red staining and polycationic ferritin labelling, by scanning electronmicroscopy, and by light microscopy. Electronmicroscopy showed that the surface of strains of P. haemolytica biotype A was covered by irregular protrusions which were probably capsular material. The surface and general morphology of P. haemolytica biotype T were distinct from those of biotype A.

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.

Immunity of specific pathogen-free lambs to challenge with an aerosol of Pasteurella haemolytica biotype A serotype 2. Pulmonary antibody and cell responses to primary and secondary infections.

Specific pathogen-free (SPF) lambs previously exposed to an aerosol of P. haemolytica biotype A serotype 2 (A2) were immune to subsequent challenge with an aerosol of P. haemolytica A2. Untreated control lambs were not immune to this challenge. The local immune responses of the lung to these challenges were examined. High IgG and IgA titres to P. haemolytica and high levels of opsonizing antibody against P. haemolytica were present in the lung washings from previously infected immune lambs at autopsy, seven days after the second infection. Lung washings from control lambs, 7 days after challenge with P13 virus and P. haemolytica A2, had no IgG titres, very little opsonizing activity but did have IgA titres which were significantly higher than in unchallenged control lambs. The cellular response of animals challenged with P13 virus and P. haemolytica was significantly greater than that of unchallenged controls or of lambs exposed only to P. haemolytica. However, this finding was complicated by the response to P13 virus. Lymphocytes from lung washings of all lambs failed to respond in a lymphocyte stimulation test to phytohaemagglutinin while blood lymphocytes did respond. There was little specific response to P. haemolytica antigen in the test.

The specificity of the fluorescent antibody test using the sera of rabbits inoculated with strains of myocobacteria.

Sera from experimentally infected rabbits were used to test the specificity of the fluorescent antibody test. It was possible using mono-specific sera to differentiate antigenically Mycobacterium phlei, M fortuitum, M smegmatis, M avium, M intracellulare, M bovis (BCG) and M johnei. The cross-reactivity within the M avium and M intracellulare group was such that one antigen from these groups would detect infection within that group and exclude M johnei infection. The M phlei growth factor independent strain M johnei 316F was shown to be antigenically distinct from a M phlei dependent strain 9N96. There was loss of specificity when M avium infection was superimposed on a previous M johnei infection and when M johnei infection was superimposed on M avium infection.

The identification of Moraxella bovis and Neisseria ovis from the eyes of cattle and sheep.

The cultural characteristics of Moraxella bovis and Neisseria ovis from eyes of cattle and sheep were examined to determine which tests precisely identified the isolates. The elongation test to distinguish the bacillary M vovis from the coccal N ovis, the nitrate reduction and the litmus milk tests were found to be the most reliable.

The specificity and sensitivity of the fluorescent antibody test for Mycobacterium johnei infection in abattoir and culled cattle.

In a series of 100 cattle in which there was no bacteriological or histopathological evidence of Mycobacterium johnei infection there were four positive reactions to the fluorescent antibody (FA) test with M johnei antigen and 20 to the complement fixation (CF) test. In a second series of 118 culled adult cattle M johnei infection was found in 26. The FA test was positive in 16 and the CF in 12 of those infected cattle. In 92 cattle with no evidence of M johnei infection the FA test was positive in seven and the CF test in 22.

The specificity and sensitivity of the fluorescent antibody test in cattle experimentally infected with Mycobacterium avium and Mycobacterium johnei.

In five calves experimentally infected with Mycobacterium avium and in 10 with M johnei it was shown that the serological response by the fluorescent antibody (FA) test was specific. The serological response in the M avium infected calves was transitory and lasted up to five months. The first evidence of a serological response with the FA test in the M johnei dosed calves occurred at four months, and all calves had reacted by nine months after dosing. At the 23 tests carried out between four and 32 months after dosing, when the experiment was terminated, an average of 5-8 animals was positive at any one test. Johne's disease was confirmed bacteriologically and histologically in six of the 10 calves though none had shown clinical signs.

Serotypes of Pasteurella haemolytica in ovine pasteurellosis.

The serotypes of 406 strains of Pasteurella haemolytica were determined. These came from cases of pasteurellosis in sheep of all ages. Serotype A2 was the most frequent and comprised 33 per cent of all strains recovered. Serotypes T3, T4 and T10 comprised 16, 14 and 12 per cent of the total respectively. Serotypes A1 and A6 contributed 5 per cent each, A7, 18, A9, A11 and A12 a total of 8 per cent collectively, serotype A5 was not isolated at all while 6 per cent of strains were serologically untypable. More strains were received during September, October and November than at any other three-month period and in these months the T biotype predominated.

Characterisation of a new serotype of P haemolytica isolated in Hungary.

Three strains belonging to a new serotype of Pasteurella haemolytica (A16) were isolated from lambs and a wild boar in Hungary. The identity and validity of the new serotype was proved by biochemical tests and by the indirect haemagglutination test using unabsorbed and absorbed hyperimmune sera raised in rabbits.

Susceptibility of specific pathogen-free lambs to concentrations of Pasteurella haemolytica serotype A2 in aerosols.

A 100-fold reduction in the numbers of organisms in an aerosol of Pasteurella haemolytica used to infect specific pathogen-free lambs did not alter the number of cases of pneumonia which resulted. In a separate experiment a further 10-fold reduction in the number of organisms in the aerosol did not cause fewer cases of pneumonia.