Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.

Effect of thermal/pressure processing and simulated human digestion on the immunoreactivity of extractable peanut allergens.

Peanut allergy is one of the most widespread types of food allergies especially affecting developed countries. To reduce the risk of triggering allergic reactions, several technological strategies have been devised to modify or remove allergens from foods. Herein we investigated the combination of high temperature and pressure on the modulation of peanuts immunoreactivity after simulated gastro-duodenal digestion. Extractable proteins of raw and autoclaved peanuts were separated on SDS-PAGE and immunogenicity was assessed by ELISA and Western Blot analyses. Proteins surviving the heat treatment and reacting towards allergic patients' sera were analysed and attributed to Ara h 3 and Ara h 1 proteins by untargeted LC-high resolution-MS/MS. A progressive reduction in the intensity of the major allergen proteins was also highlighted in the protein fraction extracted from autoclaved peanuts, with a total disappearance of the high molecular allergens when samples were preliminary exposed to 2 h hydration although the lower molecular weight fraction was not investigated in the present work. Furthermore, raw and processed peanuts underwent simulated digestion experiments and the IgE binding was assessed by using allergic patients' sera. The persistence of an immunoreactive band was displayed around 20 kDa. In conclusion, the synergistic effects of heat and pressure played a pivotal role in the disappearance of the major peanut allergens also contributing to the significant alteration of the final immunoreactivity. In addition, the surviving of allergenic determinants in peanuts after gastrointestinal breakdown provides more insights on the fate of allergenic proteins after autoclaving treatments.

Decreased viability and proliferation of CHANG conjunctival epithelial cells after contact with ultraviolet light-irradiated pollen.

CONTEXT: Contact with pollen is the major reason for the development of allergic symptoms on the ocular surface leading to a significant increase of allergic diseases worldwide. Environmental changes such as increased ultraviolet (UV) radiation and air pollution are discussed as contributory causes for this increase. OBJECTIVE: We investigated the effect of UV light on the histamine content of pollen and examined if an irradiation of pollen affects the viability and proliferation of conjunctival cells. MATERIALS AND METHODS: Alder (Alnus glutinosa) and hazel (Corylus avellana) pollen were irradiated for different time periods with sunlight, UV-A or UV-B light and the histamine content was analysed and compared with non-irradiated pollen. Conjunctival epithelial cells (CHANG cells) were exposed to irradiated and non-irradiated pollen followed by an assessment of cell viability with the colorimetric MTS test and the impedance-based measurement of cell proliferation using the xCELLigence real-time analysis system. RESULTS: UV light irradiation increased the histamine level of alder and hazel pollen in a dose-dependent manner. CHANG cells treated with irradiated pollen induced a statistically significant higher decrease of cell viability than treatment with non-irradiated pollen. DISCUSSION AND CONCLUSIONS: Our results indicate that UV light is able to alter pollen thus making them more harmful for conjunctival cells.

Influence of Gamma Irradiation on Porcine Serum Albumin Structural Properties and Allergenicity.

Pork provides an ideal source of food energy; however, pork can elicit an allergic reaction, and porcine serum albumin (PSA) has been identified as a major allergen. This study examined the impact of gamma irradiation on the allergenicity and structural qualities of PSA; the PSA solution was gamma-irradiated at 1, 2, 4, 6, and 8 kGy. Allergenicity was investigated by immunoblotting and competitive indirect ELISA using serum from patients who are allergic to pork, and conformational changes in irradiated PSA were measured by circular dichroism, sulfhydryl group detection, and fluorescence emission spectra. The secondary and tertiary structures of gamma-irradiated PSA were altered, and the allergenicity of PSA was lowered by boosting the amount of irradiation. In addition, there is high correlation between depletion in the α-helix and immunoglobulin E-binding capability of PSA. The results show a new possibility in using gamma irradiation to reduce the allergenicity of pork products.

Eosinophilic esophagitis: demographic, clinical, endoscopic, histologic, and atopic characteristics of children and teenagers in a Region in Cental Spain

Background and objective: Eosinophilic esophagitis (EoE) is an increasingly prevalent chronic inflammatory disease of the esophagus with an immunoallergic etiology. Few studies have been published on EoE in children and adolescents. The objective of this study was to analyze the demographic, clinical, serologic, endoscopic-histologic, and atopic characteristics of pediatric patients with EoE and to identify atopic and digestive comorbidities. Methods: We conducted a prospective observational study in children and adolescents (<16 years) assessed in a specialized multidisciplinary EoE unit in a tertiary referral hospital in a central region of Spain between 2011 and 2015. Results: Thirty-five patients were included in the study. Twenty-eight (80%) were male. The mean age was 9.6 years, 83% were atopic, and 28% reported a family history of atopy. The most common symptom was dysphagia (51%). Eosinophilia was detected in the blood of 60% of patients. Eosinophil cationic protein and total IgE were elevated in 88% and 77% of patients, respectively. The most frequent endoscopic finding was linear grooves (57%). Skin tests with aeroallergens were positive in 82% of patients (pollen 62% and food 60%). The main atopic comorbidities were asthma (48%) and rhinoconjunctivitis (37%). Digestive diseases were more often associated with gastritis and Helicobacter pylori infection (17%). Conclusions: Our results are similar to those previously reported. EoE is more common in boys and in individuals with a history of atopy and sensitization to airborne allergens and food. These results support the consideration of EoE as an atopic disease and underline the important role of allergists in early diagnosis and treatment (AU)

Introducción y objetivos: La esofagitis eosinofílica (EoE) es una enfermedad inflamatoria crónica, emergente del esófago, con etiología inmunoalérgica. Hasta ahora, pocos estudios sobre esta enfermedad en niños y adolescentes han sido publicados. El objetivo de este estudio era estudiar las características epidemiológicas, clínicas, serológicas y endoscópico-histológicas de estos pacientes y las comorbilidades alergológicas y digestivas. Metodología: Es un estudio observacional descriptivo en niños y adolescentes (<16 años), estudiados en una unidad multidisciplinar en un hospital de tercer nivel en una región del centro de España. Resultados: Se incluyeron en el estudio 35 pacientes. 28 eran hombres (80%). La edad media era 9,6 años, el 83% eran atópicos y tenían antecedentes familiares de atopia el 28%. El síntoma más común fue la disfagia (51%). Se detectó eosinofilia en sangre en el 60% de los pacientes. La proteína catiónica del eosinófilo y la Ig E total estaban elevadas en el 78% y 77% de los pacientes, respectivamente. El hallazgo endoscópico más frecuente fueron los surcos lineales (62%). Loa tests cutáneos fueron positivos en el 82% de los pacientes (polen 62%, alimentos 60%). Las comorbilidades atópicas fueron, asma (48%) y rinoconjuntivitis (37%). Las enfermedades digestivas más asociadas fueron gastritis e infección por Helicobacter pylori (17%). Conclusiones: Nuestros resultados son similares a los publicados previamente. La EoE es mas frecuente en niños, con historia de atopia, sensibilizados a aeroalérgenos o alimentos. Estos resultados apoyan que la EoE pueda considerarse una enfermedad atópica y el importante papel del alergólogo en el diagnóstico y tratamiento precoz de esta enfermedad (AU)

Potential allergenicity response to structural modification of irradiated bovine α-lactalbumin.

Bovine α-lactalbumin (α-La) is a major food allergen found in milk and is characterized by high conformational stability because of its four disulfide bridges and being calcium bound. This study aimed to describe the influence of gamma irradiation on the structure and potential allergenicity of α-La. The prepared α-La was irradiated at doses of 1-10 kGy. The changes in structure were characterized through SDS-PAGE, circular dichroism spectroscopy, ultraviolet absorption spectroscopy, and fluorescence spectroscopy. The potential allergenicity of the irradiated α-La was evaluated in vitro through IgG/IgE inhibition ELISA and the human basophil KU812 degranulation assay. The results showed that the secondary and tertiary structures of α-La significantly changed and caused extensive protein denaturation and aggregation. IgG and IgE binding properties remarkably decreased, and the degranulation capacity of basophils weakened. The results suggested that structural damage of α-La induced by irradiation significantly reduces the potential allergenicity of α-La.

Control of airborne and liquid-borne fungal and pet allergens using microwave irradiation.

In this study, the dog, cat allergens (Can f 1 and Fel d 1) and fungal allergens (Alt a 1 and Asp f 1) were aerosolized and exposed to the microwave irradiation (2450 MHz) at different output powers for up to 2 min. The allergen bioaerosols were collected by a BioSampler, and analyzed using enzyme-linked immunosorbent assay (ELISA). Control and microwave-irradiated Asp f 1 allergens were also tested with IgEs in human blood sera samples. For airborne Asp f 1 and Alt a 1 allergens, the allergenicity was shown to decrease about 50% when exposed to microwave irradiation at 385 and 119 W and relatively no change at 700 W. For airborne Can f 1 allergen, the allergenicity was shown to increase about 70% when exposed to the irradiation at 385 W, but remained relatively unchanged at 700 and 119 W. In contrast, airborne Fel d 1 allergen was observed to lose allergenicity completely at 700 W, and retained about 40% and 80% at 385 and 119 W, respectively. Radioallergosorbent (RAST) tests showed that changes detected in IgE levels in human blood sera mixtures were not statistically significant for the control and microwave-irradiated waterborne Asp f 1 allergens. This study implies that although certain allergenicity reductions were observed for some allergens in certain cases, particular care should be taken when the microwave irradiation is used to disinfect food, water, and air because of its complex effects.

Soft-X-ray-enhanced electrostatic precipitation for protection against inhalable allergens, ultrafine particles, and microbial infections.

Protection of the human lung from infectious agents, allergens, and ultrafine particles is difficult with current technologies. High-efficiency particulate air (HEPA) filters remove airborne particles of >0.3 µm with 99.97% efficiency, but they are expensive to maintain. Electrostatic precipitation has been used as an inexpensive approach to remove large particles from airflows, but it has a collection efficiency minimum in the submicrometer size range, allowing for a penetration window for some allergens and ultrafine particles. Incorporating soft X-ray irradiation as an in situ component of the electrostatic precipitation process greatly improves capture efficiency of ultrafine particles. Here we demonstrate the removal and inactivation capabilities of soft-X-ray-enhanced electrostatic precipitation technology targeting infectious agents (Bacillus anthracis, Mycobacterium bovis BCG, and poxviruses), allergens, and ultrafine particles. Incorporation of in situ soft X-ray irradiation at low-intensity corona conditions resulted in (i) 2-fold to 9-fold increase in capture efficiency of 200- to 600-nm particles and (ii) a considerable delay in the mean day of death as well as lower overall mortality rates in ectromelia virus (ECTV) cohorts. At the high-intensity corona conditions, nearly complete protection from viral and bacterial respiratory infection was afforded to the murine models for all biological agents tested. When optimized for combined efficient particle removal with limited ozone production, this technology could be incorporated into stand-alone indoor air cleaners or scaled for installation in aircraft cabin, office, and residential heating, ventilating, and air-conditioning (HVAC) systems.

An automated multiplex specific IgE assay system using a photoimmobilized microarray.

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10 µL of serum within a period of 20 min.

In vitro gastric and intestinal digestions of pulsed light-treated shrimp extracts.

Pulsed ultraviolet light (PUV), a novel technology most commonly used for microbial inactivation, has recently been employed to effectively mitigate food allergens in peanuts, soybean, shrimp, and almond. Putative mechanisms for the efficacy of PUV in reducing allergen reactivity include photothermal, photochemical, and photophysical effects. To date, there are no published data highlighting the effects of in vitro simulated gastric and intestinal digestion on the stability of PUV reduced allergen reactivity of food. In this study, PUV-treated shrimp extracts were subjected to simulated gastric fluid containing pepsin and simulated intestinal fluid containing trypsin and chymotrypsin, and then tested for changes in allergen potency. SDS-PAGE showed no major band deviation between undigested and digested PUV-treated shrimp extracts. IgE binding to tropomyosin remained markedly decreased as seen in Western blot analysis. Total shrimp allergen reactivity remained unchanged following in vitro peptic digestion and was markedly reduced following in vitro intestinal digestion as illustrated in indirect ELISA. The PUV reduced shrimp allergens remained at a low level under the in vitro simulated digestive conditions. The results inferred that PUV could be a potential method to create less allergenic shrimp products that would remain at a low allergen level under human gastric and intestinal digestive conditions.

Pulsed ultraviolet light reduces immunoglobulin E binding to Atlantic white shrimp (Litopenaeus setiferus) extract.

Pulsed ultraviolet light (PUV), a novel food processing and preservation technology, has been shown to reduce allergen levels in peanut and soybean samples. In this study, the efficacy of using PUV to reduce the reactivity of the major shrimp allergen, tropomyosin (36-kDa), and to attenuate immunoglobulin E (IgE) binding to shrimp extract was examined. Atlantic white shrimp (Litopenaeus setiferus) extract was treated with PUV (3 pulses/s, 10 cm from light source) for 4 min. Tropomyosin was compared in the untreated, boiled, PUV-treated and [boiled+PUV]-treated samples, and changes in the tropomyosin levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE binding of the treated extract was analyzed via immunoblot and enzyme-linked immunosorbent assay (ELISA) using pooled human plasma containing IgE antibodies against shrimp allergens. Results showed that levels of tropomyosin and IgE binding were reduced following PUV treatment. However, boiling increased IgE binding, while PUV treatment could offset the increased allergen reactivity caused by boiling. In conclusion, PUV treatment reduced the reactivity of the major shrimp allergen, tropomyosin, and decreased the IgE binding capacity of the shrimp extract.

Effects of pulsed UV-light on peanut allergens in extracts and liquid peanut butter.

Pulsed ultraviolet (PUV) light, a nonthermal technology, was used to treat both the peanut extracts and liquid peanut butter. The objective was to determine if such treatment would lead to a reduction in the allergenic properties of the peanut extract and butter. Peanut samples were PUV treated using a Xenon RS-3000C under the following conditions: 3 pulses/s, 14.6 cm from the central axis of the lamp, 4 min (extract) or 3 min (liquid peanut butter). After the treatment, the peanut samples were centrifuged and the supernatants analyzed by SDS-PAGE and competitive inhibition enzyme-linked immunosorbent assay (ciELISA). For comparison, boiling treatments were also performed. SDS-PAGE showed that while boiling treatment had little effect on the peanut allergens, PUV-light-treated samples displayed a reduced solubility or level of peanut allergens (63 kDa). Solubility of another allergen (18 to 20 kDa) was unaffected. Insoluble aggregates formed were responsible for the reduced level of allergens in PUV-light-treated samples. ciELISA showed that untreated samples exhibited an IgE binding 7-fold higher than the PUV-treated samples. It was concluded that PUV light was effective in reducing IgE binding of peanut extracts and liquid peanut butter. The current study provides an approach to the development of a possibly less allergenic peanut product. However, the reduction in actual allergenicity needs to be confirmed by clinical studies.

Microbial population, physicochemical quality, and allergenicity of molluscs and shrimp treated with cobalt-60 gamma radiation.

Frozen molluscs (squid, octopuses, and cuttlefish) and crustaceans (shrimp) were irradiated using a cobalt-60 gamma source, at different doses, in order to investigate the effects of gamma radiation on their microbial population, organoleptic characteristics, lipid profile, and tropomyosin content. Irradiation of shrimp and squid with either 2.5 or 4.7 kGy reduced mesophilic bacteria contamination to low or nondetectable levels, respectively, whereas irradiation of octopus and cuttlefish with the same doses reduced the bacterial population. Irradiation treatment had no significant (P > 0.05) effect on the total lipid content and the major detected classes of polar and neutral lipids, whereas it significantly (P < 0.05) increased the contents of neutral lipids in octopus mantle and in shrimp muscle and cephalothorax samples. The total fatty acid content and the omega-3: omega-6 fatty acid ration was not affected. A dose-dependent significant (P < 0.05) decrease in the ratio of polyunsaturated fatty acids:saturated fatty acids was observed. With the increase in radiation dose, redness (a) and yellowness (b) values showed a variation, whereas the lightness (L) value was significantly (P < 0.05) decreased in mollusc mantles and shrimp muscle and increased in shrimp cephalothorax. The total of color changes ( delta E) increased (P < 0.05) as the dose increased. Significant (P < 0.05) changes in textural properties were observed with radiation treatment in octopus tentacles and in squid and cuttlefish mantle. The amount of tropomyosin, which is the major mollusc and crustacean allergen in the irradiated organisms, was reduced by gamma radiation, depending on the dose.

[Results and assessment of photopatch testing in Spain: towards a new standard set of photoallergens]. / Resultados y evaluación del fotoparche en España: hacia una nueva batería estándar de fotoalergenos.

INTRODUCTION: While the standardization of exploration with photoallergy tests or photopatch testing runs its course in Europe, we have carried out an epidemiological study about the current situation of photoallergy in our country. MATERIAL AND METHODS: We have gathered the results of photopatch testing in seven hospital centres of Madrid, Cataluña, Galicia and Comunidad Valenciana during the years 2004 and 2005. The exploration has included, at least, the standard set of the Spanish Photobiology Group (GEF), with 16 (photo) allergens, that have been irradiated with 10 joules/cm(2). We have assessed the total number of explored patients, their sex, present, past or unknown relevance of positive photopatch testing, cross reactions, and allergens responsible for photosensitization. RESULTS: Of 224 patients explored by photopatch testing, 39.3 % show one or more positive tests. Seventy-one percent (103) were considered relevant with respect to clinical history, 14 cases (9.6 %) were cross reactions, and 28 (19.3 %) were considered of unknown relevance. The most prevalent allergens were nonsteroidal antiinflammatory drugs, specially ketoprophen (43 patients), followed by bencydamine (7 patients) and etofenamate (5 patients). The mixture of four sunscreens from the standard set of the GEF only detected 10 of 16 patients with photoallergy to sunscreens. Photopatch testing of unknown relevance was mainly due to antiseptics (fenticlor) and topical antihistamines. CONCLUSIONS: We propose the modification of the standard set of photoallergens from the GEF, that should include the majority of nonsteroidal antiinflammatory drugs and sunscreens available in Spain. Ketoprophen continues to be the most frequent photoallergen in our country. It is also important for the cross sensitizations that may present. Sunscreens should be explored separately and not in form of a mixture.

Ovalbumin modified by gamma irradiation alters its immunological functions and allergic responses.

It is well known that gamma (gamma)-ray irradiation results in the alteration of biological function of bioactive materials such as proteins, saccharides and lipids. In this study the effect of gamma-irradiation on the chemical and immunological property of an allergen, ovalbumin (OVA), was investigated. Irradiation of more than 10 kGy resulted in the alteration of the structure of OVA. However, OVA treated with 10 kGy irradiation (10 kGy-OVA), but not 100 kGy-OVA, fully maintained immunological reactivity to a monoclonal antibody specific to the intact allergen (clone 14). Mice immunized with 10 kGy- as well as 100 kGy-OVA showed significantly lower antibody response to the allergen than those with intact OVA in a gamma-ray dosage-dependent manner. Especially immunization of both 10 kGy- and 100 kGy-OVA induced a significant decrease of OVA-specific IgE. Splenocytes of mice immunized with irradiated OVA showed a significant reduction in OVA-specific T cell proliferation and the secretion of Th1-type (IFN-gamma and IL-2) and Th2-type cytokines (IL-4 and IL-6). The expression of T cell activation markers such as CD25 and CD44 was also down-regulated in T cells of mice immunized with irradiated OVAs. These results suggest that gamma-ray irradiation of OVA suppress humoral and cellular immune responses specific to the allergen OVA, and the modification method with gamma-irradiation may be available for the control of allergy.

Deleterious effects of electron beam radiation on allergen extracts.

BACKGROUND: The recent threat to the public posed by the dissemination of Bacillus anthracis through the US postal system has resulted in increased security measures, including electron beam irradiation for the sterilization of some mail. The deleterious effects of electron beam radiation on biological products are not fully understood. OBJECTIVE: The purpose of this investigation was to assess the effect of electron beam radiation, as currently used to sterilize packages and mail in the United States, on several standardized or characterized allergen extracts. METHODS: Selected irradiated extracts were analyzed for allergen content and potency by SDS-PAGE, immunoblot, and ELISA (including inhibition) and compared with untreated extracts. RESULTS: The compositions and immunochemical potencies of these products were altered significantly by irradiation treatment. Physical changes to native protein structures observed after electrophoretic separations coincided with near-complete loss of allergenic and antigenic epitopes present on major and minor allergens, according to ELISA and immunoblot comparisons with untreated extracts. CONCLUSIONS: These results indicate that extracts subjected to electron beam sterilization conditions are likely to contain modified component structures and properties that might compromise the clinical effectiveness of these products.

Effects of gamma radiation on the allergenic and antigenic properties of milk proteins.

This study was carried out to evaluate the application of food irradiation technology as a method for reducing milk allergies. Bovine alpha-casein (ACA) and beta-lactoglobulin (BLG) were used as milk proteins. Using milk-hypersensitive patients' immunoglobulin E (IgE) and rabbit IgGs individually produced to ACA and BLG, the changes of allergenicity and antigenicity of irradiated proteins were observed by competitive indirect enzyme-linked immunosorbent assay. Allergenicity and antigenicity of the irradiated proteins were changed with different slopes of the inhibition curves. The disappearance of the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increase of the turbidity showed that solubility of the proteins decreased by radiation, and this decrease might be caused by agglomeration of the proteins. These results indicated that epitopes on milk allergens were structurally altered by gamma irradiation.

Effects of gamma radiation on the conformational and antigenic properties of a heat-stable major allergen in brown shrimp.

This study was performed to evaluate the application of food irradiation technology as a method for reducing shrimp allergy without adverse effects. Shrimp heat-stable protein (HSP) was isolated and gamma irradiated at 0, 1, 3, 5, 7, or 10 kGy in the condition of solution (1 mg/ml), and fresh shrimp was also irradiated. Conformational change of irradiated HSP was monitored by means of spectrometric measures, enzyme-linked immunosorbent assay with mouse monoclonal antibody, or human patients' sera and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ability of the immunoglobulin E of patients allergic to shrimp to bind to irradiated HSP was dose dependently reduced. The amount of intact HSP in an irradiated solution was reduced by gamma irradiation, depending on the dose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the main band disappeared and the traces induced from coagulation appeared at a higher molecular weight zone. The binding ability of immunoglobulin E to allergens in the extracts from irradiated shrimp decreased, depending on the dose. The results provide a new method so that food irradiation technology can be applied to reduce allergenicity of shrimp.

Use of the occlusive patch to evaluate the photosensitive properties of chemicals in guinea-pigs.

Guinea-pig tests were conducted on a known photocontact allergen, tetrachlorosalicylanilide (TCSA), a known phototoxin, 8-methoxypsoralen, two reportedly weak photoallergens, musk ambrette and 6-methylcoumarin, and a negative control, octylphenoxy polyethoxyethanol (Triton X-15). The data show that under the test conditions used, photosensitivity responses can be produced, and combinations of these as well as the other biological responses can be readily defined. The results indicate that musk ambrette is photoallergenic, that 8-methoxypsoralen is phototoxic and that Triton X-15 is only a slight irritant. On the other hand, results with TCSA suggest that it is a strong contact allergen and photoallergen, while 6-methylcoumarin would be considered to be a weak contact allergen with weak phototoxic properties. Previous reports that barrier destruction or adjuvanticity is necessary to produce photoallergy to musk ambrette were not confirmed; by ensuring occlusion using standard methods, the photoallergic nature of the response to this material was clearly demonstrated. A device described elsewhere (Newmann & Parker, Fd Chem. Toxic. 1985, 23, 683) has made it possible to develop methods that can be used to differentiate clearly among the possible biological responses that can occur in guinea-pigs when photoreactive materials are applied to their skin and irradiated. The probable biological responses that need to be defined, under the above conditions, are primary irritation, delayed contact hypersensitivity, phototoxicity and/or photoallergenicity.