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C L Galli

Publications and source records attributed to C L Galli.

At least 55 records · Page 3Linked to original sources

Meat allergy: III--Proteins involved and cross-reactivity between different animal species.

BACKGROUND: Although relatively infrequent, meat allergy represents a serious problem for children both because it is generally associated with intolerance to other protein sources and because of the suggested role of meat in stimulating the gastrointestinal development during weaning. OBJECTIVE: With these considerations, the aim of our work was to improve biological-biochemical knowledge of meat allergy. METHODS: This study was performed using in vivo skin prick test (SPT) and in vitro (electrophoresis associated with the immunoblotting technique) tests. RESULTS: Bovine serum albumin (BSA) and actin were the proteins most frequently involved in binding with the circulating IgE. BSA involvement was confirmed by SPT; the high number of positive responses observed with actin in immunoblotting was not confirmed by SPT data. Cross-reactivity between serum albumins from different animal species was demonstrated. Our studies show that in this group of children, the correspondence between the percentage of sequence identity (phylogenetic similarity) and the number of positive responses was surprisingly high. CONCLUSIONS: Although further studies are necessary, the data reported here provide new biochemical data on meat allergy.

Actins↗

Induction of tumor necrosis factor-alpha in vivo by a skin irritant, tributyltin, through activation of transcription factors: its pharmacological modulation by anti-inflammatory drugs.

Skin irritant reactions are under the control of a network of cytokines and lipid mediators. This study characterized the production of tumor necrosis factor-alpha (TNF) induced by a skin irritant treatment, tributyltin (TBT), in mice through transcription factor activation and its pharmacologic modulation by anti-inflammatory agents. The ears of BALB/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. At different times thereafter, TNF production was analyzed both at the mRNA and protein level, by semiquantitative RT-PCR and L929 cytotoxicity assay, respectively. TBT induced rapid (1 h) TNF gene expression and protein synthesis. Maximal TNF production was observed 2 h after treatment. The production of TNF was paralleled by accumulation of skin water; this was partially prevented by intraperitoneal injection of antibody against murine TNF. These data indicate that skin irritation induced by TBT is attributable, in addition to the actions of other inflammatory mediators, to the action of keratinocyte-derived TNF. TNF production was preceded by a rapid (5 min) activation of nuclear factor-kappaB (NF-kappaB), which was also maximal 30 min after treatment. TBT-induced accumulation of skin water and TNF production were significantly reduced by topical treatment with dexamethasone and pentamidine, two anti-inflammatory agents. Interestingly, dexamethasone, but not pentamidine, decreased TBT-induced NF-kappaB activation, confirming in vivo that the glucocorticoid receptor interacts functionally within the nucleus with other transcription factors opposing one another's activity.

Animals↗

Monoclonal and polyclonal antibodies against casein components of cow milk for evaluation of residual antigenic activity in 'hypoallergenic' infant formulas.

BACKGROUND: Hydrolysed casein and whey protein formulas have been developed with the aim of preventing sensitization in infants at high risk of cow milk allergy. Subsequently these products have also been used for treatment of children with cow milk allergy. However, severe reactions have occurred in some allergic infants fed with these formulas raising doubts about their absolute safety and suggest the need for developing in vitro techniques for detection of eventual residual allergenic activity in such preparations. OBJECTIVES: Our purpose was to evaluate the usefulness of monoclonal and polyclonal antibodies against casein components (alpha, beta and kappa casein) as reagents for the detection of the residual antigenic activity of casein components in several hydrolysed formulas. METHODS: The monoclonal and polyclonal antibodies were produced according to standard procedures by immunizing female Balb/c mice with casein fraction (a mixture of alpha, beta and kappa casein). ELISA assays were developed to test the specificity of the antibodies and to detect and evaluate the amount of residual antigenic activity of the casein components in hydrolysed formulas. RESULTS: Use of polyclonal antiserum specific for casein allowed detection of residual antigenic activity of casein components in all partial hydrolysates and in the two extensive whey protein hydrolysates in the amounts ranging from 0.05 to 0.67% of total protein. No such activity was detectable in either the two extensive casein hydrolysates tested or the aminoacid based formula. The polyclonal antiserum proved to be more suitable than monoclonals for detecting residual antigenic activity in the hydrolysates. The monoclonal antibodies were directed against epitopes expressed on different casein components. CONCLUSIONS: In this study the ELISA inhibition assay with polyclonal antibodies specific for casein components of cow milk proved to be a sensitive method for estimating residual antigenicity in the hydrolysed formulas commercially available for infants with cow milk allergy suggesting their potential application for the quality control of hypoallergenic infant formulas.

Animals↗

NF-kappaB activation by triphenyltin triggers apoptosis in HL-60 cells.

Trisubstituted organotin pesticides are lethal for different cell types. In this study we investigated whether triphenyltin chloride (TPT) causes apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We report that 5 microM TPT increased intracellular Ca2+ in HL-60 cells within seconds; concomitantly actin depolymerization was detected 30 s and 1 min after the treatment. This was followed 15 min later by NF-kappaB activation, and apoptotic bodies and DNA fragmentation were evident after 3 and 6 h, respectively. At these times TPT also induced the release of tumor necrosis factor-alpha (TNF-alpha). Prior treatment of the cells with a polyclonal antibody to human TNF-alpha abolished TPT-induced DNA fragmentation, which suggests that the ultimate effect of TPT may be mediated by TNF-alpha. Prior treatment of the cells with 100 microM pyrrolidine dithiocarbamate, an antioxidant and potent inhibitor of NF-kappaB activation, prevented actin depolymerization, NF-kappaB activation, and DNA fragmentation, although it did not affect TPT-induced Ca2+ mobilization. These findings suggest that TPT increases intracellular Ca2+, alters actin polymerization and the cytoskeleton, and induces NF-kappaB activation, TNF-alpha synthesis, DNA degradation, and apoptosis. Reactive oxygen species seem to be essential to NF-kappaB activation, TNF-alpha synthesis, and the subsequent steps.

Actins↗

Is actin polymerization relevant to neurosecretion? A study on neuroblastoma cells.

We have investigated the relevance of actin rearrangement to neurotransmitter release, in neuroblastoma cell line SH-SY5Y stimulated with carbachol (1 mM). Carbachol reversibly polymerizes actin and releases norepinephrine in undifferentiated SH-SY5Y cells as well as in tetradecanoylphorbol 13-acetate (16 nM) and retinoic acid (5 microM) differentiated SH-SY5Y cells. Microscopic analysis of F-actin distribution indicates polymerization of cortical actin. Prior treatment with iota toxin E from Clostridium perfringens inhibits both carbachol-induced actin polymerization and norepinephrine release, slightly affecting the basal actin network. These data suggest that actin polymerization is associated with norepinephrine release.

Actins↗

Effect of pesticide mixtures on in vitro nervous cells: comparison with single pesticides.

The toxicity of dimethoate, azinphos-methyl, diazinon, pirimiphos methyl, organophosphorus insecticides, and benomyl (a benzimidazole fungicide) singly and in mixture was studied in a human neuroblastoma cell line, SH-SY5Y. The cells were incubated for 30 min and 4 h with pesticides at concentrations ranging from 0.4 to 100 micrograms/ml, or with the same compounds mixed as follows: (a) dimethoate-diazinon-azinphos; (b) benomyl-pirimiphos; (c) all together. Pesticides in the mixtures were at the same concentration used when tested singly. Diazinon, azinphos-methyl and pirimiphos, but not dimethoate and benomyl, inhibited acetylcholine esterase (AchE) activity, whereas all the compounds inhibited protein synthesis in the following order: benomyl > azinphos > diazinon >> pirimiphos = dimethoate. The mixtures showed a toxicity on AchE activity at a maximum equal to that of the most active compound in the mixture. On the contrary, the mixture were more toxic than the single compounds on protein synthesis, and in certain cases potentiation occurred. Therefore, we can conclude that it is not feasible to predict the toxicity of pesticide mixtures on the basis of the results of the toxicity of single components.

Acetylcholinesterase↗

Endogenous interleukin-1 alpha associated with skin irritation induced by tributyltin.

Tributyltin (TBT) salts are well-known skin irritants in both human and rodents. This study investigated the role of interleukin-1alpha (IL-1alpha) in the process in mice and in murine keratinocytes. The ears of Balb/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. Two hours later there was dose-related production of IL-1alpha along with ear swelling and accumulation of skin water, all of which were partially prevented by intraperitoneal injection of antibody against murine IL-1alpha. By reverse transcription-polymerase chain reaction we were able to show that the neutralizing antibody also partially prevented TBT-induced in vivo IL-6 expression but no TBT-induced TNF-alpha expression, suggesting a paracrine effect of IL-1alpha on IL-6 production but not TNF-alpha expression and indicating that other inflammatory mediators are involved. TBT induced both intracellular production of IL-1alpha and its release into culture medium in a murine keratinocyte cell line (HEL30). IL-1alpha production was inhibited by addition of a neutralizing antibody against IL-1alpha, which suggests an autocrine effect of IL-1alpha on its own production. The intracellular production of IL-1alpha could he significantly inhibited by prior treatment with antioxidants, which strongly suggests a role for oxidative species in the mechanism of action of TBT in IL-1alpha induction. The complex-1 inhibitor rotenone also significantly inhibits IL-1alpha production. Since TBT causes disturbances in the respiratory chain in mitochondria, the mechanism of its action may be the production of reactive oxygen intermediates at the ubiquinone site, which activate transcription factors and promote IL-1alpha synthesis.

Animals↗

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Animal Testing Alternatives↗

Role of mitochondria in tributyltin-induced interleukin-1alpha production in murine keratinocytes.

Tributyltin (TBT) salts are well known skin irritants in rodents and humans. TBT induced both the intracellular production of interleukin-1alpha (IL-1alpha) and its release into culture medium in a murine keratinocyte cell line (HEL30). Here, we report that mitochondria are important for TBT-induced IL-1alpha production. Confluent cells were treated with increasing concentrations of TBT (0--2.5 microM) or dimethylsulfoxide as vehicle control. At different times thereafter (0--24 h), nuclear extracts were analyzed for nuclear factor-(kappa)B (NF-(kappa)B) binding activity by electrophoretic mobility shift assay, and the released and cell-associated IL-1alpha was measured by enzyme-linked immunosorbent assay. TBT induced a direct and concentration-related activation of NF-(kappa)B, which peaked at 2 h and was blocked by pyrrolidinedithiocarbamate, a potent NF-(kappa)B inhibitor, and rotenone, an inhibitor of the electron entry from complex I to ubiquinone. Rotenone also induced a concentration-related inhibition of IL-1alpha synthesis induced by TBT, but rotenone did not completely abrogate TBT-induced IL-1alpha production, which suggests that other transcription factors may be involved in IL-1alpha production. Prolonged treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, was used to partially deplete cells of functional mitochondria. After 5 d of treatment, mitochondrial conversion of tetrazolium bromide to formazan was reduced by 50%, and IL-1alpha release was decreased by 65%, whereas no induction of intracellular IL-1alpha was observed. This effect was not due to inhibition of protein synthesis, because identical incorporation of [3H]leucine into protein in control and ethidium bromide-treated cells was identical. This impairment of mitochondrial metabolism inhibited NF-(kappa)B activation by TBT. These findings indicate that mitochondria may be the source of second messenger molecules important for TBT-induced IL-1alpha production.

Animals↗

Use of immunoblotting and monoclonal antibodies to evaluate the residual antigenic activity of milk protein hydrolysed formulae.

BACKGROUND: Partial and extensive hydrolysed protein formulae have been developed to lower or eliminate the antigenicity of milk proteins. Although normally well tolerated, extensive hydrolysates have been reported to induce serious allergic reactions in very sensitive children. Moreover, clinical practice has often raised concern about the role of partial hydrolysates in cow's milk allergy prevention. OBJECTIVE: Starting from these considerations, we used anti-casein monoclonal antibodies to evaluate the presence of residual antigenic activity in both partially and extensively protein hydrolysates. METHODS: Electrophoretic analyses associated with immunoblotting technique were performed using nine protein-enriched commercial formulae. RESULTS: The presence of different amounts of residual intact cow's milk proteins and/or polypeptidic material with conserved antigenic activity (according to the extensive or partial hydrolysis) was verified in most milk-based samples considered. CONCLUSION: The use of monoclonal antibodies and immunoblotting could be useful for the quality of commercial 'hypoallergenic' formulae.

Antibodies, Monoclonal↗

Cloning of a new FRTL5-derived cell line stably expressing active human thyroid peroxidase.

A full-length cDNA clone for human thyroid peroxidase inserted into the mammalian cell expression vector pECE was stably transfected into the rat thyroid cell line FRTL5. Clones expressed immunologically and enzymatically assessed human thyroid peroxidase protein. Methimazole (25 microM) inhibited the thyroid peroxidase activity dose-dependently and this effect was completely antagonised by 100 microM NaI. Ethylenethiourea, metabolite of dithiocarbamate pesticides, inhibited the enzyme at 50 microM. Thus, we have obtained thyroidal cells stably expressing enzymatically active human thyroid peroxidase which can be pharmacologically modulated and studied.

Animals↗

Effect of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol, on protein content, protein synthesis and LDH leakage in mouse epidermal cells.

The cytotoxicity of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol was investigated in murine epidermal cell line, HEL-30. Lactate dehydrogenase (LDH) leakage, protein synthesis and protein content were determined after exposure of cell monolayers to the compounds ranging from 0.1-200 microM for 2, 6, or 24 h. 26-Aminocholesterol affected all the parameters studied time and concentration dependently; 25-hydroxycholesterol and 26-hydroxycholesterol were not toxic for HEL-30 cells. The cellular target for 26-aminocholesterol was primarily the membrane, since the LDH leakage was already detectable 10 min after exposure. On the other hand, for the other two oxysterols a protective role on this structure might be postulated. In fact 25-hydroxycholesterol and 26-hydroxycholesterol decreased the natural LDH leakage due to the ageing of the culture. In addition, 20 microM 25-hydroxycholesterol reversed the effect of moderately lytic doses of 26-aminocholesterol and Triton X-100, but not of sodium dodecyl sulfate.

Analysis of Variance↗

Meat allergy: I--Specific IgE to BSA and OSA in atopic, beef sensitive children.

OBJECTIVE: The use of lamb meat products has been suggested as an alternative diet for polyallergic children, although until now this clinical practice has not been supported by in-depth biochemical/immunological studies. The aims of this research were: to evaluate cross-reactivity between lamb and beef; to evaluate the role of BSA and OSA as allergens in beef allergic children; and to evaluate cross-reactivity between BSA and OSA. METHODS: 16 children suffering from atopic dermatitis (AD), aged 12 months-8 8 years (mean age 2.61 +/- 1.93 years) were found skin prick test (SPT)--positive to bovine meat; all of them were also SPT-positive to ovine meat and to milk. After a period of restricted diet, the selected 16 children were recalled; 12 AD-free children (8 males and 14 females, aged 12 months-4.33 years (mean age 2.21 +/- 1.05 years) were evaluated by SPT and radioallergosorbent test (RAST) for the following allergens: bovine meat, ovine meat, BSA 1 mg/ml, OSA 1 mg/ml. Double-blind, placebo-controlled food challenge (DBPCFC) with bovine serum albumin (BSA) and ovine serum albumin (OSA) were performed. For SPT, the results were expressed in mm of wheal, and 3 mm was considered as the end point; correlation between wheal diameters was calculated by Spearman rank test. For DBPCFC, according to the Sampson's experimental procedure, BSA and OSA were given in pear juice (the dermal negative response to the pear juice was verified by fresh food SPT before starting the oral challenge test). The total dose administered to the children corresponded to the amount of albumin present in 180 g of calf or lamb meat (90 and 63 mg respectively, as calculated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SPS-PAGE). The administration of pear juice (containing placebo or albumin) and symptom evaluation were entrusted to medical people who did not know what the child received. RESULTS: All children tested SPT positive to bovine and ovine meat, and to BSA and OSA. Significant correlations were observed between the following diameters of wheal: BSA vs OSA (R = 0.846, p < 0.0001); ovine meat vs OSA (R = 0.769, p < 0.005); b.meat vs o.meat (R = 0.771, p < 0.005); and ovine meat vs BSA (R = 0.594, p < 0.043). In RAST, 6 of 12 children were positive to bovine meat, 3 to lamb meat, 4 to BSA and 3 to OSA. DBPCFC showed an immediate reaction to BSA or OSA in 2 and 3 children, respectively. One other child developed severe dyspnea, cough and asthma 3 hours after OSA challenge. CONCLUSIONS: BSA and OSA are important beef and lamb allergens; they share not only proteic sequences, but also allergenic properties. Clinical tolerance to BSA and OSA can be present in beef and lamb SPT-positive children.

Animals↗

Meat allergy: II--Effects of food processing and enzymatic digestion on the allergenicity of bovine and ovine meats.

OBJECTIVES: This study was designed to evaluate whether peptic treatment of BSA and OSA affects their allergenicity and to evaluate the effect of technological treatments of meat for infant feeding on the allergenicity of these proteins. SUBJECTS: Twelve children (8 males and 14 females) suffering from atopic dermatitis (AD), aged 12 months to 4.33 years (mean age 2.21 +/- 1.05 years). STUDY DESIGN AND METHODS: Children suffering from atopic dermatitis (AD), (AD), clinically reactive and SPT-positive to beef, underwent skin prick testing (SPT) and radioallergosorbent test (RAST) with BSA and OSA, before and after peptic treatment. They were tested also with commercially available homogenized bovine meat (HBM) and with freeze-dried bovine (FDBM) and ovine (FDOM) meats. Peptic digestion of BSA and OSA was carried out in a Dubnoff's water bath containing 0.05 mg/ml of pepsin. SPT and RAST were performed for 1 mg/ml of BSA and OSA digested 5 minutes, 2 hours and 4 hours with pepsin; homogenized bovine meat, and FDBM and FDOM. SPT results were expressed as diameters (in mm) of the wheal obtained after SPT; a diameter > or = 3 mm was considered as positive. RAST was considered positive for IgE values 5 > UI/ml. RESULTS: SPT to native BSA and OSA were positive in all patients. Only 2 of the 12 children were SPT-positive to HBM, FDBM, and FDOM. After digestion, SPTs and were positive as follows: for BSA, 4/12 after 5 minutes peptic treatment, 2/12 after 2 hours and 2/12 after 4 hours; for OSA, 3/12 after 5 minutes peptic treatment, none after 2 and 4 hours. None of RASTs was positive after peptic attack. CONCLUSIONS: Both proteolytic digestion and technological treatment reduced the allergenic potential of meat products.

Animals↗

Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas.

BACKGROUND: Extensively hydrolysed protein formulas are widely used as an alternative diet for children with cow's milk allergy. Partially hydrolysed protein formulas have been noted in some studies as useful in the prevention of allergy in infants at high risk of atopy. Although normally well tolerated, these 'hypoallergenic' products have been reported to cause serious immunological reactions in very sensitive subjects. OBJECTIVE: Starting from these considerations, we studied some commercial hydrolysed formulas in search of biological data supporting the observed clinical reactions. METHODS: We set up an electrophoretic method sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which allowed us to study the molecular weight of peptides contained in hydrolysed products. Then, using the immunoblotting technique we evaluated the reactivity of circulating IgE (from serum of children allergic to cow's milk proteins) with the residual intact proteins and with the peptides present in these formulas. RESULTS: Both group of milk proteins (caseins and whey proteins) were important allergens for children included in this study. The presence of high-molecular polypeptides was shown in partial hydrolysed formulas as such and in extensive hydrolysed products after protein enrichment by trichloroacetic acid (TCA) precipitation. Intact residual proteins were mainly responsible for the formation of IgE-antigen complexes observed in immunoblotting. More rarely, polypeptides of partial hydrolysed formulas were involved in immunological responses. CONCLUSIONS: Both partial and extensive hydrolysed formulas could induce clinical reactions in very sensitive subjects. These responses are mainly associated with allergy to the small amounts of residual intact proteins.

Animals↗

In vitro mechanism(s) of ultraviolet-induced tumor necrosis factor-alpha release in a human keratinocyte cell line.

It has been demonstrated that ultraviolet (UV) irradiation is able to induce both in vivo and in vitro, tumor necrosis factor-alpha (TNF) release. The purpose of the present study was to evaluate, using a human keratinocyte cell line NCTC 2544, the mechanism(s) of UV-induced TNF release and the ability of commonly used sunscreens to modulate UV-induced TNF release. TNF release can be partially prevented both by adding an anti-human IL-1 alpha antibody after irradiation, suggesting an autocrine effect of IL-1 alpha in inducing TNF release, and by adding antioxidants indicating also a role of oxidant species. TPCK, a I kappa-B alpha protease inhibitor, was able to virtually abolish UV-induced TNF release, indicating that UV-induced TNF release requires NF-kappa B activation. Anti-human IL-1 beta antibody was ineffective as expected, considering that keratinocytes are unable to process pre-IL-1 beta to its active form. To evaluate the sunscreen's modulation on UV-induced TNF release, confluent cells were irradiated, in the presence or absence of the tested sunscreens (Uvinul MS40, Uvinul P25 and Uvinul DS49). Different IC50 values could be calculated, which may be related to different UV absorption spectrums: Uvinul MS40 offers great protection by virtue of its broader absorption spectrum, closely followed by Uvinul P25 and finally by Uvinul DS49.

4-Aminobenzoic Acid↗

In vitro keratinocytes responses to chemical allergens.

An early event during the induction of skin sensitization is the production by keratinocytes (kc) of cytokines. Considering the role of kc in skin inflammatory and immune responses, we investigated the effect of three skin allergens (oxazolone, nickel sulfate and mixture of methyisothiazolinone and methylcholoisothiazolone) on IL-1 alpha production by a murine kc cell line (HEL30). Confluent cells were treated for different times with various concentrations of oxazolone, NiSO4 and mixture of methyisothiazolinone and methylcholoisothiazolone. IL-1 alpha was measured by ELISA both in conditioned media and cell-associated. All chemicals were able to induce a dose-response production of IL-1 alpha. This increase was related to an increase in protein synthesis as measured by 3H-leucine incorporation. At high concentrations all chemicals were cytotoxic as measured by LDH release. In the same cell system we also measured the effect of lanolin, a non irritant compound, on IL-1 alpha release. Even at the concentration of 300 micrograms/ml no IL-1 alpha release could be detected in the conditioned media. We have previously found a good rank correlation between in vitro surfactants induced IL-1 alpha release and in vivo Draize test. We now extend the possibility to use IL-1 alpha production for preliminary screening of chemicals for their irritative/allergenic potential, reducing the need for in vivo models.

Allergens↗

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II) oil/water emulsions.

The Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program is an evaluation of the relationship between Draize ocular safety test data and comparable data from a selection of in vitro tests. In Phase II, 18 representative oil/water-based personal-care formulations were subjected to the Draize primary eye safety test and 30 in vitro assay protocols (14 different types of in vitro endpoints were evaluated; the remainder were protocol variations). Correlation of in vitro with in vivo data was evaluated using analysis of sensitivity/specificity and statistical analysis of the relationship between maximum average Draize score (MAS) and in vitro endpoint. Regression modelling is the primary approach adopted in the CTFA Program for evaluating in vitro assay performance. The objective of regression analysis is to predict MAS for a given test material (and to place upper and lower prediction interval bounds on the range in which the MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves: the narrower the prediction interval, the more predictive of the Draize score is the in vitro test result. 16 assays were shown to have the greatest agreement with the Draize procedure and were therefore selected for regression analysis. Based on the magnitude of the 95% prediction bounds of each of the 16 selected assays over the range of test data, it may be inferred that prediction of MAS values from experimentally determined in vitro scores is more accurate for oil/water-based formulations with lower rather than higher irritancy potential. The assays selected for modelling in Phase II generally exhibited weaker relationships with MAS than those selected in Phase I (evaluated using hydroalcoholic formulations), even though several assays were common to both Phases.

3T3 Cells↗