Performance and fouling characteristics of a submerged anaerobic membrane bioreactor for kraft evaporator condensate treatment.

Submerged anaerobic membrane bioreactor (SAnMBR) technology was studied for kraft evaporator condensate treatment at 37 +/- 1 degrees C over a period of 9 months. Under tested organic loading rates of 1-24 kg COD/m3/day, a chemical oxygen demand (COD) removal efficiency of 93-99% was achieved with a methane production rate of 0.35 +/- 0.05 L methane/g COD removed and a methane content of 80-90% in produced biogas. Bubbling of recycled biogas was effective for in-situ membrane cleaning, depending on the biogas sparging rate used. The membrane critical flux increased and the membrane fouling rate decreased with an increase in the biogas sparging rate. The scanning electron microscopy images showed membrane pore clogging was not significant and sludge cake formation on the membrane surface was the dominant mechanism of membrane fouling. The results suggest that the SAnMBR is a promising technology for energy recovery from kraft evaporator condensate.

Sludge properties and their effects on membrane fouling in submerged anaerobic membrane bioreactors (SAnMBRs).

Two submerged anaerobic membrane bioreactors (SAnMBRs) (thermophilic vs. mesophilic) were operated for a period of 3.5 months with kraft evaporator condensate at a feed chemical oxygen demand of 10,000 mg/L. The results show that the filtration behavior of the two systems was significantly different. The filtration resistance in the thermophilic SAnMBR was about 5-10 times higher than that of the mesophilic system when operated under similar hydrodynamic conditions. Comparison of sludge properties and cake layer structure from the two systems was made to elucidate major factors governing the different filtration characteristics. There were more soluble microbial products (SMP) and biopolymer clusters (BPC) produced and a larger portion of fine flocs (<15 microm) in the thermophilic SAnMBR. Analysis of bound extracellular polymeric substances (EPS) showed that the thermophilic sludge had a higher protein/polysaccharide ratio in EPS, as compared to that in the mesophilic sludge. A series of analyses, including Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-ray spectroscopy (EDX), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), atomic force microscopy (AFM) and particle size analyzer showed that the cake layer formed in the thermophilic SAnMBR contained higher levels of both organic and inorganic foulants, smaller particle sizes, and especially, a denser and more compact sludge cake structure. These results indicate that floc size, SMP, BPC, bound EPS as well as cake layer structure are the major factors governing membrane fouling in SAnMBR systems.

Measurement of H2 consumption and its role in continuous fermentative hydrogen production.

To maximise the yield from fermentative H(2) production, H(2) consumption must be minimised. This work demonstrated for the first time that H(2) consumption exists in an established continuous-flow biohydrogen system. The rate of H(2) consumption was found to be related to the concentration of CO(2), with H(2) consumption inhibited at both low and high CO(2). N(2) sparging of the continuous reactor at 31 mL/min/L-liquid increased the H(2) yield from 1.31 to 1.87 mol H(2)/mol glucose, but did not significantly change the in-situ rate of H(2) consumption (0.07-0.09 mM/h). Assuming sparging completely inhibited H(2) consumption, it could only account for 2-11% of the H(2) yield increase during sparging, based on H(2) consumption rates measured in the reactor and in vials. Therefore, H(2) consumption may be of minor concern for continuous biohydrogen systems.

Fermentative hydrogen production in a system using anaerobic digester sludge without heat treatment as a biomass source.

Hydrogen produced from anaerobic fermentation of organic matter is a sustainable energy source. Anaerobic hydrogen-producing systems have been typically seeded with heat-treated inocula to eliminate hydrogen-consuming methanogens. This can be both energy- and economically-intensive. In this work, operational parameters were modified to determine if operating a reactor at low pH (5.5) and low SRT (10 hours) would result in a hydrogen-producing system free of methanogens using anaerobic digester sludge with no heat treatment as an inoculum. Initially, the reactor exhibited a hydrogen productivity of approximately 7.9% when fed glucose. After purging was begun with 10% CO2/90% N2, the hydrogen productivity increased to > 20% for the first day. Hydrogenotrophic methanogens then established themselves in the reactor, reducing the hydrogen productivity in the second non-purged phase by 80%. The operational controls examined were not sufficient to eliminate hydrogen-consuming methanogens for longer than approximately one week, and thus further methods must be developed.

A review of biofouling and its control in membrane separation bioreactors.

Membrane separation technology is increasingly becoming an important innovation in biological wastewater treatment. Biofouling of the membrane is a major factor affecting the efficient and economic operation of membrane separation bioreactors (MBRs). This review summarizes the state-of-the-art progress in understanding the mechanisms and factors affecting membrane biofouling and the strategies for biofouling control. Biofouling mechanisms include the adsorption of soluble and suspended extracellular polymers on membrane surfaces and in membrane pores, the clogging of membrane pore structure by fine colloidal particles and cell debris, and the adhesion and deposition of sludge cake on membrane surfaces. Design and operating conditions of membrane modules and materials, hydrodynamic conditions in MBRs, process and environmental conditions of activated sludge systems, and the physicochemical properties of the wastewater are the dominant factors determining membrane biofouling. Current strategies to control biofouling include periodic relaxation, backwashing, chemical cleaning, and possible manipulation of hydrodynamic conditions and sludge properties. Achieving full integration of MBRs in wastewater treatment technology requires further research and development. Fundamental information on the bacteria, colloid, and membrane interaction, developed through multimethod and multiscale approaches, is particularly needed.

Anaerobic degradation and methanogenic inhibitory effects of oleic and stearic acids.

Oleic acid, an 18 carbon acid with one double bond (C18:1) was degraded anaerobically to palmitic (C16:0) and myristic (C14:0) acid by-products at 21 C by a culture unacclimated to long-chain fatty acids. These by-products were degraded to acetate and ultimately to methane. In comparison, no long-chain fatty acid by-products were observed in unacclimated anaerobic cultures receiving stearic (C18:0) acid although slow removal of stearic acid occurred. Oleic acid concentrations above 30mg l(-1) inhibited acetate degradation but stearic acid up to 100 mg l(-1) did not inhibit aceticlastic methanogenesis. Hydrogenotrophic methanogenesis was slightly inhibited by oleic and stearic acids. A thermodynamic basis for comparing anaerobic C18 acid degradation and predicting by-products is presented.

Effect of patch type on the cumulative irritation potential of 4 test materials.

BACKGROUND: Many different patch systems are available for predicting contact dermatitis. It is important to determine the ideal patch to meet the objective of the testing method. OBJECTIVE: The 21-day cumulative irritation test is well accepted for predicting irritation after repeated exposures. The patch type must allow separation of materials to predict irritation potential in the marketplace. Three patch systems were compared to determine which best provides this separation and prediction. METHODS: Four test materials were evaluated using 3 patch systems in a 21-day cumulative irritation test. Tested were water, 0.06% sodium lauryl sulfate (SLS), and 2 underarm products (UAP), one having lower and one having higher irritation potential. The patch types were; Webril pad and 8-mm and 12-mm Finn Chambers. RESULTS: Both the 12-mm Finn Chamber and Webril pad showed the ability to differentiate the higher irritating UAP and the 0.06% SLS from the lower irritation UAP product and water. The 8-mm Finn chamber was less discriminating, showing the 0.06% SLS to be the same as water and the lower-irritating UAP. CONCLUSION: The Webril pad and the 12-mm Finn Chamber are better at discriminating irritation potential than is the 8-mm Finn Chamber. The 12-mm Finn Chamber might also allow discrimination with a lower degree of irritation.

Clinical evidence for the lack of triclosan accumulation from daily use in dentifrices.

PURPOSE: To demonstrate through clinical pharmacokinetic studies that triclosan does not accumulate in blood or plasma in human subjects who regularly use triclosan-containing dentifrice. MATERIALS AND METHODS: Three clinical pharmacokinetic studies were conducted to assess the blood or plasma levels of triclosan following toothbrushing with dentifrice formulations containing triclosan. In Study 1, both a single-dose and a multiple-dose phase were conducted. In the single-dose phase, subjects brushed one time with 1.25 g dentifrice containing 0.3% triclosan (3.75 mg triclosan dose) and ingested all of the dentifrice. Blood samples were collected at multiple time points from pre-dose to 72 hrs post-dose and analyzed for total triclosan levels. In the multiple-dose phase, these same subjects brushed three times daily as in the single-dose phase. This pattern was followed for 12 consecutive days. Blood samples were taken for triclosan analysis at multiple time points up to 48 hrs after the first dose of day 12. Study 2 was a parallel, open-labeled clinical study to compare triclosan blood levels from twice daily brushing with 1 gm of dentifrice containing 0.2% triclosan to twice daily ingestion of 20 ml of a 0.01% triclosan aqueous solution over a period of 21 days. Blood samples were taken for triclosan analysis at baseline and at 4 hrs after the morning dose on days 7, 14, and 21. Study 3 was a parallel, double-blind, 12-wk brushing study with dentifrice containing 0.2% triclosan or a matching placebo. Blood samples were taken for triclosan analysis at baseline and at 3 and 12 wks at 4 hrs after the morning dose. RESULTS: In the single-dose study, Triclosan was absorbed into the systemic circulation with a T(1/2) of the terminal plasma concentration ranging between 6-63 hrs. The mean AUC(0-inf) after a single dose was found to be 2,809 ng x hr/ml. After 12 days of three times daily toothbrushing and ingestion of the dental slurry, the mean triclosan plasma concentration was 352 ng/ml in the steady state period, and the mean AUC in a 24-hr period (AUC24) was found to be 8,460 ng x hr/ml. This AUC24 was normalized for the number of brushings for comparison to the AUC(0-inf) after a single brushing. There was no significant (P = 0.93) difference between these AUC values suggesting a complete elimination of daily triclosan dose and no increase in the triclosan level during repeated brushing/ingestion. In the two other dentifrice studies, the triclosan blood concentration appeared to reach a steady state level by day 7 and was maintained at the steady state level (14 to 21 ng/ml) for up to 12 wks. These results support the conclusion that the elimination of a daily triclosan dose is complete and no accumulation of triclosan was observed even after three times daily toothbrushing with 1.25 g dentifrice containing 0.3% triclosan and full ingestion of the dentifrice.

Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study.

The chorioallantoic membrane vascular assay (CAMVA) is an alternative to the Draize rabbit eye irritation method. The CAMVA employs the vascularized membrane of a fertile hen's egg to assess eye irritation potential. This irritation potential is a function of alterations in the vasculature following the administration of test material. Because of the history of use of the CAMVA it was selected as one of the methods for a validation study organized and sponsored by COLIPA. For this validation study mathematical prediction models (PMs) were developed to convert the CAMVA results into predicted Draize eye irritation scores known as a modified maximum average Draize score (MMAS). These predicted scores were statistically compared with the observed scores to assess the relevance of the CAMVA. The assay was conducted on the same set of test materials by two independent laboratories. These two sets of data were compared to assess the interlaboratory reproducibility of the assay. The results of this validation study of the CAMVA show that for test materials with MMASs in the 0 to 5 range or the 55 to 110 range, the CAMVA did not give a good prediction. The predictions were better for samples of mild to moderate irritation (MMAS 5-55). The difficulty in predicting at the low end of the irritation scale appears to be due to the biological variability of the test system and the subjective nature of the CAMVA evaluation. For those samples with an MMAS above 55, the CAMVA appeared to be limited in demonstrating the more severe response. This may be due to the fact that the PMs were developed using historical data sets of test materials with MMASs below this range. Two approaches for improving the CAMVA for eye irritation prediction are (1) to decrease the variability at the low end by reducing the subjectivity in the scoring and (2) to develop better prediction models using more data in the range of severe irritants.

Eye irritation: updated reference chemicals data bank.

A list of 55 chemicals for which comprehensive rabbit eye irritation data were available was published by ECETOC in 1992. Similar data for a further 77 chemicals are now available. The total of chemicals included in the enlarged data bank is 132, assessed in 149 in vivo studies in rabbits. 28 of the chemicals were tested as solids, 24 as aqueous solutions. No new in vivo testing was carried out in order to qualify a chemical for inclusion in the data bank. The chemicals are available at known, high, consistent purity and are expected to be stable in storage. The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three animals evaluated at the same time, involving instillation of 0.1ml (or equivalent weight) into the conjunctival sac, and in which observations were made at least 1, 2 and 3 days after instillation. The chemicals are ranked for eye irritation potential on the basis of a 'modified maximum average score'. The reference chemicals data bank should be of use in validation tests of promising alternatives to the in vivo rabbit eye irritation test.

IRAG working group 2. CAM-based assays. Interagency Regulatory Alternatives Group.

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsor's product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearson's linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

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.

Skin irritation: Reference chemicals data bank.

A list of 176 chemicals, all of high or consistent purity and stable on storage, has been developed using available comprehensive in vivo rabbit skin irritation data. No new in vivo testing was conducted to qualify a chemical for inclusion in the list. The chemicals were tested undiluted in in vivo studies, apart from those chemicals where high concentrations could be expected to cause severe effects. The in vivo data were generated in studies carried out since 1981 according to OECD Test Guideline 404 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving application of 0.5 g or 0.5ml to the flank under semi-occlusive patches for 4 hr, and in which observations were made at least 24, 48 and 72 hr after removal of the patch. The chemicals represent a range of chemical classes [acids, acrylates/methacrylates, alcohols, aldehydes, alkalis, amines, brominated derivatives, chlorinated solvents, esters, ethers, fatty acids and mixtures, fragrance oils, halogenated aromatics, hydrocarbons (unsaturated), inorganics, ketones, nitrites, phenolic derivatives, S-containing compounds, soaps/surfactants, triglycerides] and different degrees of irritancy. They are ranked for skin irritation potential on the basis of a 'primary irritation index'. These chemicals could be used in validation tests of promising alternatives to the in vivo rabbit skin irritation/corrosion test. This is an essential step in the progression to regulatory acceptance of alternative procedures.

Chloroform degradation in methanogenic methanol enrichment cultures and by Methanosarcina barkeri 227.

The effects of methanol addition and consumption on chloroform degradation rate and product distribution in methanogenic methanol enrichment cultures and in cultures of Methanosarcina barkeri 227 were investigated. Degradation of chloroform with initial concentrations up to 27.3 microM in enrichment cultures and 4.8 microM in pure cultures was stimulated by the addition of methanol. However, methanol consumption was inhibited by as little as 2.5 microM chloroform in enrichment cultures and 0.8 microM chloroform in pure cultures, suggesting that the presence of methanol, not its exact concentration or consumption rate, was the most significant variable affecting chloroform degradation rate. Methanol addition also significantly increased the number of moles of dichloromethane produced per mole of chloroform consumed. In enrichment cultures, the number of moles of dichloromethane produced per mole of chloroform consumed ranged from 0.7 (methanol consumption essentially uninhibited) to 0.35 (methanol consumption significantly inhibited) to less than 0.2 (methanol not added to the culture). In pure cultures, the number of moles of dichloromethane produced per mole of chloroform consumed was 0.47 when methanol was added and 0.24 when no methanol was added. Studies with [14C]chloroform in both enrichment and pure cultures confirmed that methanol metabolism stimulated dichloromethane production compared with CO2 production. The results indicate that while the addition of methanol significantly stimulated chloroform degradation in both methanogenic methanol enrichment cultures and cultures of M. barkeri 227, the prospects for use of methanol as a growth substrate for anaerobic chloroform-degrading systems may be limited unless the increased production of undesirable chloroform degradation products and the inhibition of methanol consumption can be mitigated.

Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the Draize rabbit eye irritation test.

The chorioallantoic membrane (CAM) of a fertilized hen's egg has been studied extensively as a promising alternative model for predicting eye irritation potential. The specific methodology used with this model has varied among investigators but the basic premise of applying test material to the membrane surface and evaluating changes in the vasculature is relatively consistent. The CAM vascular assay (CAMVA) has shown high correlation with in vivo rabbit eye irritation data. This method uses the CAM of a 14-day-old egg and the response at 30 min after treatment as the endpoint. The primary CAM methods being evaluated in Europe use 9-10-day-old eggs because older eggs are considered 'live animals'; the possibility of using 10-day-old eggs to make the method more globally acceptable as a non-animal test was therefore investigated. By keeping the original CAMVA dosing and evaluation procedures the same, and only altering the age of the eggs from 14-day to 10-day, the results were found to be nearly identical for the two methods and both produce equivalent correlations to the in vivo eye irritation test results. Maintaining the original CAMVA methodology but using a younger egg, therefore, provides a good alternative method for predicting eye irritation potential that is more globally acceptable as a non-animal test.

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.

Safety profile of Colgate Platinum Professional Toothwhitening System.

Colgate Platinum, a professional tooth-whitening paste containing 10% urea peroxide as the active ingredient, was evaluated for potential acute oral toxicity, genotoxicity, and irritation to oral mucosa. Oral administration to rats of a single dose of 5 g/kg of Colgate Platinum did not induce any adverse effects. Colgate Platinum was not mutagenic in Ames/Salmonella Plate Incorporation assay and did not induce primary DNA damage in the bone marrow hematopoietic cells of rats that were given oral doses of up to 1 g/kg for 5 consecutive days. Results of the oral mucosa irritation study in rats indicated that Colgate Platinum did not induce damage to soft and hard tissues of oral cavity after repeated applications for 28 days. Collectively, the data from these studies document the safety of the product for the intended use.

Safety profile of Colgate Platinum Professional Toothwhitening System.

Colgate Platinum, a professional tooth-whitening paste containing 10% urea peroxide as the active ingredient, was evaluated for potential acute oral toxicity, genotoxicity, and irritation to oral mucosa. Oral administration to rats of a single dose of 5 g/kg of Colgate Platinum did not induce any adverse effects. Colgate Platinum was not mutagenic in Ames/Salmonella Plate Incorporation assay and did not induce primary DNA damage in the bone marrow hematopoietic cells of rats that were given oral doses of up to 1 g/kg for 5 consecutive days. Results of the oral mucosa irritation study in rats indicated that Colgate Platinum did not induce damage to soft and hard tissues of oral cavity after repeated applications for 28 days. Collectively, the data from these studies document the safety of the product for the intended use.

An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo.

Five alternative techniques, each of which had been successfully used by one of the participating companies, were evaluated in the assessment of the eye-irritation potential of 32 samples. The 32 samples included chemical ingredients and preparations from household cleaning product, personal care, and cosmetic categories. Historical data from rabbit eye irritation tests in vivo existed for each sample; it was therefore not necessary to carry out any tests in vivo as part of this evaluation exercise. The five alternative methods used were the silicon microphysiometer test, the Microtox test, the neutral red uptake assay, the chorioallantoic membrane vascular assay (CAMVA) and the hen egg test-chorioallantoic membrane assay (HETCAM). Three of the assays were conducted in two laboratories, allowing an interlaboratory comparison of performance to be made. The CAMVA assay was carried out on 10-day-old as well as on 14-day-old fertile eggs. Correlations between the data sets in vivo and in vitro were determined for the five assays. The results demonstrated that for the materials tested, all of the assays show some promise as alternative methods to the rabbit eye test in vivo in the prediction of eye irritation, and that the reproducibility of results of those techniques carried out in two laboratories was very good. The results from 14-day and 10-day CAMVA assays were virtually identical. It is recommended that a larger-scale validation exercise be carried out to demonstrate the ultimate usefulness of these alternative procedures in the safety evaluation process.