The use of non-animal alternatives in the safety evaluations of cosmetics ingredients by the Scientific Committee on Consumer Safety (SCCS).

In Europe, the safety evaluation of cosmetics is based on the safety evaluation of each individual ingredient. Article 3 of the Cosmetics Regulation specifies that a cosmetic product made available on the market is to be safe for human health when used normally or under reasonably foreseeable conditions. For substances that cause some concern with respect to human health (e.g., colourants, preservatives, UV-filters), safety is evaluated at the Commission level by a scientific committee, presently called the Scientific Committee on Consumer Safety (SCCS). According to the Cosmetics Regulations, in the EU, the marketing of cosmetics products and their ingredients that have been tested on animals for most of their human health effects, including acute toxicity, is prohibited. Nevertheless, any study dating from before this prohibition took effect is accepted for the safety assessment of cosmetics ingredients. The in vitro methods reported in the dossiers submitted to the SCCS are here evaluated from the published reports issued by the scientific committee of the Directorate General of Health and Consumers (DG SANCO); responsible for the safety of cosmetics ingredients. The number of studies submitted to the SCCS that do not involve animals is still low and in general the safety of cosmetics ingredients is based on in vivo studies performed before the prohibition.

Aggregation properties of diacyl lysine surfactant compounds: hydrophobic chain length and counterion effect.

In this paper, we report on the study of aqueous solution and aggregation properties of diacyl Lysine surfactant salts with several surfactant counterions at a fixed hydrophobic chain length. They present a critical micellar concentration nearly independent of the counterion. The area per surfactant molecule is around 1.3 nm (2) also independent of the counterion. We have also studied the dry state crystallization of these surfactant salts. We show that small counterion systems tend to form bicontinuous cubic structures and that the increase in counterion size tends to form lamellar structures. We have compared this behavior with the dry state crystallization of the diacyl Lysine surfactants as a function of hydrophobic chain length. For long hydrophobic chains, the crystal structure is lamellar, while for intermediate, length is cubic. Among the structures studied, the one with the shortest chain length crystallizes in a hexagonal inverse phase.

Alternative methods for eye and skin irritation tests: an overview.

The evaluation of eye and skin irritation potential is essential to ensuring the safety of individuals in contact with a wide variety of substances designed for industrial, pharmaceutical or cosmetic use. The Draize rabbit eye and skin irritancy tests have been used for 60 years to attempt to predict the human ocular and dermal irritation of such products. The Draize test has been the standard for ocular and dermal safety assessments for decades. However, several aspects of the test have been criticised. These include: the subjectivity of the method; the overestimation of human responses; and the method's cruelty. The inadequacies of the Draize test have led to several laboratories over the last 20 years making efforts to develop in vitro assays to replace it. Protocols that use different types of cell cultures and other methods have been devised to study eye and skin irritation. Different commercial kits have also been developed to study eye and skin irritation, based on the action of chemicals on these tissues. This article presents a review of the main alternatives developed to replace the use of animals in the study of chemical irritation. Particular attention is paid to the reproducibility of each method.

Potential irritation of lysine derivative surfactants by hemolysis and HaCaT cell viability.

Surfactants represent one of the most common constituents in topical pharmaceutical and cosmetic applications or cleansers. Since adverse skin and ocular reactions can be caused by them, it is important to evaluate damaging effects. Amino acid-based surfactants deserve particular attention because of their low toxicity and environmental friendly properties. New lysine derivative surfactants associated with heavy and light counterions were tested. The ocular irritancy was assessed by hemolysis, and photohemolysis was employed to evaluate their phototoxicity. Cytotoxicity on HaCaT cells was determined by neutral red uptake and MTT assay to predict skin irritation. All lysine derivative surfactants were less hemolytic and thus less eye-irritating than the commercial surfactants used as model irritants. No phototoxic effects were found. All surfactants presented cytotoxic effects as demonstrated by decrease of neutral red uptake and reduction of MTT salt, with clear concentration-effect profiles. However, the rates of cytotoxicity on HaCaT for the new surfactants suggested that they were less cytotoxic and then, less skin-irritating than the reference ones; surfactants with heavy counterions were the less cytotoxic. The anionic surfactants investigated in the present work may constitute a promising class of surfactants given their low irritancy potential for pharmaceutical and cosmetic preparations.

Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays.

Arginine-derivative surfactants constitute a novel class of surfactants, which can be regarded as an alternative to conventional surfactants. Prior to human exposure, it is necessary to assess their irritation potential. The classical in vivo evaluation of the irritancy potential via the Draize test has been extensively criticized. In that regard, a great number of in vitro alternatives have been developed. Erythrocytes were chosen as the target cells for eye irritation assessment and hemolysis and hemoglobin denaturation were selected as appropriate endpoints. For skin irritancy assessment, the keratinocyte cell line NCTC 2544 was used and different in vitro endpoints were measured: two cytotoxicity assays (NRU and MTT) and the synthesis of the proinflammatory cytokine IL-1alpha. The eye and skin Draize tests were also performed for comparative purposes. The results point out that, according to in vivo and in vitro assays, the new arginine-derivative surfactants have lower eye and skin irritation potential than the synthetic surfactant SDS. Furthermore, in vitro methods were also able to detect differences in irritancy among the new surfactants not noticeable by the Draize tests, indicating that in vitro methods can be more sensitive than the in vivo test, offering the opportunity to detect subtle differences in irritancy.

Determination of interleukin-1alpha in human NCTC 2544 keratinocyte cells as a predictor of skin irritation from lysine-based surfactants.

Lysine derivative surfactants are a class of amino acid-based surfactants synthesized as lecithin analogues that deserve particular attention because of their low toxicity and high biocompatibility. To complete the toxicological profile of these surfactants, IL-1 alpha production (cell-associated and release to the culture medium) was determined as an in vitro method for predicting skin irritation. In addition, an MTT assay was used as a viability marker in keratinocytes NCTC 2544. Keratinocytes are a biologically relevant target for developing in vitro techniques to assess skin irritants: moreover, they are the principal source of the proinflammatory cytokine IL-1 alpha in the epidermis. Lysine derivatives proved to be less potent in stimulating IL-1 alpha synthesis and induced a lower release of this cytokine into the culture medium when compared to the anionic surfactant sodium dodecyl sulfate. Due to their low irritancy potential, lysine-based surfactants may offer promising applications in pharmaceutical and cosmetic preparations.

The chorioallantoic membrane test as a model to predict the potential human eye irritation induced by commonly used laboratory solvents.

The purpose of this study was to investigate the potential eye irritation of a range of solvents, extensively used in industry and laboratory and the capacity of the chorioallantoic membrane test to predict this eye irritation. The irritation has been evaluated by an in vitro method using the chorioallantoic membrane as an alternative to in vivo Draize rabbit test. All the solvents studied are potentially strongly irritants, even though diluted, except dimethyl sulfoxide which was moderately irritant at a concentration of 10% v/v. In some cases there is a correlation between the concentration of the solvent and the potential eye irritation induced. The method allows prediction of the potential eye irritation of the solvents studied.

Oligomers are not the limiting factor in the absorption of DL-2-hydroxy-4-(methylthio)butanoic acid in the chicken small intestine.

The methionine hydroxy analogue DL-2-hydroxy-4-(methylthio)butanoic acid (HMB) is commonly used as a supplemental source of methionine in commercial animal diets. The HMB free acid is an aqueous solution that contains 88% product in an equilibrium mixture of monomer, dimer, and polymeric compounds. The present study examines whether the presence of these nonmonomeric forms reduces the absorption of the hydroxy analogue in the chicken small intestine. In vivo and in vitro methodologies were used to compare the intestinal absorption of an HMB product containing mainly monomer (HMB-PCM) with commercial HMB. The results from the in vivo perfusion of the jejunum showed no significant differences between the 2 hydroxy analogue sources in monomer absorption from the intestinal lumen, tissue accumulation, or plasma concentration. The results also indicate that the nonmonomeric forms are hydrolyzed during perfusion. Moreover, monomer tissue accumulation in everted sacs showed no significant differences between substrates, either in the presence or in the absence of a H+-gradient; a higher value was observed in the jejunum and ileum in comparison with the duodenum. Similarly, serosal appearance in H+-gradient conditions did not differ significantly between substrates, and it showed the same regional profile as in tissue accumulation. Oligomer hydrolysis was confirmed in vitro without significant differences between segments. In conclusion, the presence of nonmonomeric forms is not a limiting factor in HMB absorption, apparently because of the hydrolytic capacity of intestinal mucosa, as confirmed by experiments in vivo and in vitro.

New cationic vesicles prepared with double chain surfactants from arginine: Role of the hydrophobic group on the antimicrobial activity and cytotoxicity.

Cationic double chain surfactants have attracted much interest because they can give rise to cationic vesicles that can be used in biomedical applications. Using a simple and economical synthetic approach, we have synthesized four double-chain surfactants with different alkyl chain lengths (LANHCx). The critical aggregation concentration of the double chain surfactants is at least one order of magnitude lower than the CMC of their corresponding single-chain LAM and the solutions prepared with the LANHCx contain stable cationic vesicles. Encouragingly, these new arginine derivatives show very low haemolytic activity and weaker cytotoxic effects than conventional dialkyl dimethyl ammonium surfactants. In addition, the surfactant with the shortest alkyl chain exhibits good antimicrobial activity against Gram-positive bacteria. The results show that a rational design applied to cationic double chain surfactants might serve as a promising strategy for the development of safe cationic vesicular systems.

Gelatin-based nanoparticles as DNA delivery systems: Synthesis, physicochemical and biocompatible characterization.

The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery systems, rendering them promising DNA vehicles to be used as non-viral gene delivery systems.

Age differences on ileal glucose absorption in rats.

Ileal absorption of D-glucose was studied in fasted 21-day-old and 18-month-old rats for a 5-min period using a perfusion technique in vivo. D-Glucose initial concentrations were: 2.5, 5, 10, 20 and 40 mmol/l. Phloridzin (0.5 mmol/l) was used as inhibitor of the active transport mechanism, inducing a 65 and 70% inhibition in the ileum of the young and old rats, respectively, at 40 mmol/l initial sugar concentration. Saturation was at 10 mmol/l in the two groups studied. A reduction in the rate of total sugar absorption, active transport and diffusion coefficient was observed with aging.

Age influence on D-glucose absorption depending on sodium concentration in the lumen of rat small intestine.

The influence of decreased sodium concentration on D-Glucose jejunal and ileal absorption has been investigated in rats of three groups of different ages: 21-23 days, 2-3 months and 18 months old, using a perfusion system in vivo. In all the cases studied there was a decrease of D-Glucose absorption with reduced sodium concentrations. The ileum of 21-23 days old animals showed the greatest inhibition of D-Glucose absorption when sodium was substituted partially or totally by Tris-HCl.

Preliminary studies of the toxic effects of non-ionic surfactants derived from lysine.

The toxic effects of new synthetic monodisperse non-ionic long-chain N alpha, N epsilon-diacyl lysine polyoxyethylene glycol amide compounds with a structural resemblance to natural lecithin phospholipids were studied by the haemolytic method and the test of the chorioallantoic membrane of the hen's egg (HET-CAM). The following compounds were tested: symmetrical N alpha,N epsilon-diacyl lysine homologues (N alpha,N epsilon-dihexanoyl, N alpha,N epsilon-dioctanoyl and N alpha,N epsilon-didecanoyl lysine) with one methyl ether polyoxyethylene glycol chain of different oxyethylene units (dioxyethylene glycol, tetraoxyethylene glycol and hexaoxyethylene glycol) as headgroup; symmetrical N alpha,N epsilon-diacyl lysine homologues with two methyl ether dioxyethylene glycol chains and the asymmetrical N alpha-butanoyl, N epsilon-dodecyl lysine with two hydrophilic methyl ether dioxyethylene glycol chains as headgroup. A commercial (polydisperse) oleoyl polyoxyethylene glycol diethanolamide with an average of eight units of ethylene oxide was used as control. All the synthesized tested compounds appeared to be less haemolytic and less irritant than the control. The synthesized products were studied with regard to their hydrophobic and hydrophilic chains in order to evaluate the influence of their structure on their haemolytic and irritative action. The results of this study show that the acyl chain distribution of these compounds greatly influence toxic effects: the asymmetrical compound N alpha-butanoyl,N epsilon-dodecyl lysine-bis[methyl ether diethylene glycol]amide was found to be the most haemolytic and irritating compound. Among the symmetrical homologues, the shortest-chain compounds N alpha,N epsilon-dihexanoyl lysine methyl ether polyoxyethylene glycol amides present the least haemolytic and irritating activity, independently of the number and length of the hydrophilic methyl ether polyoxyethylene glycol chains. Taking into account their surface activity properties and their less haemolytic and irritant action, the compound N alpha,N epsilon-dioctanoyl lysine-bis[methyl ether diethylene glycol]amide would be the most suitable for practical purposes.

Assessment of primary eye and skin irritants by in vitro cytotoxicity and phototoxicity models: an in vitro approach of new arginine-based surfactant-induced irritation.

Extensive efforts have been made, recently, to find surfactants with lower irritation potential than those presently commercially available, for use in pharmaceutical and cosmetic preparations. Cytotoxic and phototoxic effects of a novel family of dicationic arginine-diglyceride surfactant compounds, 1,2-diacyl,3-O-(l-arginyl)-rac-glycerol with alkyl chain lengths in the range from 8 to 14 carbon atoms, were compared to three commercial surfactants. The end-points used to assess toxicity were the red blood cell lysis assay and uptake of the vital dye neutral red 24h after dosing (NRU), respectively. Two immortalized cell lines, murine fibroblast cell line, 3T3, and one human keratinocyte cell line, HaCaT, were used as in vitro models to predict the potential phototoxicity which could result in irritation, determined by resazurin reduction to resorufin and neutral red uptake (NRU). All tested surfactants had cytotoxicity effects as demonstrated by and decrease of NR uptake, which showed a clear concentration-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) range from 1 microM(-1) (hexadecyl trimethyl ammonium bromide) to 565 microM(-1) (12,12-l-arginine). Erythrocyte haemolysis also showed a clear concentration-response relationship, the 50% of haemolysis ranged from 37 microM(-1) (10,10-l-arginine) to 151 microM(-1) (sodium lauryl sulphate). Phototoxicity was performed with 12,12-l-acetyl-arginine, the most stable chemical structure. The validated 3T3 NRU photoxicity assay was used and revealed a phototoxic potential.

Erythrocyte hemolysis and shape changes induced by new lysine-derivate surfactants.

The effects of new synthetic lysine-derived anionic surfactants on human and rat erythrocytes were studied. The surfactants were salts of Nalpha,Nepsilon-dioctanoyl lysine with different counterions: lysine (77KK), tris (trishydroxymethyl amminomethane) (77KT), sodium (77KS), and lithium (77KL). 77KK and 77KT showed a biphasic hemolytic behavior in the erythrocytes. The surfactants 77KS and 77KL showed concentration-dependent hemolysis with a CH50 of about 3.4 and 2.6 mmol/l, respectively. 77KK and 77KT induced protection against hypotonic hemolysis in rat erythrocytes at the concentration which showed the least hemolytic activity under isotonic conditions. With human erythrocytes, 77KT did not show biphasic behavior in isotonic medium, but under hypotonic conditions biphasic behavior was present. Changes in shape of the erythrocyte, from discocytic to stomatocytic were observed after incubation with the anionic surfactants studied. Such shape changes occurred progressively over time, with total alteration in shape occuring after about 20 min of incubation.

Comparative study of the HET-CAM test and the Draize eye test for assessment of irritancy potential.

The ocular irritancy potential of different substances used as vehicles has been tested by the Draize test method and the alternative hen's egg test-chorioallantoic membrane (HET-CAM) test. With the latter test, carboxymethylcellulose was not found to be irritant and can be used to suspend insoluble substances to be assessed by this method. The ocular irritancy potential of six commercial disinfectants was also tested by the Draize test method and the alternative HET-CAM test. Products were assigned to categories of irritancy, according to two different sets of criteria for the Draize test results, and according to irritancy potential assessed by the HET-CAM test. When both sets of results for Draize categories were compared with those for the HET-CAM test, four of the six products tested showed similar irritancy potential. The remaining two products gave false positive results on the HET-CAM compared with the Draize test.

The quantitive chlorioallantoic membrane test using trypan blue stain to predict the eye irritancy of liquid scintillation cocktails.

The chorioallantoic membrane-trypan blue staining assay (CAM-TBS) is used to evaluate the potential ocular irritation caused by liquid scintillation cocktails constituted by complex mixtures, including surfactants and other potential irritants. The harmful effect of these substances is determined by the amount of trypan blue adsorbed by the CAM. In the membrane previously treated with the scintillation liquids this amount was concentration dependent only in certain cases, irrespective of the water solubility of the mixtures. In general, it showed a high correlation (r=0.986) with the scores obtained in the Draize eye irritation test. In the present study, only two cocktails presented values of trypan blue adsorption higher than those recorded for their eye irritation in vivo, irrespective of the solvent nature. Unlike the classical HET-CAM procedure, this assay is objective and allows the evaluation of opaque and coloured substances without interfering in the determination of irritancy. Despite these advantages, the method is not suitable for complex mixtures of products that induce ocular irritation in small quantities.

Irritancy potential induced by surfactants derived from lysine.

The ocular irritancy potential of surfactants of the anionic and non-ionic type, derived from lysine has been tested by the hen's egg test-choriollantoic membrane (HET-CAM) to correlate the potential irritation with the structure of the surfactant, in order to synthesize the less irritant surfactant for their use in the pharmaceutical and cosmetics industry. The anionic compounds, independently of the counterion, showed an irritant action higher than non-ionic surfactants. Among the anionic surfactants the presence of lysine as cation reduced the degree of irritation; nevertheless, the salt of lysine of lauric acid was a severe irritant. The non-ionic surfactant with two chains was less irritant than the corresponding compound with one chain and represented the best compound for ocular application. The time of appearance of haemorrhage, vasoconstriction and coagulation is used to evaluate the degree of irritation. There was a close relationship between the concentration and the time of the appearance of vasoconstriction at the concentrations tested.

Potential eye irritation of some "biodegradable" liquid scintillation cocktails determined in vitro.

The potential eye irritation of a range of liquid scintillation cocktails has been evaluated by an in vitro method using the chorioallantoic membrane as an alternative to the Draize rabbit test. All the cocktails studied are potentially moderately to strongly irritant. The only slightly irritant product was Solventgreen, which is a solvent used in some of the cocktails. There is a correlation between the concentration of the cocktail and the potential eye irritation induced. Manufacturers of chemicals including liquid scintillation cocktails should accept responsibility for disclosing data about the composition and toxicity of their products with regard to waste disposal and safety of laboratory staff.

Elucidation of intestinal transport results as a function of age.

Duodenal, jejunal, ileal and caecal morphometrics were determined in chickens from hatching to the age of 15 weeks. The ratios of fresh weight/surface area and dry weight/surface area showed age-dependent changes in all the intestinal segments studied. The percentage of mucosa in each segment was also age-dependent in the first three weeks of life with a higher participation in the proximal intestine. Transport of 3-oxy-methyl-D-glucose was determined in the jejunum and ileum using a perfusion technique in vivo and expressed as a function of fresh weight, dry weight and surface area. The age-dependent differences observed in weight and mucosa composition of the intestine make it difficult to interpret the results of transport capacity during development. The optimum expression of results in the field of intestinal absorption may be the surface area, since it involves the smallest errors.