Health safety of parabens evaluated by selected in vitro methods.

Seven selected parabens (4 allowed, 3 banned in cosmetics) were tested in order to confirm and expand historical data on their toxicological properties and safety. The aim was to apply novel in vitro methods, which have been sufficiently technically and scientifically validated for the purposes of toxicological testing of chemicals. The study included several toxicological endpoints such as skin/eye irritation, skin sensitization, endocrine disruption and genotoxicity. The battery of selected methods comprised regulatory accepted EpiDerm™ skin model (OECD TG 439); EpiOcular™ corneal model (OECD TG 492) and scientifically valid test method HET-CAM (DB-ALM Protocol No. 47); in chemico test DPRA (OECD TG 442C); in vitro test LuSens (OECD TG 442D) and in vitro test h-CLAT (OECD TG 442E); Ames MPF™ (Xenometrix) and XenoScreen YES/YAS (Xenometrix). Overall, none of the 4 allowed parabens exhibited skin/eye irritation or genotoxicity. However, all allowed parabens in cosmetics were predicted as samples with potentially sensitizing properties in the LuSens and h-CLAT test methods, but not confirmed by DPRA. Endocrine disruption was recorded only at high concentrations, whereas methyl paraben and ethyl paraben exhibited the lowest activity. This study confirmed the safety of use of the allowed parabens in the highest recommended concentrations in cosmetics or pharmaceuticals.

Characteristics of titanium dioxide microdispersions with different photo-activity suitable for sunscreen formulations.

The aim of the study was the comparison of photo-activity of three types of titanium dioxide (TiO2) micro-dispersions intended for use as UV filters for cosmetic sunscreen products. The dispersions were also investigated with regard to their influence on the stability of photo-protective systems in cosmetic emulsions, their skin penetration/absorption and their photo-toxicity for humans and skin bacterial flora. All the tested micro-dispersions of rutile TiO2 type (agglomerates with diameter 120-150 nm), with primary particle size lower than 100 nm, demonstrated no phototoxic effect and insignificant antimicrobial behaviour. On the other hand, TiO2 with insufficient deactivation of photo-activity had significant negative impact on the stability of other organic UV filters and therefore on the stability of declared UV protective factors (SPF, UVA-PF). The study demonstrated that the level of deactivation of TiO2 is one of the highly important factors for evaluation of UV filters used as sunscreens.

In vitro cytotoxicity and phototoxicity study of cosmetics colorants.

The aim of the work was early identification of preventable risk factors connected with the consumers usage of products of everyday use, such as cosmetics, toys and children products, and other materials intended for contact with human skin. The risk factor is represented by substances with irritation potential and subsequent possible sensitisation, resulting in negative impact on human physical and psychical health with social and societal consequences. The legislation for cosmetics, chemical substances and other products requires for hazard identification the application of alternative toxicological methods in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were employed for identification of early morphological and functional changes on cellular level. Four colorants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV irradiation (dose 5 J cm(-2)). Fluorescence methods for the study of cell damage using fluorescence probes offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellular production of ROS investigated by molecular probe CM-H(2)DCFDA, which is primarily sensitive to the increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yellow soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using Chlorophyllin and Unicert Red K 7054-J.

Oxidative stress may modify zinc protoporphyrin/heme ratio in hematofluorometry.

Washed red blood cells (RBCs), supplemented or non-supplemented with sodium azide (to inhibit catalase activity), were exposed to different concentrations of hydrogen peroxide as well as ascorbic acid. Strikingly, catalase within RBCs protected the cells against exogenic hydrogen peroxide even at millimolar concentrations. However, the activity of the erythrocytic catalase failed to protect the RBCs when they were exposed to an oxidative burst of stimulated polymorphonuclear cells (PMNCs) in the presence of several reactive species in addition to peroxide. Oxyhemoglobin, with an excess of hydrogen peroxide, formed oxidized hemoglobin species and caused protein denaturation as well as the rise of heme degradation products which was suspected to falsify zinc protoporphyrin/heme (ZPP/heme) ratio as assessed by hematofluorometry. Our experiments may thus imply that the non-fluorescent hemoglobin background can be modified by reactive oxygen species (ROS) and this can lead to a spurious ZPP/heme ratio. We discuss this phenomenon with respect to ZPP quantification in clinical practice.

Study of cytotoxic effect of photodynamically and sonodynamically activated sensitizers in vitro.

High resolution imaging of biological structures and their changes induced by different agents such as drugs are commonly performed by confocal and electron microscopy. The past decade has witnessed an emersion of the atomic force microscopy (AFM) from solid-state physics into cell biology and even medical applications. For these reasons, we used this relatively new microscopic technique to study the morphology of cell lines. We imaged the cells by atomic force microscopy before and after the photodynamic therapy (PDT) using the photosensitizer ClAlPcS(2). We also compared the impact of the photosensitizer in combination with silymarin antioxidant on cancer and non-cancer cell lines by measuring the kinetic production of reactive oxygen species (ROS). PDT was induced by LED source with total irradiation dose of 15 J cm(-2) and SDT was induced by therapeutic ultrasound with frequency of 1 MHz, intensity 2 W cm(-2) and time of exposition 10 min. The results show ROS kinetic production within the cells during PDT, sonodynamic therapy (SDT) and modification of morphological features investigated by AFM. The combination of a sensitizer and the specific light source can lead to the loss of surface rigidity and eventually to dramatic changes of the cell shape, which we can study by AFM.

Quartz plates for determining sun protection in vitro and testing photostability of commercial sunscreens.

Testing plate made of optical quartz has been developed for the purpose of determination of sun protection factor (SPF)(in vitro) by the method of diffusion transmission spectroscopy; the plates were coarsened by sanding and grinding to surface roughness values (Ra) of 18 mum. The plate was coated with a film of sunscreen by an application of 2 mg cm(-2) as that used for determination of SPF(in vivo) by the COLIPA method. The transmission values measured were converted into the SPF(in vitro) and the protection factor in ultraviolet A light, UVAPF(in vitro). The testing plate was tested with commercial sunscreens. The found values of SPF(in vitro) fit well with the values determined by means of the COLIPA method in vivo. The plates coated with sunscreen film were irradiated with light simulating the sun radiation. The values of protection factors obtained before and after irradiation were compared, and the differences were used for estimation of photostability of the UV filters included.

In vitro approaches to evaluation of Sun Protection Factor.

The efficacy of sunscreen products has been recognized as an important public health issue. Adequate methods for assessment of the level of protection should be developed and standardised. While the SPF COLIPA testing method in vivo has been used for years, preference should be given to in vitro testing methods as in vivo methods raise ethical concern. The present study aims to assess possible in vitro approaches based on diffuse transmission spectroscopy, published previously by Diffey, and two methods based on measurements of UVB transmission through a defined layer of a sunscreen product applied on various UV-transparent substrates. The attenuated UVB intensity, using different UV light sources, is detected radiometrically and transformed to real SPF value by means of a calibration curve, which is based on an extensive number of measurements performed using both in vivo and in vitro method The outcome of the three in vitro methods employed in the study showed great differences in the obtained SPF values in comparison with reference SPF determined by means of the COLIPA method in vivo. The high variability of in vitro results suggests that main attention should be focused on substrate selection simulating the human skin surface and homogenous product application. The in vitro screening methods may represent a fast and reasonable tool reducing the number of in vivo experiments and risks related to UV exposure of human subjects, when the technical test parameters are adjusted and optimized.

Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests.

The aim of this study was to clarify the differences in the phototoxicity of bergamot oil obtained from four different suppliers. Spectral and chemical analyses were performed to identify presence of photoactive compounds in the test samples. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D PT). Confirmatory photopatch tests in a group of volunteers were performed using the first non-phototoxic concentration determined in the H3D PT. The spectral and chemical analyses revealed, that two samples of bergamot oil exhibited a potential for photoactivation. These oils were subsequently classified as phototoxic in the 3T3 NRU PT, however, only on the basis of borderline results and depending on the solvent used. H3D PT revealed clear classifications, correlating well with the findings of spectral and chemical analysis. The test was, however, not yet capable of precise prediction of safe, non-phototoxic concentrations. Additional endpoints, e.g. interleukin determination might be employed to increase the sensitivity of the test. Although the study showed the usefulness of the tiered testing strategy, currently, the extrapolation of in vitro results to human situation may be performed only to a limited extent.

In vitro photodynamic therapy on melanoma cell lines with phthalocyanine.

Photodynamic therapy (PDT) is a new treatment modality of tumours. The photochemical interactions of sensitizer, light, and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxid anion. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was tested as an inducer of photodamage. We report the production of reactive oxygen species (ROS) and the phototoxicity of ClAlPcS(2) assessed using G361 melanoma cells. A semiconductor laser (lambda=675nm, output power 21mW) was used as a source for evocation of the photodynamic effect. ROS generation and H(2)O(2) release after PDT on G361 cells were detected using probe CM-H(2)DCFDA and recorded by luminescence spectrometer. Viability studies show, that the optimum phototoxic effect tested on G361 melanoma cells was determined in the combination of laser dose of 25Jcm(-2) and phthalocyanine ClAlPcS(2) concentration of 5microg/ml. This combination of phthalocyanine concentration and corresponding radiation dose was lethal for melanoma cells.

Phototoxicity of bituminous tars-correspondence between results of 3T3 NRU PT, 3D skin model and experimental human data.

Bituminous tars (Ichthammol and Ichthyol Pale) are widely used in pharmaceutical, veterinary and cosmetic industries for their anti-microbial, anti-inflammatory and anti-pruritic effects. In contrast to coal tar, no phototoxicity of bituminous tars has been reported in man, although both Ichthammol and Ichthyol Pale exhibit UV absorption which is higher and broader for the former. The validated 3T3 NRU phototoxicity test indicated phototoxic potential of both substances. The phototoxicity test in a 3D human skin model (EpiDerm) only confirmed phototoxicity for Ichthammol. Human data on Ichthammol phototoxicity are missing. A photopatch test in human volunteers was performed in order to clarify the discrepancy between the phototoxicity found in the skin model and the absence of reported human phototoxicity. Following 4h exposure to 5% and 10% aqueous solutions of Ichthammol and Ichthyol Pale the test sites were irradiated with a UVA dose of 5 J/cm(2). Early phototoxic reaction (erythema) within 4-6h after irradiation was only elicited by Ichthammol and not by Ichthyol Pale. These data correspond well with those from the 3D skin model test and suggest the necessity to employ several test systems for final phototoxicity assessment. In addition to the results obtained in 3T3 NRU PT, further testing on 3D skin models may better reflect bioavailability of a given chemical in the skin, relevant to the situation in humans.

Hydrophilic polymers--biocompatibility testing in vitro.

Biocompatibility is one of the main prerequisites for safe use of medical devices. Estimation of cytotoxicity is a part of the initial evaluation laid down in ISO standards on biological evaluation of medical devices. Hydrophilic polymers (based on 2-hydroxyethyl methacrylate HEMA) doped by addition of selected additives with antioxidant and/or free radical scavenging potential (vitamin C and hindered amine stabilizer N-(2,2,6,6-tetramethylpiperidin-4-yl)methacrylamide) were tested in different in vitro systems (3T3 Balb/c cell culture and a 3D human skin model) for biocompatibility and suitability for use as wound dressings. The results of the 3T3 NRU cytotoxicity test using both the direct and indirect contact approaches and a 3D skin model modified irritation test (EpiDerm) confirmed high biocompatibility and good skin tolerance of both the basic polymers and those enriched with specific additives up to a balanced level. HEMA polymer showed a beneficial effect against cytotoxicity of an irritant (sodium dodecyl sulfate). The in vitro biocompatibility test results were confirmed by human local skin tolerance testing.

In vitro toxicity testing of supramolecular sensitizers for photodynamic therapy.

We report the phototoxicity of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) and zinc metallocomplex (ZnTPPS4) sensitizers in the presence or absence of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on G361human melanoma cells. Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope and image analysis. Viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit- double staining with Calcein AM and Ethidium Homodimer). The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. We found that the most effective sensitizer is ZnTPPS4 bound to HP-beta-CD, since the IC50 value was 12.5 g/ml at the dose of light radiation of 10 J/cm2.

The benefits of the 3T3 NRU test in the safety assessment of cosmetics: long-term experience from pre-marketing testing in the Czech Republic.

We have introduced the 3T3 NRU cytotoxicity test for methodological, economical and ethical reasons as a regular part of tier pre-marketing testing to assess local tolerance of raw materials for cosmetics, household chemicals and final cosmetic products. Using the 3T3 cell line according to the standard INVITTOX protocol No.64 (NRU Assay) the borderline concentration, relevant to the highest tolerated dose, is determined for each material. The toxic effect is reached at different concentration levels specific for individual cosmetics categories, depending on their chemical characteristics. Typical ranges of cytotoxicity for specific categories of cosmetics were established after testing of hundreds of materials. The range lies between 1 microg/ml (anti-dandruff shampoos), up to 2000 microg/ml (toothpastes and mouthwashes). The 3T3 NRU cytotoxicity test is a sensitive tool able to identify more aggressive products, that are also more likely to evoke irritation in human skin. It was even possible to detect protective effects of one natural herbal ingredient. The comparative study of cytotoxicity test results and human patch test results from a group of essential oils is presented. Cytotoxicity tests represent a highly ethical approach for estimation of irritancy. On the basis of in vitro test results suggesting low risk we can proceed to confirmatory tests in human volunteers.

Immunotoxic effects of carbon tetrachloride--the effect on morphology and function of the immune system in mice.

Carbon tetrachloride (CCL4), a polychlorinated hydrocarbon, is known for its hepatotoxicity, neurotoxicity and skin irritancy. Some epidemiological studies suggest possible carcinogenicity of CCl4. This substance is still present in industrial wastes and in the environment. As the major role of the immune system is immunosurveillance against cancer, we decided to follow the morphological and functional changes of the immune system during acute and subchronic exposures to CCL4 in mice. Mice (A/PhJ) were exposed i.p. to 1.7 mmol CCl4/kg b.w./day administered in olive oil (total volume 0.2 ml), for 2, 7, 14, 23 days. We evaluated: morphology of thymus, spleen and peripheral lymph nodes, immunopathology (thymus and spleen weight, spleen cellularity, number of peripheral blood leukocytes), non-specific immunity (phagocytosis, NK activity), humoral immunity (number of PFC after SRBC immunization, LPS mitogen response), cell-mediated immunity (PHA, ConA mitogen response). Morphological examination showed significant activation of lymphoid tissues in T-cell dependent areas. B-cell areas were also activated, but the formation of active germinal centers in lymphatic follicles has not been observed. The natural immunity was affected in a time dependent manner. A slightly hepatotoxic dose of CCl4 had a significant stimulative effect on phagocytosis and natural killer activity when administered in short-term schedule ("acute exposure"). Subchronic administration of the same dose led to suppression of phagocytosis and Nk activity. Similarly, the lymphocyte response to non-specific mitogens was enhanced during short-term exposure and significantly impaired when CCl4 was administered in long-term schedule. Antigen specific immune response to SRBC was impaired immediately after short-term exposure to CCl4 which suggests that the substance might affect the immunoglobuline proteosynthesis at the cellular level.

Measurement of cytotoxicity of sulphonated chloroaluminium phthalocyanine on various cell systems.

Phthalocyanines (ClAlPcS) present a new generation of substances for photodynamic treatment of tumors. To find optimal therapeutic doses for i.v. or local application, it is necessary to test the maximal non-toxic concentration on model systems. As a standard testing system (cellular substrate) for definition of the in vitro cytotoxicity, human lymphocytes separated from peripheral human blood and splenocytes obtained from rabbit spleen fragments were chosen. The vitality test on human peripheral lymphocytes proved that the used concentrations in the range 0.1-1.4 mg/ml did not significantly influence vitality of the cellular substrate. The test used for determination of the migration activity did not show any influence on MI in the concentration range 0.1-0.5 mg/ml. The higher concentrations tested (range 0.6-1.4 mg/ml) lead to reduction of the migration activity presented by a decrease in the migration index MI: for c = 0.6 mg/ml, MI = 0.85; for c = 1.4 mg/ml, MI = 0.45 (normal value for MI is in the range 0.9-1.1).

Effects of orally administered vanadium on the immune system and bone metabolism in experimental animals.

Experiments were carried out to gain more information on the effects of long term exposure to low doses of vanadium administered to mice and rats in drinking water. The selective immunotoxic effects of vanadium were depression of phagocytosis, splenotoxicity, enlargement of spleen, elevation of peripheral blood leucocytes and T and B cell activation. Vanadium accumulates in hard tissues and influences the mineralisation of epiphyseal cartilage. This effect is obviously evident in young animals. Significant differences in vanadium concentration were found between young and adult animals.

The toxic effect of dermotrophic substances in tests in vitro.

In the model system of tissue culture, the effect of dermatotrophic substances, such as the tenzides and conservatory substances, was tested. The cytotoxic effect of the substances was evaluated according to the vitality of the cells and the lactate dehydrogenase (LDH) activity in the suspension culture of human lymphocytes and further according to the migration inhibition of cells from the animal spleen fragments (SF). The cells obtained from rabbits, guinea pigs, mice and man were used. The highest correlation between the concentration of the substance in the cultivation medium and its toxic effect in the culture has been proved in the tests of LDH activity from the supernatant of the human peripheral lymphocytes culture and further in the migration inhibition test from rabbit SF. Both the tests are in mutual correlation. The determination of LDH activity represents a sensitive marker of the cell damage, the least sensitive is the estimation of the cell vitality. It has been proved that the system of suspension culture of human peripheral lymphocytes and the migration inhibition test from rabbit SF is suitable for the screening determination of the effect of dermatotrophic substances. The system of tissue culture compared to classical tests of skin irritation performed in vivo secures overall objectivity in evaluating the results and the possibility of making a number of parallels without any ethical limits.

Adjuvant activity of muramyldipeptide in a guinea pig model of contact allergy to chromium.

New chemical agents encountered increasingly in the human environment underline the urgent need for a routine testing of their sensitizing potential for man and the development of a suitable experimental model appears to be essential for a reliable assessment of this potential. In our present experiment we studied a guinea pig model of contact hypersensitivity to chromium using as immunoadjuvants Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) emulsified with muramyldipeptide. The study showed that the use of adjuvants was essential for inducing the state of hypersensitivity in experimental animals. It was also demonstrated that muramyldipeptide was not only a fully potent substitute for FCA, but was even superior to it in all the parameters tested. The optimal time interval for demonstrating the induced hypersensitivity to chromium was the third week after the onset of sensitization. This animal model appears to be well suited for the experimental testing of contact allergen potentials.