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Harmonization of thresholds for primary skin irritation from results of human repeated insult patch tests and laboratory animal skin irritation tests.

Classification of a chemical or chemical product as a primary skin irritant using laboratory animal tests has been defined rigorously and codified in Consumer Products Safety Commission (CPSC) regulations. However, no regulatory agency, including the CPSC, has defined a primary skin irritant threshold for human repeat insult patch tests (RIPTs) that are typically conducted on products, such as cosmetics, anticipated to come into direct contact with humans. Further more, the protocols for animal and human tests are significantly different, as are the schemes for grading responses. Consequently, comparing the results of one type of test with those from the other type has proved to be difficult. In this short communication, we propose a procedure to harmonize the results from these two types of skin irritation tests and suggest that a score of "5" in animal tests, considering 24-h results on unabraded skin, is equivalent to a score of "3" in human RIPTs.

Animals↗

Inhibitory effects of Ramulus mori extracts on melanogenesis.

To develop an active agent for skin whitening, the inhibitory effects of 285 plant extracts on tyrosinase activity were examined, and one plant extract having tyrosinase inhibition activity was chosen. Ramulus mori (young twigs of Morus alba L.) extracts showed inhibition activity in tyrosinase and melanin synthesis in B-16 melanoma cells. To clarify the mechanism of its inhibition on melanogenesis, the effect of R. mori extracts on tyrosinase activity, synthesis, and gene expression was evaluated. R. mori extracts showed tyrosinase inhibition activity by competitive method, and there was no suppression of tyrosinase synthesis and gene expression. Further, to evaluate the inhibitory activity of R. mori in vivo, its effect on melanin production in UV-induced brown guinea pigs was examined, where a decrease of melanin production in the guinea pig model was observed. Also, R. mori extracts showed no toxicity in animal tests such as the acute toxicity test, the skin irritation test, the eye irritation test, the skin sensitization test, and the acute oral toxicity test, and no toxicity in the human skin irritation test. A single compound from R. mori extracts was purified using various column chromatography and recrystallization, and its chemical structure was identified using mass chromatography, IR spectroscopy, and NMR analysis. The chemical structure was that of 2,3',4,5'-tetrahydroxystilbene(2-oxyresveratrol) and showed inhibition activity on tyrosinase (IC(50) = 0.23 microg/ml). Also, R. mori extracts inhibited tyrosinase activity in a competitive manner (Ki = 1.5 x 10(-6) M) when L-tyrosine was used as a substrate.

Animals↗

Use of XTT-assay to assess the cytotoxicity of different surfactants and metal salts in human keratinocytes (HaCaT). A feasible method for in vitro testing of skin irritants.

Because of the increasing need of reliable skin irritation tests and in order to reduce the number of animal experiments, in vitro alternatives have to be developed. We studied four surfactants and five metal salts for their cytotoxic potency in HaCaT cells, a spontaneously immortalized human keratinocyte line. The endpoint used to assess cellular viability was metabolization of the tetrazolium salt XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide). The tested substances revealed a significant rank order of their cytotoxicity at an exposure time of 24 h. It was 1) benzalkonium chloride, 2) sodium lauryl sulphate, and 3) Tween 20 (polyoxyethylene sorbitanmonolaurate) and Tween 80 (polyoxyethylene sorbitanmonooleate), for the surfactants; and 1) potassium bichromate, 2) copper sulphate, 3) cobalt chloride and palladium chloride, and 4) nickel sulphate, for the metal salts. There is an excellent correlation to the rank order of their known irritative potency in vivo. Being practicable and effective, the presented XTT-assay on HaCaT cells would be well suitable for an initial orientating screening of substances, subsequently followed by irritation tests directly in humans.

Benzalkonium Compounds↗

[Irritation test of dexamethasone valerate (DV-17) and other steroid ointments in rabbits. Skin and eye primary irritation tests, and skin cumulative irritation test].

The skin and eye primary irritation tests, and skin cumulative irritation test were performed on male rabbits, which were topically treated with a volume of 1 g/body of 0.06% and 0.12% dexamethasone valerate (DV-17) ointments as a test drug, and 0.1% hydrocortisone butyrate (HB) and 0.12% betamethasone valerate (BMV) ointments as an active control drug in order to evaluate the topical and systemic effects. No skin and eye primary irritations except minimal skin erythema and slight conjunctival redness were seen in above four corticosteroids. Judging from the results such as skin atrophy, inhibition of body weight gains, and decreases of thymus, spleen and adrenal weights, the potency of toxicities among the four corticosteroid ointments appeared to be as follows: 0.12% DV-17, 0.12% BMV greater than 0.1% HB greater than 0.06% DV-17.

Adrenal Cortex Hormones↗

[Evaluation of biological safety of continuous carbon-fiber reinforced polyolefin as hard tissue repair].

OBJECTIVE: To evaluate the biological safety of continuous carbon-fiber reinforced polyolefin as hard tissue repair material. METHODS: Biocompatibility of the material was evaluated through hemolysis test, pyrogen test, skin irritation test, cytotoxicity test, ames test, in vitro chromosome aberration test, and bone marrow cells Micronuclei test. RESULTS: No obvious hemolysis, pyrogenic characteristics, sensitivity, cytotoxicity, and mutagenicity were observed. CONCLUSION: The continuous carbon-fiber reinforced polyolefin composite material is of good biological safety. It meets all the demand made by biological safety as hard tissue repair material.

Animals↗

[Analytical methods for mycotoxins].

Biological, physico-chemical and immunochemical methods are available today for solving the multiple problems which turn up in the course of mycotoxin diagnosis and mycotoxin research. In this review, selected analysis techniques are presented and their limits and possibilities are discussed. The biological methods (chicken-embryo-test, skin irritation test, cell cultures) indicate only toxicities, mostly without further specification for mycotoxins. In contrast, modern physico-chemical analysis (DC, HPLC, CLC, MS) allows the detection of defined mycotoxins in the picogram range; these methods should be employed or official and judicial purposes. The recently developed immunochemical methods (RIA, EIA) are characterized by a simple sample preparation, a short test duration and a high sensitivity; they could be used as a screening method in the course of mycotoxicological control of food and feed.

Animal Feed↗

Overview of animal test methods for skin irritation.

Determining the irritant effects of chemicals in experimental animals provides the public with information on and protection against possible hazards that may arise from exposure of the human skin. Furthermore, the data obtained are used for registration and classification purposes. This paper reviews the methods proposed in the most important guidelines (OECD, EEC and FIFRA guidelines) and points out the critical experimental points. It shows that the introduction of OECD Guideline No. 404 can lead to a reduction in the number of test animals used for skin irritation testing.

Animals↗

Artificial disruption of skin barrier prior to irritant patch testing does not improve test design.

BACKGROUND: Irritant patch testing is often performed as a 24- or 48-h occlusive patch test with low concentrations of sodium lauryl sulphate (SLS). OBJECTIVES: The aim of this study was to investigate potential ways to shorten this test procedure and obtain precise test results. PATIENTS AND METHODS: Thirty-six healthy volunteers underwent irritant patch testing with different pretreatments (PT) of the test fields. Occlusive test chambers were applied on the upper back with SLS 0.5%, 1%, 2% and 5% in large Finn Chambers(R). The patches were removed after 4 and 24 h, respectively, depending on the concentration used. Test fields were pretreated as follows: PT 0, field without any PT (control); PT 1, prick with lancet; PT 2, prick with test stamp; PT 3, scratch with lancet; PT 4, incision with standardized incision instrument (0.1-0.2 mm depth). Skin reactions were evaluated by transepidermal water loss (TEWL), skin erythema and skin hydration and as well by a visual score (VS) at 4, 24 and 72 h. RESULTS: Our data show an obvious distinction between PT 0-2 and PT 3-4 at all measurement methods. The average TEWL values with PT 3-4 were higher than those with PT 0-2, especially on the 4-h course. This distinction may derive from the shape and size of the skin impairment achieved by PT 3-4, leading to a mechanical barrier disruption. However, SLS may infiltrate directly into deeper skin layers supported by capillarity. Consequently, no or little penetration through the epidermis and interaction with its structures occurs, which is responsible for irritant skin reactions. The SLS dose in the upper skin layers is therefore lower at these PTs. The lower remaining dose of SLS also explains this distinction, especially for the VS. Additionally, there are presumed reactions in deeper layers of the epidermis and dermis at PT 3-4. CONCLUSIONS: In summary, all data suggest a different reaction pattern from the classical irritant response. Therefore, application without any PT seems to be best suited for irritancy skin testing, especially for visual assessment. PTs prior to irritant patch testing have been shown to be unjustifiable.

Adult↗

In vitro cytotoxicity tests on cultured human skin fibroblasts to predict skin irritation potential of surfactants.

Cultured human skin cells are a potentially useful model for skin irritancy testing. We have investigated the use of human skin fibroblasts for in vitro screening for skin toxicity. To assess the cytotoxic effects of surfactants, cell viability was measured by the NRU (neutral red uptake) assay and AB (Alamar blue) assay as in vitro methods. The skin irritation potential of surfactants by human skin patch test was assessed as in vivo methods. The close relationship was found between AB assay with human skin fibroblasts and human patch test (r=0.867). There was a relatively good agreement between the NRU and in vivo patch test (r=0.648). These results suggest that AB and NRU assay using cultured human fibroblast could be predictable methods for the irritancy of various surfactants in human.

Adult↗

Patch tests versus use tests in skin irritation risk assessment.

In the risk assessment of the ability of a substance or preparation to cause skin irritation, a patch test involving human volunteers is often seen as providing definitive information. However, the skin exposure conditions found in an occluded patch may be far removed from those in a particular use situation, not least for such reasons as the high dose per unit area, the duration of exposure and the maceration of the skin. Whilst these factors may enhance sensitivity in the identification of intrinsic hazard, they may do little to ensure accurate safety evaluation for real use. In this paper, we report data from a series of studies with an unmarketed facial skin cosmetic product. Whilst the product was unexpectedly highly irritating in a standard patch test in 30 volunteers, subsequent use tests, including a 6x daily open application to the elbow for 3 weeks and 2x daily application in a half-face test lasting 3 to 4 weeks and involving 52 volunteers failed to show any evidence of skin irritation. It is concluded that the most meaningful results for skin irritation risk assessment are likely to come from studies which involve relevant patterns of exposure.

Adult↗

An approach for development of alternative test methods based on mechanisms of skin irritation.

Recent advances in techniques for culture of human skin cells have led to their potential for use as in vitro models for skin irritation testing to augment or replace existing rabbit skin patch tests. Our work is directed towards the development of cultured human skin cells, together with endpoints that can be linked to in vivo mechanisms of skin irritation, as in vitro models for prediction of human skin irritation, and for study of mechanisms of contact irritant dermatitis. Three types of commercial human skin cell cultures have been evaluated, epidermal keratinocytes and partially or fully cornified keratinocyte-dermal fibroblast co-cultures. Human epidermal keratinocyte cultures (Clonetics) were treated with product ingredients and formulations, and the extent of cell damage was assessed by incorporation of the vital dye neutral red. Cell damage correlated with human skin patch data for ingredient chemicals with the exception of acids and alkalis, but did not correlate with skin irritation to surfactant-containing product formulations. Cultures of human skin equivalents were evaluated as potential models for measurement of responses to test materials that could not be measured in the keratinocyte/neutral red assay. We developed a battery of in vitro endpoints to measure responses to prototype ingredients and formulations in human epidermal keratinocyte-dermal fibroblast co-cultures grown on a nylon mesh ('Skin2' from Advanced Tissue Sciences) or on a collagen gel ('Testskin' from Organogenesis). The endpoints measure cytotoxicity (neutral red and MTT vital dye staining, lactate dehydrogenase and N-acetyl glucosaminidase release, glucose utilization) and inflammatory mediator (prostaglandin E2) release. Initial experiments indicate a promising correlation between responses of the Skin2 model to prototype surfactants and in vivo human skin irritation. The responses of Testskin cultures to acids and alkalis help to prove the concept that a topical application model can measure responses to these materials. These results suggest that human skin cell models can provide useful systems for preclinical skin irritation assessments, as alternatives to rabbits, for at least certain classes of test substances.

Animal Testing Alternatives↗

Retrospective appraisal of the relationship between skin irritancy and contact sensitization potential.

A retrospective analysis of the association between skin irritancy and the potential to cause contact sensitization has been performed employing a historical database for 50 chemicals and formulations. Correlations between the results of Draize skin irritation tests and skin sensitizing activity measured with the occluded patch test of Buehler have been examined. Weak, but nevertheless statistically significant, associations between contact sensitization and skin irritancy have been demonstrated. It is proposed that such correlations are consistent with the irritant properties of a material exerting an important influence on the extent to which contact sensitization is induced.

Animals↗

Validity and ethics of the human 4-h patch test as an alternative method to assess acute skin irritation potential.

For more than 50 years, the Draize rabbit skin irritation test has reigned supreme as the regulatory method of choice for the identification of skin irritant chemicals. To date no in vitro alternative test has been validated as an adequate replacement. However, one potential option, to test the endpoint of concern (skin irritation) in the species of concern (man) has been overlooked. The advent of predictive in vitro tools for the identification of substances corrosive to the skin has opened up the practical possibility of carrying out safe and ethical studies on small panels of humans. The human 4-h patch test has been developed to meet the needs of identifying chemical skin irritation potential, providing data which is inherently superior to that given by a surrogate model, such as the rabbit. This paper reviews in detail the present state of the human 4-h patch test, highlighting its advantages and noting its utility as the 'gold standard' on which to build future in vitro models.

Animal Testing Alternatives↗

Comprehensive outlook of in vitro tests for assessing skin irritancy as alternatives to Draize tests.

In vitro alternative methods have been verified for the possibility to assess cutaneous irritancy because humans cannot be direct initial experimental subjects and animal experimentation could be forbidden in the near future. Many kinds of cell cytotoxicity assays have been tried, revealing their own advantages and limitations. Cell function-based tests have been used less frequently than cytotoxicity assays. Three-dimensional culture systems are promising because they are closer to the actual in vivo skin, and some of them are commercialized nowadays. The ultimate objective of in vitro irritancy tests, which is the high degree of correlation with human in vivo test results, has been accomplished in many experimental settings. Before applying these in vitro methods we must consider several points, including cell sources, irritant characteristics, exposure time, endpoint of experiment, extrinsic factors affecting irritation, etc. In vitro skin irritancy tests have been developed continuously, and in the future they could assume a heavy responsibility of estimating the irritancy in human skin in vivo.

Cells, Cultured↗

[Study on biocompatibility of skin reproductive membrane].

OBJECTIVE: To study the biocompatibility of skin reproductive membrane. METHODS: According to ISO's standards, the extractions of the skin reproductive membrane were prepared, and the acute systematic toxicity test, primary skin irritant test, cytotoxicity test, gene expression of type I collagen and fibronectin were detected to evaluate the biocompatibility of skin reproductive membrane. RESULTS: All of those tests showed negative results. CONCLUSION: The skin reproductive membrane has excellent biocompatibility in the level of the systematic, cellular and molecular biology.

Animals↗

Reproducibility of the chamber scarification test.

The chamber scarification test is a predictive human skin irritation test developed to rank the irritation potential of products and ingredients meant for repeated use on normal and diseased skin. 12 products or ingredients can be tested simultaneously on the forearm skin of each volunteer. The test combines with the procedure scratching of the skin at each test site and subsequent closed patch tests with the products, repeated daily for 3 days. The test is performed on groups of human volunteers: a skin irritant substance or products is included in each test as a positive control, and a compound with low irritant potential as a negative control, to obtain relative characterization of the irritant potential of the unknown products. The outcome of tests with a positive and negative control product used repeatedly in 13 chamber scarification tests over a 7-year period is reported, and shows high reproducibility of the test. Further, intra-individual variation in skin reaction to the 2 control products in 26 volunteers, who participated 2x, is shown, which supports the conclusion that the chamber scarification test is a useful short-term human skin irritation test with high reproducibility.

Adult↗

The acute toxicity and primary irritancy of glutaraldehyde solutions.

Glutaraldehyde (GA, CAS Number 110-30-8), an aliphatic dialdehyde, has a wide range of industrial, scientific, and medical applications. It is available in aqueous solutions, whose concentrations vary up to 50% (w/w) and from which there is a potential during use for skin and eye contact and exposure to the vapor. The acute toxicity and primary irritancy of a wide range of GA concentrations were investigated to determine the differential hazards for such solutions. The acute peroral toxicity in the rat, expressed as ml of solution dosed, was moderate for solutions of 5% and above (LD50 range 0.88-3.25 ml/kg) and generally varied little for solutions up to 50%. Solutions less than 5% GA were of slight toxicity (LD50 range 3.34-12.30 ml/kg for 1 and 2% solutions). When lethality was expressed as absolute amount of GA dosed (mg GA/kg), there was a reciprocal relationship between the concentration of GA solution dosed and LD50. This was confirmed in the mouse, which is more susceptible than the rat to acute peroral toxicity. The acute percutaneous toxicity of GA solutions to rabbits (24 h occlusion) was moderate (LD50 range 1.59-2.71 ml/kg) for 46 and 50% solutions, and slight for 25% GA solutions (8.80-16.00 ml/kg). At 15% and less, 16.0 ml/kg was not lethal. Exposures (4-8 h) of rats to saturated vapor atmospheres of GA generated dynamically or statistically at ambient temperature (17-25 C) produced only transient peripheral sensory irritant effects to the eyes and respiratory tract. In contrast, vapor atmosphere generated dynamically at elevated temperature (60 or 65 C) produce severe effects, including mortality (4-h LC50 range 23.5-44.3 ppm). Histopathology in rats that died included exposure concentration-related acute inflammation and necrosis in the nasal mucosa, larynx, trachea, and bronchi. Standard primary skin irritation tests in the rabbit indicated severe skin irritation and necrosis at 45 and 50% GA; necrosis occurred with 1 and 4 h contact at 50% and at 4 h with 45%. Inflammation was moderate at 25%, slight to moderate with 5 and 10% GA, minor at 2%, and threshold at 1%. Standard primary eye irritation tests showed 45% GA to produce severe conjunctival and corneal injury, which was persistent. At 2% GA corneal injury was mild, and at 5% marked. The lowest concentration producing corneal injury was 1.0%, and the no-effects concentration was 0.5%. The threshold for conjunctival effects was 0.2%, and the no-effects concentration 0.1%. At 1% GA, conjunctival hyperemia and chemosis were moderate to marked, and became more severe with higher GA concentrations. The results suggest potential acute handling hazards with various concentrations of GA solutions and indicate industrial hygiene considerations.

Administration, Cutaneous↗

An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures.

We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score > or =1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the "rabbit skin irritation test") and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.

Animal Testing Alternatives↗