Summary
A new three-dimensional human skin model consisting of several layers of actively dividing and metabolically active human neonatal foreskin-derived fibroblasts and epidermal keratinocytes grown on nylon mesh has been used to assess the in vitro toxicity of test agents from various classes. Utilizing a slight modification of the published neutral red viability assay for endpoint determination, we have assayed and obtained dose-dependent toxicity curves for test agents from the following classes: detergents (n=15), alcohols (n=5), metal chlorides (n=10), perfumes and colognes (n=5), shampoos (n=4), conditioners (n=3), moisturizers (n=3), pesticides (n=3), and antimicrobial preservatives (n=4). Limited comparisons to in vivo ocular irritancy data with alcohols and detergents are encouraging. We have demonstrated the utility of this metabolically active dermal substrate containing naturally secreted collagen and other extracellular matrix proteins along with the neutral red viability assay for assessing the toxicity of a number of test agents from a variety of different classes with broad industrial applications.
Similar content being viewed by others
References
Babich, H.; Sardana, M. K.; Borenfreund, E. Acute cytotoxicities of polynuclear aromatic hydrocarbons determinedin vitro with the human liver tumor cell line, HepG2. Cell Biol. Toxicol. 4:295–309; 1988.
Babich, H.; Borenfreund, E. Structure-activity relationships for diorganotins, chlorinated benzenes and chlorinated anilines established with bluegill sunfish BF-2 cells. Fundam. Appl. Toxicol. 10:295–301; 1988.
Babich, H.; Martin-Alguacil, N.; Borenfreund, E. Comparisons of the cytotoxicities of dermatotoxicants to human keratinocytes and fibroblastsin vitro. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 7. New York: MaryAnn Liebert, Inc.; 1989:153–167.
Bohmont, B. L. The new pesticide user’s guide. Reston, VA: Reston Publishing Co., Inc.; 1983.
Booman, K. A.; Cascieri, T. M.; Demetrulias, J., et al.In vitro methods for estimating eye irritancy of cleaning products. Phase I: preliminary assessment. J. Toxicol. Cutaneous Ocular Toxicol. 7(3):173–185; 1988.
Borenfreund, E.; Puerner, J. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Methods 9(1):7–9; 1984.
Borenfreund, E.; Shopsis, C. Toxicity monitored with a correlated set of cell-culture assays. Xenobiotica 15:705–711; 1985.
Borenfreund, E.; Puerner, J. A. Cytotoxicity of metals, metal-metal and metal-chelator combinations assayedin vitro. Toxicology 39:121–124; 1986.
Borenfreund, E.; Babich, H.In vitro cytotoxicity of heavy metals, acrylamide and organotin salts to neural cells and fibroblasts. Cell Biol. Toxicol. 3:63–73; 1987.
Brown, C. D.; Li, Q.; Brabec, M. J. Cytotoxicity of six divalent metals to testicular cellsin vitro. In Vitro Toxicol. 2:129–137; 1988/1989.
Coin, P. G.; Stevens, J. B. Toxicity of cadmium chloridein vitro: indices of cytotoxicity with the pulmonary alveolar macrophage. Toxicol. Appl. Pharmacol. 82:140–150; 1986.
Gordon, V. C.; Bergman, H. C. EYTEX™, an in vitro method for evaluation of ocular irritancy. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 5. New York: MaryAnn Liebert, Inc.; 1987:293–302.
Hammond, M. E.; Goodwin, J.; Dvorak, H. F. Quantitative measurements of neutral red uptake and excretion by mammalian cells. J. Reticuloendothel. Soc. 27:337–346; 1980.
Jacques, P. J. Endocytosis. In: Dingle, J. T.; Fell, H. B., eds. Lysosomes in biology and pathology. Amsterdam: North Holland; 1973:395–420.
Muir, C. K. Surfactant-induced opacity of bovine isolated cornea: an epithelial phenomenon? Toxicol. Lett. 38:51–54; 1987.
Naughton, G. K.; Jacob, L.; Naughton, B. A. A physiological skin model forin vitro toxicity studies. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 7. New York: MaryAnn Liebert, Inc.; 1989:183–189.
Ng, T. B.; Liu, W. K. Toxic effects of heavy metals on cells isolated from the rat adrenal and testis. In Vitro Cell. Dev. Biol. 26:24–28; 1990.
North-Root, H.; Yackovich, F.; Demetrulias, J., et al. Prediction of the eye irritation potential of shampoos using thein vitro SIRC toxicity test. Food Chem. Toxicol. 23:271–273; 1985.
Sewell, R. B.; Soong Ling, T.; Yeomans, N. D. Ethanol-induced cell damage in cultured rat antral mucosa assessed by chromium-51 release. Dig. Dis. Sci. 31(8):853–858; 1986.
Shopsis, C.; Eng, B. Rapid cytotoxicity testing using a semi-automated protein determination on cultured cells. Toxicol. Lett. 26:1–8; 1985.
Shopsis, C.; Sathe, S. Uridine uptake as a cytotoxicity test: correlations with the Draize test. Toxicology 29:195–206; 1984.
Shopsis, C.; Borenfreund, E.; Stark, D. M. Validation studies on a battery of potentialin vitro alternatives to the Draize test. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 5. New York: MaryAnn Liebert, Inc.; 1987:31–44.
Silverman, J.; Pennisi, S.Tetrahymena thermophila (30377) as an indicator of ocular irritancy in rabbits. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 3. New York: MaryAnn Liebert, Inc.; 1985:673–680.
Thomson, M. A.; Hearn, L. A.; Smith, K. T., et al. Evaluation of the neutral red cytotoxicity assay as a predictive test for the ocular irritancy potential of cosmetic products. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 7. New York: MaryAnn Liebert, Inc.; 1989:297–305.
Triglia, D.; Wegener, P. T.; Harbell, J., et al. Interlaboratory validation study of the keratinocyte neutral red bioassay from Clonetics Corporation. In: Goldberg, A. M., ed. Alternative methods in toxicology, vol. 7. New York: MaryAnn Liebert, Inc.; 1989:357–365.
Verma, S. R.; Tyagi, A. K.; Pal, N., et al.In vitro effect of sodium lauryl sulfate on ATPase system in a few tissues fromChanna punctatus. Toxicol. Lett. 2:355–359; 1978.
Wallin, R. F.; Hume, R. D.; Jackson, E. M. The agarose diffusion method for ocular irritancy screening: cosmetic products, part I. J. Toxicol. Cutaneous Ocular Toxicol. 6(4):239–250; 1987.
Yu, H-S.; Pastor, S. A.; Lam, K-W., et al. Ascorbate-enhanced copper toxicity on bovine corneal endothelial cellsin vitro. Curr. Eye Res. 9(2):177–182; 1990.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Triglia, D., Braa, S.S., Yonan, C. et al. In vitro toxicity of various classes of test agents using the neutral red assay on a human three-dimensional physiologic skin model. In Vitro Cell Dev Biol - Animal 27, 239–244 (1991). https://doi.org/10.1007/BF02630923
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02630923