Human sensitization assays

Gad SC, Dunn BJ, Gavigan FA et al. (1987) Development, validation, and transfer of a new test system technology in toxicology. In Goldberg AM (ed) New Test System in Toxicology. Mary Ann Liebert, New York, pp 275-292 Van Loveren H, Kato K, Ratzlaff RE et al. (1984) Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice. J Immunol Methods 67 311-319 

VAET enhancement test is used for evaluation of ingredients of consumer products in mice. 

VAET enhancement test has been used for the evaluation of ingredients of consumer products but has not been used by a sufficient number of laboratories to be considered standard. 

The long conditioning period required prior to the study is one of the limitations of this assay. The test was never widely adopted or submitted to formal validation procedures. General comments regarding choice of micrometer for MEST also apply to VAET. 

Code of Federal Regulations (1998) Office of the Federal Registrar, National Archives of Records Service. General Services Administration Title 40, part 162.10, part 163.31, part 771 

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Human Sensitization Assays allow chemicals to be tested for their ability to induce contact hypersensitivities on the skin of human volunteers with obtained informed consent. Human studies should only be undertaken for a new compound after the results of predictive tests in animals for such compound are available. If a compound contains significantly increased levels of common ingredients, it should also undergo predictive tests in animals prior to humans. 

Table 2 Principal features of human sensitization assays ...

There are 4 basic predictive human sensitization tests in current use (1) single induction/ single challenge patch tests (2) repeated insult patch tests (RIPT) (3) RIPT with continuous exposure (modified Draize) and (4) the maximization test. Principal features of human sensitization assays are summarized in Table 2. 

There are four basic predictive human sensitization tests in current use (1) a single-induction/single-challenge patch test (2) repeated-insult patch test (RIPT) (3) RIPT with continuous exposure (modified Draize) and (4) the maximization test all of these use similar customized patches (Frosch and Kligman 1979 Kaminsky et al. 1986). Principal features of human sensitization assays are summarized in Table 1, and further details can be found in MarzuUi and Maibach (1996). For assays other than maximization, 150-200 subjects are usually tested. Henderson and Riley (1945) statistically showed that if no positive reactions are observed in 200 randomly selected subjects, as many as 15/1000 of the general population may react (95% confidence). As sample size is reduced, the likelihood of unpredicted adverse reactions in the general population increases. 

Table 1. Principal features of human sensitization assays. Table modified from Patrick and Maibach (1991)... 

Schneider, K. and Akkan, Z., Quantitative relationship between the local lymph node assay and human skin sensitization assays. Regul. Toxicol. Pharmacol., 39, 245, 2004. 

Rotshteyn Y, Weingarten B. 1996. A highly sensitive assay for the simultaneous determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatography with electrochemical and fluorescence detection. Ther Drug Monit 18(2) 179-188. 

The simplest and most sensitive assays for detecting clastogenic (i.e. chromosomal breaking) effects involve the use of mammalian cells. Cultures of established cell lines (e.g. Chinese hamster ovary) as well as primary cell cultures (e.g. human l)nnphocyte) may be used. After exposure to a range of chemical concentrations in the presence and absence of an appropriate metabolic activation system, the cell cultures are treated with a spindle inhibitor (e.g. vinblastine) to accumulate cells in a metaphaselike stage of mitosis. Cells are harvested at appropriate times and chromosome preparations are made, stained with DNA-specific dye and the metaphase cells are analysed under the microscope for chromosome abnormalities. 

Pharmacokinetic (PK) studies in different animal species and additional in vitro studies provide information on the compound s predicted human PK parameters, including dose- and time-dependencies, its protein binding, the effect of food on its PK, and the cytochrome P450 isoenzymes responsible for its metabolism as well as the stmcture and activity of the main metabolites. Also a sensitive assay to quantify the compound and its metabolites in human blood and urine should have been developed and validated. 

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application m the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins. 

Klawitter J et al (2009) Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell culture. Biomed Chromatogr... 

Wang J, Jiang Y, Wang Y, Li H, Fawcett JP, Gu J (2007) Highly sensitive assay for tiotropium, a quaternary ammonium, in human plasma by high-performance liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom 21 1755-1758... 

Kimber I, Hilton J, Dearman RJ et al.(1995) An international evaluation of the murine local lymph node assay and comparison of modified procedures. Toxicology 103 63-73 Schneider K, Akkan Z (2004) Quantitative relationship between the local lymph node assay and human skin sensitization assays. Regul Toxicol Pharmacol 39(3) 245-255 Takeyoshi M, Noda S, Yamazaki S et al. (2004) Assessment of the skin sensitizatio potency of eugenol and its dimmers using a non-radioisotopic modification of the local lymph node assay. J Appl Toxicol 24 77-81... 

Thelestam M, Mbilby R (1975) Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. In Infect Immun 12 225-232. 

Kuntzman RG, Tsai I, Brand L, Mark LC. The influence of urinary pH on the plasma half-life of pseudoephedrine in man and dog and a sensitive assay for its determination in human plasma. Clin Pharmacol Ther 1971 12(1) 62-67. 

Napoli et al. (23) developed a sensitive assay based on negative chemical ionization mass spectrometry to quantify retinoic acid in human plasma. Endogenous levels of all trans retinoic acid in plasma were 4.9 ng/ml, using a 0.1 ml sample. The limit of detection was less than 1 ng/ml. Direct quantification of 13-cis retinoic acid was impossible due to the inability of the GC to resolve the isomers. Barua and Olson (33) described a method to quantify all trans retinoic acid in serum using reverse phase HPLC. They detected 1.8 ng/ml of the all trans isomer, using a 2 ml serum sample and a non-acidic extraction procedure. 

Heneine, W Yamamoto, S Switzer, W. M., Spira, T. J., and Folks, T. M. (1995) Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency vims type 1.7. Infect. Dis. 171,1210-1216. 

Keller C, Novotny M, Ramp S, Brashear J, Brandt D, Abbott K, A rapid, sensitive assay for human thyroid-stimulating hormone (hTSH) on the Abbott IMx automated immunoassay system. CHn Chem 1988 34 1208. 

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