An Evaluation of Performance Standards and Non-radioactive Endpoints for the Local Lymph Node Assay

Basketter, David A.; Cockshott, Amanda; Corsini, Emanuela; Gerberick, G. Frank; Idehara, Kenji; Kimber, Ian; Loveren, Henk Van; Matheson, Joanna; Mehling, Annette; Omori, Takashi; Rovida, Costanza; Sozu, Takashi; Takeyoshi, Masahiro; Casati, Silvana

doi:10.1177/026119290803600211

Cited by 17 publications

(12 citation statements)

References 43 publications

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“…1,2 Consequently, the development of in vitro test methods has been encouraged, and substantive efforts made to identify such alternatives in this area of toxicology, alongside other end points. 3,9,10 The decision criteria for this hazard identification are those already fully set out in regulatory guidelines, such as those of the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). 3,9,10 The decision criteria for this hazard identification are those already fully set out in regulatory guidelines, such as those of the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).…”

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confidence: 99%

Categorization of Chemicals According to Their Relative Human Skin Sensitizing Potency

et al. 2014

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Although adoption of skin sensitization in vivo assays for hazard identification is likely to be successful in the next few years, this does not replace their use in potency prediction. Notably, measurement of potency of skin sensitizers in the local lymph node assay has been important. However, this local lymph node assay potency measure has not been formally assessed against a range of substances of known human sensitizing potential, because the latter is lacking. Accordingly, criteria for human data have been established that characterize 6 categories of human sensitizing potency, with 1 the most potent and 5 the least potent; category 6 represents true nonsensitizers. The literature has been searched, and 131 chemicals assigned into these categories according to their intrinsic potency judged only by the available human information. The criteria and data set generated provide a basis for examination of the capacity of nonanimal approaches for the determination of human sensitization potency.

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confidence: 99%

Categorization of Chemicals According to Their Relative Human Skin Sensitizing Potency

et al. 2014

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“…Generally, the assay of first choice to meet this toxicity endpoint is the LLNA (Cockshott et al , ; ECHA, ; Basketter and Kimber, ). However, the requirement for the use of radioactive thymidine in the endpoint measurement in the LLNA has considerably limited the uptake of this method in a number of countries, which, given that the assay confers animal welfare benefits compared with the previously used guinea pig assays, is not a desirable situation (Vohr et al , ; Takeyoshi et al , ; Lee et al , ; Basketter et al , ).…”

Section: Discussionmentioning

confidence: 99%

Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements

Basketter

Kolle

Schrage

et al. 2011

J of Applied Toxicology

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The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach.

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“…The pellets were resuspended in 1 ml TCA and transferred to 10 ml of scintillation fluid. Incorporation of [ 3 H]-TdR was measured by b-scintillationcounting as disintegrations per minute (dpm) for each mouse and expressed as dpm/mouse (13).…”

Section: Methodsmentioning

confidence: 99%

“…Results for each treatment group were expressed as the mean SI according to an equation: SI ¼ (mean test dpm/node)/(mean vehicle dpm/ node). If one or more concentrations of the test chemical resulted in an SI of 3 or greater, the chemical was considered to have a significant potential to cause contact sensitization (11)(12)(13).…”

Section: Methodsmentioning

confidence: 99%

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Investigation of the Dermal Sensitizing Potential of Traditional Medical Extracts in Local Lymph Node Assays

Yan¹,

Zhao²,

He³

et al. 2009

Exp Biol Med (Maywood)

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Traditional medical extracts are commonly used as complex mixtures, which may contain naturally occurring contact sensitizers. In this investigation, the mice local lymph node assay (LLNA) was performed to evaluate the dermal sensitization potential of Myrrh, Borneolum, Olibanum, Moschus and Cassia Bark, which are widely used in topical traditional medication. In the radioactive LLNA, the stimulation index (SI) values were calculated for each medical extract. Myrrh, Borneolum, Olibanum and Moschus induced dose-dependent cell proliferation and SI was more than 3. Cassia Bark showed no positive response over the range of test concentrations. In the flow cytometry analysis, the total number of CD3(+), CD4(+), and CD8(+) cells in local lymph nodes was increased in Moschus-, Olibanum-, Myrrh- and Borneolum-treated mice. The ratio of the B220(+)/CD3(+) (B/T cell ratio) and the percentage of I-A(k+) cells that was also positive for the CD69 marker (I-A(k+)/ CD69(+)) were increased in the Moschus-, Olibanum- and Myrrh-treated mice. However, no ofbvious change was observed in Borneolum-treated mice. Cassia Bark did not induce changes in the lymphocyte subpopulations. These results indicate that Moschus, Olibanum and Myrrh can be regarded as sensitizers, and Borneolum regarded as an irritant. Cassia Bark is neither a sensitizer nor an irritant. The combination of radioactive and flow cytometric LLNA can be used for the prediction of sensitizing potential of medical extracts which lead to allergic contact dermatitis in humans.

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An Evaluation of Performance Standards and Non-radioactive Endpoints for the Local Lymph Node Assay

Cited by 17 publications

References 43 publications

Categorization of Chemicals According to Their Relative Human Skin Sensitizing Potency

Categorization of Chemicals According to Their Relative Human Skin Sensitizing Potency

Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements

Investigation of the Dermal Sensitizing Potential of Traditional Medical Extracts in Local Lymph Node Assays

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