An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round

Abstract

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products, Swissmedic. Statistical analyses of all data were performed by an independent centre at the University of Bern, Department of Statistics. Ear-draining, LN weight and cell count were used to assess proliferation instead of radioactive labeling of lymph node cells. In addition, the acute inflammatory skin reaction was measured by ear swelling and weight of circular biopsies of the ears to identify skin irritating properties of the test items. Hexylcinnamaldehyde (HCA) and three blinded test items were applied to female, 8--10 weeks old NMRI and BALB/c mice. Results were sent via the independent study coordinator to the statistician. The results of this first round showed that the alternative endpoints of the LLNA are sensitive and robust parameters. The use of ear weights added an important parameter assessing the skin irritation potential, which supports the differentiation of pure irritative from contact allergenic potential. There were absolute no discrepancies between the categorisation of the three test substances A--C determined by each single participating laboratories. The results highlighted also that many parameters do have an impact on the strength of the responses. Therefore, such parameters have to be taken into consideration for the categorisation of compounds due to their relative sensitizing potencies.

Use of an Ex Vivo Local Lymph Node Assay to Assess Contact Hypersensitivity Potential

“…In summary, the results of the current study and others (van Och et al, 2000;Woolhiser et al, 2000;De Jong et al, 2002;Ehling, et al, 2005;Takeyoshi et al, 2004Takeyoshi et al, , 2005van den Berg et al, 2005;Piccotti et al, 2006) collectively demonstrate the robustness of the murine LLNA, in that the hypersensitivity potential of compounds could be evaluated in different mouse stains and by measuring lymph node cell proliferation by multiple methods. Although additional positive and negative chemicals need to be tested in the ex vivo LLNA, the current study also provided some evidence indicating that this alternative approach could be used to estimate allergenic potency.…”

“…We selected BALB/c mice when initiating assay development due to the limited availability of CBA mice in the United States. Importantly, BALB/c mice have been shown to be a suitable alternative to CBA mice in the LLNA (Woolhiser et al, 2000;De Jong et al, 2002;Ehling et al, 2005). One potential advantage of using an albino strain such as BALB/c mice is the ability to evaluate erythema on the ears by visual inspection, which is more difficult with pigmented CBA mice.…”

Comparative Studies of Lymph Node Cell Subpopulations and Cytokine Expression in Murine Model for Testing the Potentials of Chemicals to Induce Respiratory Sensitization

“…The development of models for assessment of the potentials of LMW chemicals to induce allergic reactions is an important task for occupational health care professionals and the decisions they undertake establishing safe exposure limits. The local lymph node assay (LLNA) is now a validated method for assessing the potentials of chemicals to induce skin contact allergy [3][4][5][6]. However, until now, there is no validated method to assess the potentials of chemicals to induce respiratory sensitization.…”

Effects of Strain Differences and Vehicles on Results of Local Lymph Node Assays

“…In this study, mouse strains and different vehicles which are commonly used in Europe and those described in the OECD Guideline 429 [3,4] were used. Two strains of female mice aged 8 to 12 weeks at the time of treatment were used for the experiments.…”

“…The objectives of Experiment 2 were not to identify an appropriate vehicle, but rather to demonstrate that the local lymph node response is different among vehicles and to provide important fundamental knowledge allowing researchers to more accurately compare dpm/LN levels obtained with different vehicles in different laboratories or dpm/LN levels of similar pharma- 0 334 211 437 296 364 1055 5 504 512 1096 1229 578 1614 10 755 960 2627 1705 1736 1952 25 2804 2764 2869 2589 3649 4984 HCA in 6 different vehicles, AOO, EtOH 70%, DMF, BN, PG, and DMSO, was topically applied to CBA/CaOlaHsd mice at concentrations of 0, 5, 10, and 25% (v/v) (n=4 each), and the incorporation of 3 HTdR (dpm/LN) was determined. The dpm/LN level was determined in lymph nodes pooled on an experimental group basis.…”