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

Ehling, G.; Hecht, Monica; Heusener, A.; Huesler, Juerg; Gamer, Armin; Loveren, Henk Van; Maurer, T.; Riecke, Kai; Ullmann, Ludwig; Ulrich, Peter; Vandebriel, Rob J.; Vohr, Hans-Werner

doi:10.1016/j.tox.2004.12.038

Cited by 45 publications

(2 citation statements)

References 29 publications

(25 reference statements)

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“…the 2-fold rule 102 or 1.5-fold for cellularity in BALB/c mice local lymph node assay. 103 The concept of a relevance threshold represents an alternative to the usual p-value criterion as a measure for a positive assay. The common use of p < 0.05 as a criterion leads to the contradiction between statistical significance and biological relevance.…”

Section: In Vivo and In Vitro Mutagenicity Assaysmentioning

confidence: 99%

Statistical evaluation of toxicological bioassays – a review

Hothorn

2014

Toxicol. Res.

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The basic conclusions in almost all reports on new drug applications and in all publications in toxicology are based on statistical methods. However, serious contradictions exist in practice: designs with small samples sizes but use of asymptotic methods (i.e. constructed for larger sample sizes), statistically significant findings without biological relevance (and vice versa), proof of hazard vs. proof of safety, testing (e.g. no observed effect level) vs. estimation (e.g. benchmark dose), available statistical theory vs. related userfriendly software. In this review the biostatistical developments since about the year 2000 onwards are discussed, mainly structured for repeated-dose studies, mutagenicity, carcinogenicity, reproductive and ecotoxicological assays. A critical discussion is included on the unnecessarily conservative evaluation proposed in guidelines, the inadequate but almost always used proof of hazard approach, and the limitation of data-dependent decision-tree approaches.

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Section: In Vivo and In Vitro Mutagenicity Assaysmentioning

confidence: 99%

Statistical evaluation of toxicological bioassays – a review

Hothorn

2014

Toxicol. Res.

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“…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.…”

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confidence: 99%

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

Anzai

Ullmann²,

Hayashi

et al. 2010

Exp. Anim.

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Abstract:The Local Lymph Node Assay (LLNA) is now regarded as the worldwide standard. The analysis of accumulated LLNA data reveals that the animal strains and vehicles employed are likely to affect LLNA results. Here we show that an obvious strain difference in the local lymph node response was observed between DMSO-treated CBA/CaOlaHsd and CBA/ CaHsdRcc mice. We also show that a vehicle difference in the response was observed when CBA/CaHsdRcc mice were exposed to 6 vehicles; 4:1 v/v acetone/olive oil (AOO), ethanol/ water (70% EtOH), N,N-dimethylformamide (DMF), 2-butanone (BN), propylene glycol (PG), and dimethylsulfoxide (DMSO). The dpm/LN level was lowest in the 70% EtOH group and highest in the DMSO group. When alpha-hexylcinnamaldehyde (HCA) was used as a sensitizer for the LLNA, HCA was a weak sensitizer when AOO or DMSO was used as a vehicle, but a moderate sensitizer when the other 4 vehicles were used. This study showed that there are vehicle differences in the local lymph node response (dpm/LN level) in the LLNA and that the sensitization potency of HCA may be classified in different categories when using different vehicles. This suggests that careful consideration should be exercised in selecting a vehicle for the LLNA. A further comprehensive study will be needed to investigate why vehicle differences are observed in the LLNA. Key words: animal strain, local lymph node assay, vehicle-effect The local lymph node assay (LLNA) is the most commonly used among several alternatives of skin sensitization tests [1,8,10], including the human cell line activation test (h-CLAT), the peptide-binding test [7], and the quantitative structure-activity relationship (QSAR) system. Furthermore, a three-dimensional human skin model is now being actively developed as an in vitro model for the assessment of primary skin irritation [15], and put into trials for use as an in vitro model for the evaluation of skin sensitization. These newly developed alternative methods are expected to be implemented for practical use in the future. However, a considerable time will be required for these in vitro test methods to become reliable and accepted international standard test methods.In 2001, Takeyoshi et al. developed the non-radioisotope (non-RI) LLNA [12,13] in Japan. The novel test method was expected to become an alternative to the tra--Note-

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Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2′-deoxyuridine incorporation

et al. 2010

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The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5-bromo-2'-deoxyuridine (BrdU) incorporation (LLNA:BrdU-ELISA). The LLNA:BrdU-ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU-ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut-off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU-ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively.

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

Cited by 45 publications

References 29 publications

Statistical evaluation of toxicological bioassays – a review

Statistical evaluation of toxicological bioassays – a review

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

Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2′-deoxyuridine incorporation

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