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

Abstract

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.

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA)

“…Non-radioactive endpoints, such as lymph node weight, cell proliferation and ear swelling, have been described by several authors (Ehling et al 2005a, b;Suda et al 2002;for review: Anderson et al 2011). Two versions of the LLNA with non-radioactive endpoints, the LLNA: DA and the BrdU-ELISA-version, are currently validated and available as draft OECD guidelines (Omori et al 2008;Kojima et al 2011). Ulrich et al (2001 described a two-tiered murine local lymph node assay and proposed that it is useful to discriminate between contact allergenic potential and skin irritation potential.…”

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers

“…). EC 2 values obtained from our experiments using the LLNA:BrdU‐ELISA (Table ; 10%, 8.5% and 6.1%) were in good agreement with those from a previously published study (12%, 12% and 2.2%) for HCA, EUG and CA, respectively (Kojima et al ., ).…”

“…The sensitizing potential of each of these substances has been previously reported to be strong (TMA), moderate (HCA and CA) or weak (EUG) (Loveless et al ., ). IP and LA are non‐sensitizers (Kojima et al ., ). NiS, a known human sensitizer, is often regarded as an LLNA false negative as it is only occasionally correctly identified as a sensitizer in this assay (Kimber et al ., ).…”

“…The EC 2 is the effective concentration of test article to elicit an SI of 2 (Kojima et al ., ). The EC 2 was calculated by linear interpolation using the equation c + [(2 – d )/( b – d )] × (a – c) where a and c are the doses immediately above and below the SI of 2 and b and d are the SI values immediately above and below the SI of 2.…”

“…In 1999, the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) recommended the development and utilization of alternative LLNA endpoints (ICCVAM, 1999). One area selected for improvement involves the development of an LLNA protocol that does not require the use of radioisotope (Idehara et al, 2008;Jung et al, 2010;Kojima et al, 2011;Piccotti et al, 2006;Suda et al, 2002;Takeyoshi et al, 2001Takeyoshi et al, , 2003Takeyoshi et al, , 2005Ulker et al, 2013). A promising approach is to replace 3HTdR with its non-radioactive analog, 5-bromo-2′-deoxyuridine (BrdU).…”