Performance of the Neutral Red uptake assay in the COLIPA international validation study on alternatives to the rabbit eye irritation test

AbstractThe Hyacinthaceae family (sensu APGII), with approximately 900 species in around 70 genera, plays a significant role in traditional medicine in Africa as well as across Europe and the Middle and Far East. The dichloromethane extract of the bulbs of Massonia pustulata (Hyacinthaceae sensu APGII) yielded two known homoisoflavonoids, (R)-5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromanone 1 and 5-hydroxy-3-(4-hydroxybenzyl)-7-methoxy-4-chromone 2 and four spirocyclic nortriterpenoids, eucosterol 3, 28-hydroxyeucosterol 4 and two previously unreported triterpenoid derivatives, (17S,23S)-17α,23-epoxy-3β,22β,29-trihydroxylanost-8-en-27,23-olide 5, and (17S, 23S)-17α,23-epoxy-28,29-dihydroxylanost-8-en-3-on-27,23-olide 6. Compounds 1, 2, 3, and 5 were assessed for cytotoxicity against CaCo-2 cells using a neutral red uptake assay. Compounds 1, 2, and 5 reduced cell viability by 70% at concentrations of 30, 100, and 100 µM, respectively. Massonia bifolia yielded three known homoisoflavonoids, (R)-(4′-hydroxy)-5-hydroxy-7-methoxy-4-chromanone 1, (R)-(4′-hydroxy)-5,7-dihydroxy-4-chromanone 7 and (R)-(3′-hydroxy-4′-methoxy)-5,7-dihydroxy-4-chromanone 9, two previously unreported homoisoflavonoids, (E)-3-benzylidene-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 8 and (R)-(3′,4′-dihydroxy)-5-hydroxy-7-methoxy-4-chromanone 10, and a spirocyclic nortriterpenoid, 15-deoxoeucosterol 11. Compounds 1, 1Ac, 7, 8, 9, and 10 were screened for antiangiogenic activity against human retinal microvascular endothelial cells. Some compounds showed dose-dependent antiproliferative activity and blocked endothelial tube formation, suggestive of antiangiogenic activity.

Download Full-text

The Neutral Red Uptake Assay: Comments on the Results of the Istituto Superiore di Sanità in the EC/HO Validation Study

Alternatives to Laboratory Animals ◽

10.1177/026119299802600110 ◽

1998 ◽

Vol 26 (1) ◽

pp. 61-68

Author(s):

Annalaura Stammati ◽

Franco Zampaglioni ◽

Cristiana Zanetti

Keyword(s):

Validation Study ◽

Rank Correlation ◽

Neutral Red ◽

Home Office ◽

Uptake Assay ◽

Neutral Red Uptake ◽

British Home ◽

Chemical Groups

The neutral red uptake (NRU) assay was included, among others, in a validation study sponsored by the European Commission/British Home Office (EC/HO) study, for its reliability as an in vitro alternative to the Draize eye irritancy test. The test was performed in parallel by four laboratories (Istituto Superiore di Sanità [ISS], Microbiological Associates, Hatano Research Institute and Kurabo Industries) on 60 selected chemicals. The results obtained by the ISS are reported in this paper. A poor rank correlation was obtained between the in vivo endpoint and the ISS in vitro results for the full set of chemicals and for the subsets, with the exception of surfactants, by an independent statistics group. The same unsatisfactory results were obtained by the ISS group when the rank correlation was calculated for compounds divided into chemical groups. The performance of the NRU assay, as an alternative to the Draize eye irritancy test, is discussed.

Download Full-text