Cutaneous toxicity testing in organ culture: Neutral red uptake and reduction of tetrazolium salt (MTT)

1993 ◽  
Vol 7 (1) ◽  
pp. 81-86 ◽  
Author(s):  
J.J.M. van de Sandt ◽  
A.A.J.J.L. Rutten ◽  
H.B.W.M. Koëter
Keyword(s):  
Organ Culture ◽  
Toxicity Testing ◽  
Neutral Red ◽  
Tetrazolium Salt ◽  
Cutaneous Toxicity ◽  
Neutral Red Uptake

scholarly journals Advances in acute toxicity testing: strengths, weaknesses and regulatory acceptance

2018 ◽  
Vol 11 (1) ◽  
pp. 5-12 ◽  
Author(s):  
Earnest Oghenesuvwe Erhirhie ◽  
Chibueze Peter Ihekwereme ◽  
Emmanuel Emeka Ilodigwe
Keyword(s):  
Toxicity Testing ◽  
Neutral Red ◽  
Test Methods ◽  
Systemic Toxicity ◽  
Regulatory Approval ◽  
Fixed Dose ◽  
Regulatory Body ◽  
Cytotoxicity Test ◽  
Food Ingredients ◽  
Neutral Red Uptake

Abstract Safety assessment of chemicals, pharmaceuticals, food and food ingredients, cosmetics, industrial products is very crucial prior to their approval for human uses. Since the commencement of toxicity testing (about 500 years ago, since 1520), significant advances have been made with respect to the 3Rs (reduction, refinement and replacement) alternative approaches. This review is focused on the update in acute systemic toxicity testing of chemicals. Merits and demerits of these advances were also highlighted. Traditional LD50 test methods are being suspended while new methods are developed and endorsed by the regulatory body. Based on the refinement and reduction approaches, the regulatory body has approved fixed dose procedure (FDP), acute toxic class (ATC) method and up and down procedure (UDP) which involves few numbers of animals. In terms of replacement approach, the regulatory body approved 3T3 neutral red uptake (NRU), the normal human keratinocyte (NHK), and the 3T3 neutral red uptake (NRU) phototoxicity test for acute phototoxicity. However, other promising replacement alternatives such as organ on chip seeded with human cells for acute systemic toxicity and 3T3 neutral red uptake (NRU) cytotoxicity test for identifying substances not requiring classification, as well as the in silico approaches are yet to receive regulatory approval. With this backdrop, a collaborative effort is required from the academia, industries, regulatory agencies, government and scientific organizations to ensure speedily regulatory approval of the prospective alternatives highlighted.

Skin Organ Culture as an Alternative to In Vivo Dermatotoxicity Testing

1993 ◽  
Vol 21 (4) ◽  
pp. 443-449
Author(s):  
Johannes J.M. van de Sandt ◽  
Jacqueline van Schoonhoven ◽  
Wilfred J.M. Maas ◽  
Alphons A.J.J.L. Rutten
Keyword(s):  
Organ Culture ◽  
Chloroacetic Acid ◽  
Tetrazolium Salt ◽  
Dependent Manner ◽  
Cutaneous Toxicity ◽  
Culture Model ◽  
The Relationship ◽  
Dose Dependent ◽  
Skin Organ Culture

Various aspects of acute cutaneous toxicity were studied in a skin organ culture model. Chemicals were applied topically for four hours, after which cytotoxicity was assessed by measuring the conversion of the tetrazolium salt, MTT. The relationship between pKa and cytotoxicity was investigated for a homologous series of benzoic acids. In this series, salicylic acid had the lowest pKa and proved to be the most toxic compound. Furthermore, the pH of the carrier solution was shown to influence the toxicity of chloroacetic acid and acetic acid in a different way. Using skin discs of both human and rabbit origin, we found that human skin was more resistant to toxicity induced by the irritants benzalkonium chloride and formaldehyde. As an additional aspect of dermal toxicology, the percutaneous absorption of testosterone was studied. After topical application to rabbit skin discs, testosterone was absorbed in a dose-dependent manner and concurrent metabolism was demonstrated.

The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol

1990 ◽  
Vol 17 (4) ◽  
pp. 325-333
Author(s):  
Paul J. Dierickx ◽  
Virginia C. Gordon
Keyword(s):  
Neutral Red ◽  
Alternative Methods ◽  
Inhibition Assay ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
In Vitro Methods ◽  
Neutral Red Uptake

The neutral red uptake inhibition assay and the EYTEX™ system were investigated as alternative methods for the assessment of eye irritation, determined according to the EEC protocol. The 17 test chemicals used were mainly organic solvents. The xenobiotics were applied to Hep G2 cells for 24 hours at different concentrations. Neutral red uptake inhibition was then measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The same chemicals were also tested as coded samples by the EYTEX™ test according to the manufacturer's directions. A nearly identical quantitative correlation was found for both in vitro methods with corneal opacity scores: r = 0.84 for EYTEX™ scores and r = 0.83 for log NI50, expressed in μg/ml. Whilst these correlations are certainly not perfect, it is clear that both in vitro methods can be used as valuable prescreening methods.

scholarly journals Biological Innocuousness of a Device developed to select the Optimum Spermatozoa to be used in the Treatment of Infertility

2021 ◽  
Author(s):  
Marisa A Cubilla ◽  
Hector A Guidobaldi ◽  
Laura C Giojalas
Keyword(s):  
Colony Formation ◽  
Physiological State ◽  
Neutral Red ◽  
Sperm Selection ◽  
Neutral Red Uptake

The sperm selection assay (SSA) is a method based on chemotaxis to obtain spermatozoa at the optimum physiological state to successfully fertilize the egg. It consists of a device made of acrylic and an attractant solution which includes progesterone. We evaluate potential cytotoxicity interactions by means of Neutral Red uptake, the MTT and colony formation assays, according to ISO normative. Here we showed that even stressing the conditions of the assays, the SSA device alone or together with the progesterone solutions employed, showed to be innocuous for the cells. Suggesting that SSA could be incorporated into the ART procedures.

The effect of hexaaza-and hexathia–macrocyclic ligands on transition metal cytotoxicity in human hepatoma-derived cultured cells

2002 ◽  
Vol 21 (8) ◽  
pp. 421-427 ◽  
Author(s):  
P W Smet ◽  
T F Pauwels ◽  
P J Dierickx
Keyword(s):  
Transition Metal ◽  
Cultured Cells ◽  
Copper Ions ◽  
Transition Metal Ions ◽  
Neutral Red ◽  
Macrocyclic Ligands ◽  
Human Hepatoma ◽  
Uptake Inhibition ◽  
Copper Zinc ◽  
Neutral Red Uptake

The effect of macrocyclic ligands on cytotoxic concentrations of the transition metal ions of copper, zinc, and cadmium was investigated. For this purpose, a hexaaza-[3,6,9,17,20,23-hexaazatricyclo[23.3.1.111,15] triaconta–1(29),11(30),12,14,25,27–hexaene (L2)] and hexathia-chelating ligand [1,4,7,10,13,16-hexathiacyclooctadecane (L3)] were used in the human hepatoma-derived HepG2 cell line. The cytotoxicity was measured by the neutral red uptake inhibition assay. First, the NI50 of the ligands, i.e., the concentration of the ligand inducing a 50% inhibition in neutral red uptake compared to control cells, was determined. In several metal/ligand combination experiments, the effects for L2 were difficult to interpret, whereas for L3 in combination with copper ions, a severe increase–and for zinc ions, a significant decrease of cell toxicity–relative to the metal control was observed. To further examine the different effects observed with L3 in combination with, respectively, Cu2+ and Zn2+, the glutathione (GSH) content was measured. The relative GSH content decreased as the concentration of L3 increased. It was proposed that the increased toxicity of the combination Cu2+ /L3 could be caused by the depletion of GSH and a subsequent inability to scavenge the produced reactive oxygen species (ROS). This hypothesis was supported by experiments during which vitamin E or C was added to the Cu2+ / L3 system.

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