Development and performance evaluation of a positive reference material for hemolysis testing

Abstract

This study deals with the development and performance evaluation of a positive reference material for hemolysis testing, which is used for evaluating the biological safety of medical devices. Genapol X-080, a nonionic detergent, was selected as a candidate hemolytic substance in a survey of 23 chemical compounds; it showed significant hemolytic activity against rabbit defibrinated blood at concentrations more than 20 µg/mL. A polyvinyl chloride (PVC) sheet spiked with 0.6% (w/w) of the compound exhibited weak hemolytic activity in direct contact and/or extract-based assays after 4 h incubation at 37°C. A PVC sheet containing 5.8% (w/w) Genapol X-080 induced complete hemolysis in both assays. The amount of Genapol X-080 eluted from each PVC sheet during hemolysis testing using the direct contact method increased time-dependently and reached 25.6 (former sheet) or 1154 (later sheet) µg/mL after 4 h incubation, which was similar to or much higher than the critical micelle concentration, respectively. Similar elution behavior was observed using the extract-based method, and the Genapol X-080 content in test solutions prepared by autoclave extraction of both sheets was 22.5 and 358 µg/mL, respectively, indicating a clear relationship between the degree of hemolytic activity and the eluted amount of Genapol X-080. Thus, a PVC sheet spiked with a compound exhibiting different hemolytic activity depending on its concentration may be useful as a positive reference material to validate the hemolysis tests.

Proof of concept testing of a positive reference material for in vivo and in vitro skin irritation testing

“…Previously, we found that Y‐2 and Y‐4 sheets elicit weak or strong hemolysis, respectively, in direct contact and/or extract‐based assays . There is a clear relationship between the degree of hemolytic activity and elution volume for Genapol X‐080 .…”

“…Previously, we found that Y-2 and Y-4 sheets elicit weak or strong hemolysis, respectively, in direct contact and/or extract-based assays. 7 There is a clear relationship between the degree of hemolytic activity and elution volume for Genapol X-080. 7 Using the direct contact method, the amount of Genapol X-080 eluted from Y-2 and Y-4 sheets into blood samples increases over time, reaching 25.6 or 1154 lg/mL after incubation at 378C for 4 h, corresponding to approximately the CMC, and substantially higher than the CMC, respectively.…”

“…T‐die molded PVC sheets (thickness = 0.4 mm) containing 55 parts of DEHP, 8 parts of ESBO, and various amounts of Genapol X‐080 (0, 1, 1.5, and 10 parts, referred to as Y‐1, Y‐2, Y‐3, and Y‐4, respectively) per 100 parts of PVC by weight, along with other additives, were mixed according to a previously reported method . The contents of Genapol X‐080 in Y‐1, Y‐2, Y‐3, and Y‐4 sheets are 0, 0.61%, 0.91%, and 5.8% (w/w), respectively.…”

“…Negative and positive reference materials, and positive control substances, have been developed for various biological safety evaluation methods, including cytotoxicity, sensitization, genotoxicity, and implantation tests; however, reference materials are lacking for other types of tests. Recently, we developed negative and positive reference materials constructed from plasticized polyvinyl chloride (PVC) sheets, which demonstrated variable hemolytic activity depending on their content of Genapol X‐080 for use in three official test methods conducted in Japan and the USA . Since Genapol X‐080 is a non‐ionic surfactant, we hypothesized that this positive reference for hemolysis testing may also induce skin irritation activity.…”

“…Recently, we developed negative and positive reference materials constructed from plasticized polyvinyl chloride (PVC) sheets, which demonstrated variable hemolytic activity depending on their content of Genapol X-080 for use in three official test methods conducted in Japan and the USA. 7 Since Genapol X-080 is a non-ionic surfactant, we hypothesized that this positive reference for hemolysis testing may also induce skin irritation activity. In the present study, we evaluated whether the reference materials developed for hemolysis testing can also be applied as reference materials for in vivo and in vitro skin irritation testing.…”