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

In vivo and in vitro irritation testing is important for evaluating the biological safety of medical devices. Here, the performance of positive reference materials for skin irritation testing was evaluated. Four reference standards, referred to as Y-series materials, were analyzed: a polyvinyl chloride (PVC) sheet spiked with 0 (Y-1), 1.0 (Y-2), 1.5 (Y-3), or 10 (Y-4) parts of Genapol X-080 per 100 parts of PVC by weight. Y-1, Y-2, and Y-3 did not induce skin irritation responses in an in vitro reconstructed human epidermis (RhE) tissue model, as measured by tissue viability or interleukin-1α release, or in an in vivo intracutaneous response test using rabbits. In contrast, Y-4 extracts prepared with saline or sesame oil at 37°C and 50°C clearly elicited positive irritation responses, including reduced viability (< 50%) and significantly higher interleukin-1α release compared with the solvent alone group, in the RhE tissue model and an intracutaneous response test, where substantial necrosis was observed by histopathology. The positive skin irritation responses induced in vitro under various extraction conditions, as well as those elicited in vivo, indicate that Y-4 is an effective extractable positive control material for in vivo and in vitro skin irritation tests of medical devices. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2807-2814, 2018.

Alternative plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester, for blood containers with protective effects on red blood cells and improved cold resistance.

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC), is eluted from PVC-made blood containers and protects against red blood cell (RBC) hemolysis. However, concerns have arisen regarding the reproductive and developmental risks of DEHP in humans, and the use of alternative plasticizers for medical devices has been recommended worldwide. In this study, we propose that the use of a novel plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH), could help produce more useful and safe blood containers. PVC sheet containing DL9TH and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH) provides comparable or superior protective effects to RBCs relative to PVC sheet containing DEHP or di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH® , an alternative plasticizer that has been used in PVC sheets for blood containers). The total amount of plasticizer eluted from DOTH/DL9TH-PVC sheets is nearly the same as that eluted from DEHP-PVC sheets. In addition, DOTH/DL9TH-PVC has better cold resistance than DEHP- and DINCH® -PVC sheets. In vitro and in vivo tests for biological safety based on International Organization for Standardization guidelines (10993 series) suggest that the DOTH/DL9TH-PVC sheet can be used safely. Subchronic toxicity testing of DL9TH in male rats in accordance with the principles of Organisation for Economic Co-operation and Development Test Guideline 408 showed that DL9TH did not induce adverse effects up to the highest dose level tested (717 mg/kg body weight/day). There were no effects on testicular histopathology and sperm counts, and no indications of endocrine effects: testosterone, thyroid-stimulating hormone, follicle-stimulating hormone, and 17ß-estradiol were unchanged by the treatment, compared with the control group. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1052-1063, 2018.

Pilot study on novel blood containers with alternative plasticizers for red cell concentrate storage.

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC) blood containers, is eluted from the blood containers and exerts protective effects on red blood cells. However, a concern for detrimental effects of DEHP on human health has led to the development of potential DEHP substitutes. Here, we compared the red blood cell preservation ability of two types of non-DEHP blood containers with safe alternative plasticizers to that of DEHP blood containers. Red cell concentrates in mannitol-adenine-phosphate solution (MAP/RCC) were stored for 6 weeks in PVC blood bags containing DEHP, di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH) and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH), or 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH) and DOTH. There was no significant difference in the total amount of plasticizer eluted into MAP/RCC (till 3 weeks from the beginning of the experiment), hemolysis of MAP/RCC, and osmotic fragility of MAP/RCC between the non-DEHP blood containers and DEHP blood containers. Hematological and blood chemical indices of MAP/RCC in all containers were nearly the same. Thus, DOTH/DINCH and DOTH/DL9TH blood containers demonstrate the same quality of MAP/RCC storing as the DEHP blood containers. Since DOTH, DINCH, and DL9TH were reported to be safe, DOTH/DINCH and DOTH/DL9TH blood containers are promising candidate substitutes for DEHP blood containers.

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

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.

Screening study on hemolysis suppression effect of an alternative plasticizer for the development of a novel blood container made of polyvinyl chloride.

The aim of this study is to identify a plasticizer that is effective in the suppression of the autohemolysis of the stored blood and can be used to replace di(2-ethylhexyl) phthalate (DEHP) in blood containers. The results of hemolysis test using mannitol-adenine-phosphate/red cell concentrates (MAP/RCC) spiked with plasticizers included phthalate, phthalate-like, trimeliate, citrate, and adipate derivatives revealed that di-isononyl-cyclohexane-1,2-dicarboxylate (Hexamoll(®) DINCH), di(2-ethylhexyl)-1,2,3,6-tetrahydro-phthalate (DOTP), and diisodecyl phthalate (DIDP) exhibited a hemolysis suppression effect almost equal to that of DEHP, but not other plasticizers. This finding suggested that the presence of 2 carboxy-ester groups at the ortho position on a 6-membered ring of carbon atoms may be required to exhibit such an effect. The hemolytic ratios of MAP/RCC-soaked polyvinyl chloride (PVC) sheets containing DEHP or different amounts of DINCH or DOTP were reduced to 10.9%, 9.2-12.4%, and 5.2-7.8%, respectively (MAP/RCC alone, 28.2%) after 10 weeks of incubation. The amount of plasticizer eluted from the PVC sheet was 53.1, 26.1-36.5, and 78.4-150 µg/mL for DEHP, DINCH, and DOTP, respectively. PVC sheets spiked with DIDP did not suppress the hemolysis induced by MAP/RCC because of low leachability (4.8-6.0 µg/mL). These results suggested that a specific structure of the plasticizer and the concentrations of least more than ∼10 µg/mL were required to suppress hemolysis due to MAP/RCC.

A development and biological safety evaluation of novel PVC medical devices with surface structures modified by UV irradiation to suppress plasticizer migration.

This study examines the chemical, physicochemical, and biological properties of PVC sheets treated with UV irradiation on their surfaces to suppress the elution of a plasticizer, di-(2-ethylhexyl) phthalate (DEHP), for developing novel polyvinyl chloride (PVC) medical devices. The PVC sheets irradiated under conditions 1 (52.5 µW/cm(2), 136 J/cm(2)) and 2 (0.45 mW/cm(2), 972 J/cm(2)) exhibited considerable toxicity in cytotoxicity tests and chromosome aberration tests due to the generation of DEHP oxidants, but no toxicity was detected in the PVC sheet irradiated under condition 3 (8.3 mW/cm(2), 134 J/cm(2)). The release of DEHP from the surface irradiated under condition 3 was significantly suppressed, and mono-(2-ethylhexyl) phthalate (MEHP) converted from a portion of DEHP could be easily removed from the surface by washing with methanol. The physicochemical properties of the surface regarding the suppression of DEHP elution remained stable through all sterilizations tested, but MEHP elution was partially recrudesced by the sterilizations except for gamma irradiation. These results indicated that UV irradiation using a strong UV-source over a short time (condition 3) followed by methanol washing and gamma sterilization may be useful for preparing novel PVC products that did not elute plasticizers and do not exhibit toxicity originating from UV irradiation.

Higher toxicity of dibutyltin and poly-L-lactide with a large amount of tin but lower toxicity of poly-L-lactide of synthetic artificial dura mater exhibited on murine astrocyte cell line.

Neurotoxicities of dibutyltin (DBT), tin(II) octylate (OT), poly-L-lactides (PLLA, molecular weight [MW]=5000, PLLA 5000), PLLA without tin (MW=3000, PLLA 3000), PLLA with a large amount (590 ppm) of tin (S3), poly(glycolic acid-co-epsilon-caprolactone) oligomer (MW=6200, PGC oligomer), and poly(L-lactic acid-co-glycolic acid-co-epsilon-caprolactone) oligomer (MW=6400, PLGC oligomer) related to artificial dura mater were examined using the murine astrocyte cell line, CRL-2534. The indices were cell viability, glutamate concentration in the cell supernatant, and cell proliferation. Lower cell viability was observed among cells exposed to 0.5 microM DBT or 10 microg/ml of S3. There were no differences in cell viability of astrocytes exposed to OT, PLLA 5000, PLLA 3000, PGC oligomer, or PLGC oligomer. Mean glutamate concentration in the supernatant of cells exposed to 0.25 muM DBT was higher than that of the control after 2 h incubation. Lower mean concentration of glutamate in the supernatant of cells exposed to 5 microg/ml of S3 was observed after 2 h incubation. Cells exposed to 50 microg/ml of PGC oligomer had a higher mean concentration of glutamate in the supernatant. OT only inhibited cell proliferation at 100 microM. Proliferation of cells exposed to 0.25 microM or 0.5 microM DBT was inhibited, as was that of cells exposed to 100 microM OT, 50 microg/ml PLLA 5000, 50 microg/ml PLLA 3000, and 5 microg/ml S3, 5 d and 7 d after exposure. Although DBT does not reach levels that induced neurotoxicity in artificial dura mater, these results suggest that DBT is neurotoxic and PLLA toxicity increases with the increase in tin concentration.