Clear, tough K-Resin styrene-butadiene copolymers are used to create attractive packaging for a broad spectrum of merchandise including household products and food (see TSM-288 “Food Packageability – K-Resin SB Copolymers”). In any case, the packaging must be chemically compatible with the product it contains. This report gives information on the resistance of K- Resin SBC to several individual reagents. Most commercial products are combinations of several chemicals and their cumulative effects cannot be predicted readily. Furthermore, certain substances can accelerate stress cracking depending on the container design and method of forming. Therefore, final packageability decisions should rely on tests of the actual product in the commercial package. The information shown here, however, can serve as general guidelines for preliminary assessment of a product’s packageability in K-Resin SBC.
The test method was ASTM D543, Standard Test Method for Resistance of Plastics to Chemical Reagents. The test specimens were Type IV tensile bars die stamped from 25 mil thick KR03 sheet.
Separate groups of test specimens were immersed in each test reagent and so conditioned for 7 days at room temperature. Additional sample groups were similarly conditioned for 7 days at 50°C (122°F). The effects of the reagents on the specimens were determined by changes in tensile strength, weight and appearance.
Test results are shown in the appended table. Oils, hydrocarbons, alcohols, and some acids attacked the K-Resin® SBC at room temperature. With the exception of aqueous materials, most of the reagents caused weight gain in the test specimens at room temperature. At 50°C most reagents, other than some dilute basic and acidic materials, affected the specimens in some manner, either by attack or change in appearance.
Though some minimal weight gain and tensile strength loss may be acceptable, K-Resin SBC are affected in some manner by most liquid chemical environments. This does not preclude the packaging of all liquids in K-Resin copolymers, but implies that adequate compatibility testing should be performed for a potential application.
Food Packageability of K-Resin SBC
By virtue of its sparkling clarity, high gloss, and impact resistance, K-Resin styrene-butadiene copolymers (SBC) are well suited for a wide variety of packaging applications. These resins are versatile and easily processed by conventional sheet extrusion, thermoforming, and injection or blow molding processes. Such versatility allows the packaging designer to maximize consumer appeal without sacrificing performance and economy. Attractive display is particularly beneficial for food products and its packaging is a major application for K-Resin® SBC. Since the package must preserve the appearance and quality of the food, certain combinations of food type, polymer, and storage conditions may prove undesirable for functional reasons. The ultimate test for suitability of a polymer for use in food packaging is the evaluation using actual conditions, which include food product for its taste and odor, the package for its performance, and representative storage conditions. This Technical Service Memorandum offers some general observations which may serve as guidelines for preliminary assessment.
K-Resin SBC perform well in packaging many diverse foods. That variety of food products is so broad, that they are difficult to categorize and each should be studied on an individual basis. The possible adverse effects of the food on its container and the container on the food are both interrelated and highly dependent on storage conditions. It is the ultimate responsibility of the producer of the package and/or the food packager to conduct actual storage tests to verify that each specific food product is compatible with the K-Resin®copolymer-based container.
Like most substances, food products can be analyzed in terms of its chemical nature. Some also contain additional chemicals used in its processing or stabilization. Obviously those combinations of chemicals must not attack the package. The resistance of K-Resin SBC to specific chemicals is detailed in PTC Report 353, “Chemical Resistance of K-Resin SB Copolymers,” but may be summarized in some general guidelines here. Water and most water- based products or powdered and granular substances do not chemically attack K-Resin SBC. Most organic solvents such as alcohols, ketones, esters, and ethers will dissolve or soften K- Resin copolymers. Most oils also affect K-Resin copolymers, but the rate and severity of their effects are highly dependent on storage conditions.
Stress Cracking As with most polymers, K-Resin SBC will crack when stressed beyond its limits, especially when its molded geometry concentrates stress, or it contacts certain deleterious chemicals. Such chemicals include the fats and oils present in many foods. The degree of stress cracking with any given product/K-Resin SBC combination also depends on the type of container. Thermoformed containers, especially those made from K-Resin SBC/crystal polystyrene blends, tested with a stress crack accelerating food have cracked almost immediately at room temperature, whereas an injection molded container tested in the same manner did not crack in over a month. Stress cracking is most heavily dependent on four factors: type of container, foodtype, storage conditions, and molded-in stresses. Injection molded containers are more resistant than thermoformed, and fats and unsaturated oils are the greatest stress crack accelerators.
The retention of volume, which is a critical factor in the long-term packaging of liquids, is highly dependent on both time and temperature. Generally, the packaging industry considers 3 percent product loss per year a reasonable maximum. Since water permeates from a K-Resin copolymer container at a rate of 9 percent per year, K-Resin SBC containers may not be suitable for long-term packaging of many aqueous products. Moisture vapor and oxygen permeation through K-Resin SBC are also relatively high which may restrict its use in certain long-term packaging applications. For some types of products, high moisture vapor transmission may be desirable to reduce condensation of moisture in the package. Some food products require oxygen to maintain their color and high oxygen permeation could be advantageous in their packaging.
To retain the quality of the food product, the containers must not induce a detectable change in the taste, aroma, color, or consistency of its contents. Of these criteria, taste and odor are probably the most sensitive to consumer acceptance and regulatory concern. Unfortunately,they are also the most difficult to predict without actual storage testing. The suitability of the polymer/food combination is highly dependent on the temperature and duration of storage. Each of these factors bears further discussion.
Food Type –Among the major constituents of food products, fats are often the most susceptible to acquiring odor and taste because so many organic chemicals are soluble in them. Noticeable changes in a total food product may be affected by the food’s fat content. Foods containing more than 5 weight percent fat and/or having free fat or oil on their surface may generally be considered fatty foods. In a few foods, the fat content may be somewhat protected by the physical nature of the food, as for example oil-in-water emulsions. Packaging fatty foods in K- Resin® SBC is not typically recommended.On the other hand, non-fatty foods are less prone to odor and taste alteration. Most non-fatty foods are economically packaged in K-Resin® SBC with functional and aesthetic success. However, fats are not the only components of food susceptible to alteration, and a few unusually sensitive non-fatty foods may acquire a detectable odor or taste from packaging materials. Suchfoods are best identified by suitable storage tests. Polymer Type –Most polymers contain trace amounts of some components used in theirmanufacture. Some of these inclusions, particularly those of high volatility, may eventuallyescape the polymer into the surrounding atmosphere. If so, they may be absorbed by certain sensitive products packaged in direct or even indirect contact with them. Residual volatiles in K-Resin® copolymers are usually present at such low levels that normal usage raises no recognized safety or health problems. Nevertheless, those minute traces may, under some conditions, affect the odor and taste of certain sensitive foods even when present in quantities as small as fractional parts per billion. Blends of K-Resin® SBC with crystal polystyrene are easy to process and exhibit attractive properties and economics, so those blends are often used in food packaging. Since residual volatiles in crystal polystyrene can contribute significantly to the odor or taste of the packaged food product, we recommend that K-Resin SBC be blended only with food grade polystyrene having low residual content of ethyl benzene and styrene monomer.As shipped by Chevron Phillips Chemical, all K-Resin® SBC grades meet the specifications of the United States FDA Food Packaging Regulation 21 CFR 177.1640 or an effective United States FDA Food Contact Notification. Regulatory compliance does not assure product compatibility. Each application should be tested under representative conditions to assess interaction with packaged food product. Longer storage duration and elevated storagetemperatures may increase packaging incompatibility.In extraction tests at 120°F (49°C), K-Resin®copolymers do exhibit a low, but detectable, level of migration into fats and oils. Therefore, K-Resin® SBC and SBC blends should not be used in containers for long-term storage of fatty foods above 40°F (4°C). On the other hand, storage tests below 40°F (4°C) indicate that migration is extremely low. Under refrigerated conditions below 40°F (4°C), therefore, K-Resin® SBC or K-Resin SBC/ crystal polystyrene blends may prove suitable for packaging fatty foods if storage tests indicate no alteration of the food product.
Based on the migration studies, K-Resin SBC /polystyrene blends are not recommended for packaging fatty foods without specific testing. Use Condition Guidelines –Both volatility and solubility are highly dependent on time and temperature. Polymer/food combinations which pick up odor or taste rapidly at high temperatures may do so slowly at low temperatures. Thus packageability depends on both temperature and duration of contact between food and polymer.Immediate use containers,such as water, soft drink or ice cream cups, are filled and served for immediate consumption (usually within two hours). Because of the short residence time of the product in the container, it is unlikely that product alteration will occur. Storage containers are defined as those not intended for immediate use and may have a shelf life ranging from several days to several years. It is essential that the producer of the package and/or the food packager conduct actual storage tests to verify that each specific food product is compatible with the K-Resin®copolymer based container.Clear, durable and versatile packaging made from K-Resin® SBC can enhance the consumerappeal of food products. Unfortunately, the mutual interaction of food and package cannot bepredicted without testing. We recommendthat the producer of the package and/or the foodpackager conduct actual storage tests to verify that each specific food product is compatiblewith the K-Resin®copolymer based container.
Medical Applications of K-Resin SBC
The medical industry has grown dramatically in the variety and sophistication of its polymer applications, particularly in the manufacture of medical devices and unit packaging. Historically, among the transparent polymers used in large volumes there has been a broad gap between the low cost resins, such as polystyrene, polyethylene and polypropylene, which are either clear or tough, but not both, and the higher priced resins, such as polycarbonate and cellulosics, which are both clear and tough. A part of this gap has been filled by clear, tough K-Resin styrene-butadiene copolymer (SBC). K-Resin® SBC is well-suited for medical applications for a number of reasons. Many medical devices must be clear enough to determine the nature, amount or condition of their contents during use, but tough enough to resist accidental breakage – particularly during those critical moments of an emergency. For that matter, routine breakage can be expensive, time consuming and hazardous. Similarly, clear packages allow accurate identification of contents and their condition. Packages composed of K-Resin® SBC are tough enough to protect their contents but can be designed to open easily. For the fabricator, K-Resin® SBC is easily and economically processed using conventional processing techniques.
Recommended processing temperatures are lower than for many transparent resins while handling, storage, and processing requirements are comparatively straightforward. Two important characteristics of K-Resin SBC are its crystal clarity and exceptional shatter resistance. In addition to this excellent clarity and impact strength, K-Resin SBC is easy to process, provides for design versatility, has the thermal stability required to permit recycling of scrap, and has the necessary physical properties for a broad spectrum of applications. More importantly, grades of K-Resin® SBC which were tested for biological performance meet U.S. Pharmacopoeia (USP) XXIII Class VI requirements, are compatible with blood, demonstrate no cytotoxic, mutagenic or irritant potential, are not sensitizers, and are sterilizable by gamma irradiation, ethylene oxide gas or electron beam irradiation. Autoclave sterilization is unacceptable for K-Resin SBC.
Injection Molding Grades
Several K-Resin® SBC grades (K-Resin®KR01, K-Resin®KR01BR, K-Resin KR03, K-Resin®KR03NW, K-Resin KR03BR and K-Resin BK10) are available for injection molding.
Sheet & Thermoforming Grades
Several K-Resin® SBC grades (K-Resin KR05, K-Resin KK38, K-Resin XK40, K-Resin® XK41 and K-Resin® XK44) have been developed for the sheet extrusion and thermoforming processes. These grades are typically blended with crystal polystyrene for use in Single Service and Rigid Packaging Markets. These grades can also be used in profile extrusion.
K-Resin® SBC grades specifically designed for blown film extrusion are K-Resin DK11 and K-Resin® DK13. K-Resin® DK11 has high stiffness, excellent clarity, and good permeability. K-Resin® DK13 is not as stiff, has greater elongation, and improved tear resistance. Both K-Resin® DK11 & K-Resin® DK13 contain a wax, and therefore will require surface treatment (such as corona discharge) before printing. K-Resin® KRDEV028B is designed for use in the uniaxial oriented film process for shrink sleeves.
Blow Molding Grades
K-Resin® KR05 is the recommended grade for blow molding and injection blow molding. K-Resin XK44 has been used as a coextruded outer gloss layer over polyethylene. All K-Resin® SBC grades as shipped by Chevron Phillips Chemical, meet the specifications of the United States FDA Food Packaging Regulation 21 CFR 177.1640 or the specifications of an effective United States FDA Food Contact Notification. By virtue of this FDA compliance, K-Resin® SBC grades may be used as a component of articles for use in contact with food. Most K-Resin® SBC grades meet the food contact requirements for EEC Directive 2002/72/EEC and all its amendments. The FDA maintains a K-Resin® SBC Drug Master File. For information regarding authorization to cite this file in support of new K-Resin® SBC medical applications, please contact your individual sales representative or one of the regional offices listed on the back of this literature.
Chevron Phillips Chemical has conducted an extensive program of biological testing to demonstrate the suitability of most K-Resin® SBC grades for medical uses. The results of these tests indicate that K-Resin® SB Copolymers, that were tested, meet USP XXIII Class VI requirements (this information is readily available to the medical device manufacturer to help in the documentation process). EVEN SO, IT IS THE ULTIMATE RESPONSIBILITY OF THE MEDICAL DEVICE MANUFACTURER TO DETERMINE THE SAFETY AND SUITABILITY OF THEIR PRODUCT IN WHICH A K-RESIN® SB COPOLYMER IS A COMPONENT.
The evaluation of a polymer for USP Class VI requires three biological tests. The AcuteSystemic Toxicity and Intracutaneous Toxicity tests are designed to determine the biological response of animals to polymers by the single dose injection of specific extracts. The implantation test is designed to evaluate the reaction of living tissue to the polymer when implanted. Additional analytical/biological testing has been performed on K-Resin SBC type KR03. The Physico-Chemical tests determine residual components and the buffering capacity of aqueous extract. Tests were conducted to measure the polymer’s ability to affect blood cells (Hemolysis) or interfere with their oxygenation (Methemoglobin formation). In the Hemolysis procedure, specific extracts of the polymer are introduced into rabbit blood and the degree of subsequent cell destruction is compared to the control. Since methemoglobin formation reduces the capacity of blood to deliver oxygen, the amount of methemoglobin formed in human blood when contacting the copolymer is compared to normal control levels. Tests such as sensitization, cell toxicity, mutagenicity and irritation were conducted to screen out other biological concerns. The Sensitization test evaluates the potential for a polymer to stimulate the production of an antibody or cause anaphylaxis (such as an allergy). The Cytotoxicity tests determine the degree of cell destruction caused by exposing certain cell cultures to an extract of the polymer as well as to the polymer itself. The Ames Mutagenicity test measures the capability of the polymer to promote cell mutation. Certain mutant strains of bacteria are incubated in the presence of extracts of the polymer and the number of colonies reverting to the original bacterial precursor is compared to the spontaneous revertant rate. For the Ocular Irritation test, specific extracts of the polymer are introduced into the eyes of rabbits which are monitored for irritation and corneal damage. The screening tests indicate that K-Resin SBC KR03 is not a sensitizer, causes no gross cytotoxic or mutagenic effects on cells and does not irritate such sensitive tissues as eyes.All tests have been conducted using independent laboratories. The test results of this complete protocol are tabulated below for easy reference. Further details of the test procedures are given in Appendix I.
Gamma irradiation and exposure to ethylene oxide (EtO) gas or electron beam (E-beam) are three typical methods for sterilizing medical products. The contact with radiation, ethylene oxide, or electron beam can affect not only the microorganisms of concern, but also potentially the medical device or package. ASTM test specimens molded from 100% K-Resin® SBC KR03 and 60% KR03 / 40% crystal polystyrene were exposed to these three types of sterilization and then tested. The physical properties were monitored to define any deleterious effect and the results for the control time are given in Tables 2 through 4. The physical properties were also tested after one and two year timeframes. These results are given in Appendix II.
K-Resin® SBC KR03 samples were exposed to different levels of gamma irradiation. Four sets of 100% KR03 and 60% KR03 / 40% crystal polystyrene blended test specimens were injection molded at the Plastics Technical Center (PTC). One set of specimens from each group was not exposed to sterilization and was the “control set.” Each of the other three sets of specimens were subjected to gamma irradiation at dosage levels of 2.5, 5.0 and 7.5 megarads (Mrad) respectively at Sterigenics located in Charlotte, North Carolina. Physical properties were determined for the exposed and control (non-exposed) specimens using standard ASTM test methods at the A2LA approved PTC Evaluation Lab. The results of these tests as tabulated in Table 2 (see below) show that at an exposure level of 2.5 Mrad, with the exception of a small decrease in flow rate and small increase in yellowness, there was no loss in physical or optical properties with the K-Resin® KR03 or K-Resin® KR03 blend. With increasing dosage levels, the only noticeable changes in physical and optical properties were a continuing decrease in flow rate, slight increase in stiffness in conjunction with a slight decrease in elongation and continuing increase in yellowness. In summation, gamma irradiation up to a 10.0 Mrad exposure level has little effect on most physical, mechanical and optical properties of K-Resin® SBC KR03 or KR03/crystal polystyrene blends, but can cause yellow color development and a decrease in melt flow.
Ethylene Oxide Gas
Four sets of 100% K-Resin copolymer KR03 and 60% KR03 / 40% crystal polystyrene blended parts were injection molded at the PTC. One set was not exposed to sterilization and was the “control set.” The other three sets were exposed to one, three, and five 100% EtO sterilization cycles, respectively, at B. Braun Medical Inc. located in Allentown, Pennsylvania. The conditions for the sterilization cycles are listed in Appendix III. Physical properties were determined for the exposed and control sets using standard ASTM test methods at the PTC Evaluation Lab. The results as tabulated below in Table 3 indicate that other than a slight loss of impact and elongation at break, no property deterioration occurred as a result of EtO sterilization.
Four sets of 100% K-Resin SBC KR03 and 60% KR03 /40% crystal polystyrene blended specimens were injection molded at the PTC. The “control set” was not exposed to any sterilization, while the other three sets were exposed to 2.5, 5.0, and 7.5 Mrads of E-beam sterilization, respectively. Sterilization occurred at E-Beam Services, Inc. located in Cranbury, NJ. Physical properties were determined for the exposed and control specimens using standard ASTM test methods at the PTC Evaluation Lab. The results as tabulated below in Table 4 indicate that other than the significant decrease in melt flow, K-Resin® SBC KR03 and blends are relatively unaffected by E-beam sterilization.
K-Resin SBC may be the resin of choice for many medical applications, but there are some applications in which a K-Resin® SBC is certainly not suitable and those applications should be avoided. Some applications demand a greater chemical and/or stress crack resistance than K-Resin® SBC can withstand. Many medical devices have fittings which attach to flexible PVC tubing. Plasticizers used in PVC tubing can migrate into K-Resin® SBC parts in which they are in direct contact, causing a wide range of effects from stress whitening, to swelling, to complete dissolution of the part.
K-Resin® SBC is attacked by many hydrocarbon solutions. Degreasing of parts in many solvents can result in stress whitening, cracking, or premature failure of K-Resin® SBC parts. Some bodily fluids contain high levels of lipids and are considered K-Resin SBC stress cracking agents. Care should be taken any time K-Resin® SBC parts are exposed to any stress crack medium to ensure suitability for use. More information
regarding the chemical resistance of K-Resin®SBC can be found in Plastics Technical Center Report #353 “Chemical Resistance of K-Resin SB Copolymers”. K-Resin® SBC grades are also stabilized with additives that have been shown to interact with certain medical parts and diagnostic testing. There has been one case of apparent incompatibility between soft implantable lenses and K-Resin® SBC, so Chevron Phillips Chemical discourages the use of K-Resin®SBC for any lens packaging applications. Also, in one case, one of the stabilizers used in K-Resin®SBC was linked to the cause of spurious results in hormonal diagnostic
analysis. Caution should be exercised when using a K-Resin® SBC in this type of application.
In all cases it is the responsibility of the customer to make the final determination of the suitability of materials used in any part. It is recommended that extensive testing under the most severe conditions be conducted (including elevated temperature) to determine possible interactions and chemical incompatibility. Chevron Phillips Chemical cannot accept liability for problems that occur as a result of incorrect material choices. K-Resin® SBC is well-suited for many medical uses for devices and packaging. Not only do the polymers process easily and perform well, but they also meet many requirements enforced by the medical industry. Most K-Resin®copolymers meet USP Class VI requirements, are compatible with blood and demonstrate no cytotoxic, mutagenic, sensitizing, or irritant potential.They are readily sterilizable by gamma irradiation, ethylene oxide gas, or electron beam with minimal effects on physical or optical properties.
Test Procedures & Evaluation Criteria for the Biological Effects of K-Resin® SBC
A. USP Class VI
A.1. Acute Systemic Toxicity
Two groups, each consisting of five mice, were used for each extract (extracts are typically: saline, alcohol in saline, polyethylene glycol 400 and cottonseed oil or sesame oil). One group was injected with extract of the K-Resin® SBC prepared by using the USP recommended 60 cm2 total surface area of K-Resin® SBC pellets per 20 ml of the extracting media. The other group of mice was injected with a blank prepared by similar conditioning of the extracting media without the test material. The animals were observed immediately after injection and then again after 4, 24, 48, and 72 hours. K-Resin® SBC met the requirements of the test if, during the observation period, none of the animals treated with the extracts showed a significantly greater reaction than the animals treated with the blank control.
A.2. Intracutaneous Toxicity
Extracts were prepared as above with the 60 cm2 total surface area of the test material per 20 ml of extracting medium. Extracts and blanks were prepared using each of the four media noted above. Two rabbits were used as test animals for each K-Resin® SBC extract. Prior to injection, the hair was closely clipped from the back and flanks of each rabbit. Exactly 0.2 ml of the extract was injected intracutaneously into ten separate sites on the left side. Injection sites were examined 24, 48, and 72 hours after injection for erythema and edema. The degree of tissue reaction was characterized according to the USP recommended numerical scale for Evaluation of Skin Reaction. The arithmetic mean of the numerical ratings was calculated and recorded as the average tissue reaction. K-Resin SBC met the requirements of the test if, during the observation period, the average reaction of tissues to the sample extract did not exceed the average reaction of tissues to the control.
A.3. Implantation Test
Two healthy, adult rabbits were used as test animals for each sample. Hair on the back of each rabbit was clipped away from both sides of the spinal column and all loose hair removed to prevent its entry into the implantation site. At least four sterilized (ethylene oxide) strips of K-Resin® SBC sheet, approximately 1 mm wide and 10 mm long, were implanted into the paravertebral muscle on the left side of the spinal column of each rabbit. At least two USP negative control strips were similarly implanted in the right paravertebral muscle of each rabbit. At five days, the rabbits were weighed and euthanized. All implants were visually located and removed. The tissue surrounding each implant was examined macroscopically for signs of hemorrhage, discoloration, encapsulation, and/or infection. K-Resin® SBC passed the test if there were no observable abnormalities or encapsulation differences between the implant sites of the K-Resin® SBC strips and the USP negative control strips.
B. Blood Compatibility
B.1. Hemolysis Test (in vitro)
B.1.a. Extraction Method
A sample of K-Resin SBC KR03 pellets having approximately 90 cm2 total surface area was placed in a flask, covered with 30 ml saline (0.9% USP sodium chloride injection) and incubated at 70°C for 24 hours. After incubation, three samples of the KR03 extract were placed in separate test tubes.
B.1.b. Direct Contact Method
Approximately 2.0 g of the test article per sample were placed directly into separate test tubes and covered with 10.0 ml of saline. Positive and negative controls were conducted in triplicate concurrently with the test samples. Each positive control tube contained 10.0 ml of deionized water (100% hemolytic) and each negative control tube contained 10.0 ml of saline (0%hemolytic). On the day of testing, whole rabbit blood was collected by cardiac puncture into tubes containing EDTA as an anticoagulant. The blood was sufficiently diluted with saline so that when 0.2 ml diluted blood was added to 10.0 ml of deionized water, the resulting hemolysis produced an absorbance reading in the range of 0.3 to 1.3 absorbance units at a wavelength of 545 nm on the spectrophotometer. The tubes were inverted gently to mix the contents then placed in a constant temperature water bath at 37°C for one hour. Each blood/saline mixture was centrifuged for five minutes at 1500 rpm. The absorbance of each sample and control solution was determined spectrophotometrically at 545 nm and recorded. The hemolysis percentage was calculated for both the extracted and direct contact samples using the following formula: K-Resin® KR03 was considered non-hemolytic if the mean hemolysis value was less than 5% for both test methods.
B.2. Methemoglobin Formation (in vitro)
Three blood samples (approximately 7.0 ml each) from four laboratory technicians (numbered 1- 4) were taken and placed in separate tubes and labeled with the technician’s number and a letter (A, B or C). Eight pieces (approximately 1 x 6.25 cm) of KR03 sheet were placed into separate screw-top containers and each container was labeled A or B with the technician number 1-4. A 3.0 ml aliquot was removed from the corresponding technician blood samples and added to the appropriately labeled container. One sample from each technician served as control and the containers were labeled “C” with the appropriate technician number 1-4. (No test article was present in any container labeled with the letter C). Each container was closed and slowly inverted five times and then horizontally incubated for four hours at 37°C. The containers were removed from the incubator once each hour, inverted slowly five times and returned to the incubator in the same horizontal position.
After incubation, all containers were sent to Diagnostic Services of Hermann Hospital (Houston, Texas) for methemoglobin analysis. The methemoglobin levels in the test blood samples werecompared to the control blood samples using a Microsoft Excel version 5.0 t-Test: Paired Two Sample for Means. K-Resin® KR03 passed if the mean methemoglobin levels in the K-Resin® SBC test samples were not significantly elevated above the mean methemoglobin levels in the control samples.
C. Screening Tests
C.1.a. MEM Elution
A monolayer of L-929 Mouse Fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium (MEM) supplemented with 31μg/ml penicillin (1650 units/mg), 50μg/ml streptomycin sulfate and 10% horse serum and then grown at 37°C in an atmosphere containing 5% CO2-in- air. The cells were sub-cultured at least once before use using dilute trypsin solution. A minimum of three replicate cultures were established for each control. Before initiating the assay, the cultures were examined using an inverted microscope to ensure that each was a sub-confluent monolayer (~80% confluent) and of near-uniform density.Four controls were used for the assay – two medium controls, a negative control, and a positive control. The medium controls consisted of complete medium (containing serum) and serum-free medium. The negative control was six 2-inch pieces of the USP Negative control Plastic Reference Standard per culture. The positive control was two 3-inch pieces of sterilized tubing per culture. An extract of the test material was prepared using serum-free (SF) culture medium. The extract was prepared by placing the 22 pieces (1 cm2 each) of sterile KR03 sheet (2.26 g) and 9.04 ml SF medium in a sterile flask. The headspace of the flask was filled with an atmospherecontaining 5% CO2-in-air and the flask was capped. Extraction was accomplished with 120-140 rpm agitation at 37°C for approximately 48 hours. The extract was then filtered and stored in a sterile container under a nitrogen blanket. The extract was mixed to ensure homogeneity and tested without dilution (100% extract) and at four additional quarter-log dilutions (56.3%, 31.5%,17.8% and 10% solutions of the extract in SF medium) in the MEM elution assay. A total of 0.8 ml of each dilution was added to each of the three cultures. The assay was initiated by aspirating the culture medium from the ~80%-confluent monolayers in each culture and by replacing the medium with the undiluted extract, a dilution of the extract or a medium, positive, or negative control. The cultures were then incubated for at least 48 hours at 37°C in an atmosphere containing 5% CO2-in-air.
At the end of the exposure period, the cells in each culture were inspected using a phase-contrast inverted microscope and evaluated according to USP reactivity grades. KR03 passed if the undiluted extract exhibited an average reactivity grade less than mild reactivity (Grade 2 of the USP reactivity scoring scale).
C.1.b. Agar Overlay
L929 cells were cultured as noted above. Then an overlay of ~2% BBL (Baltimore Biological Laboratories) agar, prepared in complete MEM and cooled to 37°C, was added to each monolayer culture.Three controls were used for the assay – an agar control, a negative control, and a positive control. The agar control consisted only of the agar overlay. The negative and positive controls were the same as noted above. One pre-sterilized test material sample (1 cm2sheet) or negative or positive control was placed on the solidified overlay surface (culture) in separate dishes. The cultures were then incubated for at least 24 hours at 37°C in an atmosphere containing 5% CO2-in-air. At the end of the exposure period, the cells beneath the agar in each culture were inspected using a phase-contrast inverted microscope and evaluated according to USP reactivity grades. K-Resin® KR03 passed if the average extent of reactivity zones around or under the specimens of the material was less than 0.5 cm (mild reactivity; Grade 2 of the USP reactivity scoring scale).
C.2. Ames Mutagenicity Test
Four mutant strains of histidine dependent Salmonella Typhimurium TA98, TA100, TA1535 and TA1537 were used to determine the mutagenic potential of K-Resin® SBC. The strains were kept frozen in nutrient broth prior to use. Before each experiment, the cultures were grown overnight with shaking at 37°C in Oxoid No. 2 broth. Approximately 2 g of KR03 pellets were placed in 13 ml of 0.85% sterile saline solution and an equal amount of K-Resin® SBC pellets was placed in 13 ml of dimethylsulfoxide (DMSO), each in labeled, capped sterile tubes. The tubes containing the test material and the extraction media were then placed in a roller drum and incubated at 37°C for at least 48 hours.Immediately after the extraction procedure, the saline extract was passed through a 0.2 micronfilter. The saline extract was then further diluted in 0.85% sterile saline solution and the DMSO extract was further diluted in sterile DMSO to form four additional quarter-log concentrations for testing. Sterile rat liver S9 homogenate was thawed and used to prepare an S9 mixture (metabolic activation mixture) immediately before thechemical exposure step of each assay. Concurrent sterility, negative (phosphate buffer and extraction media), and positive controls were used in the assay. Sterility controls included separately plating out the top agar, extraction media, test extracts, metabolic activation mixture, and buffer. The negative control preparations consisted of bacteria, 0.85% saline or DMSO, top agar, and metabolic activation mixture (for tests with metabolic activation). The following positive controls were used: sodium azide for the base-pair substitution mutants TA1535 and TA100; 9-aminoacridine for the frameshift mutant TA1537; 4-nitro-o-phenylenediamine for the frameshift mutant TA98; and 2-anthramine for all tester strains in the presence of metabolic activation.
One-tenth (0.1) ml of indicator organisms (about 108 bacteria); 0.5 ml of the metabolic activation mixture; and 0.1 ml of the appropriate dilution of an extract, the positive control or the solvent control were placed in a sterile test tube. The mixture was incubated for 30 minutes at 37°C.After the preincubation period, a molten minimal nutrient agar solution was poured onto a plate.After the top agar had set, the plates were inverted and incubated for about 48 hours before the revertant colonies were counted. A preliminary assay was not conducted. The extracts were assayed for mutagenesis utilizing the four tester strains over five quarter-log dilutions of each extract such that the highest concentration of each resulted in 100μl of undiluted extract per plate. The assay was conducted using six plates for each negative control, three plates per concentration of each extract, and three plates per positive control, in the presence and absence of the S9 metabolic activation mixture. Each extract, strain, and activation condition was evaluated separately for actual number of revertant colonies. KR03 passed if it was shown to be non-toxic or non-mutagenic to any of the four Salmonella strains in the absence and presence of S9 metabolic activation.
C.3. Ocular Irritation Study
Healthy, albino, New Zealand White rabbits were examined with and without a fluorescein sodium ophthalmic solution prior to testing. Six rabbits without eye defects or irritation were selected for testing. A K-Resin® SBC KR03 sample with approximately 60 cm2 surface area was washed in sterile water and then placed in test tubes with 20 ml of saline or cottonseed oil extraction medium added. Blanks (without the test material) of each extraction medium were also prepared as controls. All tubes were heated for 72 hours at 50°C, cooled to room temperature, and stored for less than 24 hours prior to testing. Two-tenths (0.2) ml of each extract was placed into the conjunctival sac of the left eye of three animals. A 0.2 ml dose of the saline blank was placed into the conjunctival sac of the right eye of the same animals. The eyelids were gently held together for one second to prevent loss of material. The three other animals were treated concurrently with the test material/cottonseed oil extract and the cottonseed oil blank in a similar manner.The treated eyes of all animals were examined under normal room lighting without magnification, and the grades of ocular reaction were recorded at 24, 48, and 72 hours after treatment. The corneas of all treated eyes were examined immediately after the 24-hour observation with a fluorescein sodium ophthalmic solution and observed for staining to detect possible corneal damage. K-Resin KR03 passed the test if no minimal or “positive” effects of corneal involvement, iridic irritation or conjunctival irritation (redness or chemosis) were observed in any test compared to the control during the study.
C.4. Sensitization Study
Two vehicles: 0.9% sodium chloride USP solution (SC) and NF cottonseed oil (CSO) were used for this study. For each phase of this study, a ratio of 2.3 g:12 ml (mass of the K-Resin SBC KR03 sample to volume of vehicle) was used for each test extract. The test article was extracted in SC or CSO at 50°C for 72 hours. For the challenge phase, vehicles without the test material were similarly prepared to serve as controls.
Thirty healthy, previously unused, Hartley albino guinea pigs were ear tagged and used for this study. Ten guinea pigs were maintained as control groups to be used during the challenge phase. For the Induction I stage, the hair was removed from an area of the back over the dorsocapular region of the remaining 20 guinea pigs and three rows of intradermal injections (two per row) were given to each animal within an approximate 2 x 4 cm boundary as illustrated below: For the Induction II stage one week after the injections, the areas referenced above were reclipped and the 10% sodium lauryl sulfate (SLS) suspension in petrolatum was massaged into the skin over the injection sites to provoke a mild acute inflammation. The areas were left uncovered, any remaining SLS residue was gently removed the next day, and then the process was repeated. Next, a 2 x 4 cm section of filter paper saturated with 0.3 ml of freshly prepared test article extract (SC or CSO) was topically applied to the previously injected sites and the sites were patched. The patches were removed after 48 hours. For the challenge, twelve days after the final induction patch, the hair of each guinea pig was clipped over the flank areas as needed. For each animal, a nonwoven cotton disk saturated with 0.3 ml of the control vehicle (SC or CSO) was topically applied to the left flank and the test article extract (SC or CSO) saturated disk was topically applied to the right flank. A bandage was applied to the areas to keep the sites well-occluded for 24 hours. Observations for dermal reactions were conducted and recorded at 24, 48, 72, and 96 hours after the challenge patch removal. (Prior to scoring at each interval, the sites were wiped with 35% isopropyl alcohol.) All test animal reactions were compared to any reactions in the control conditions. K-Resin® KR03 passed if no evidence of a sensitization response was noted.
D. Analytical Tests
D.1. Physico-Chemical Tests
A sample of the K-Resin® SBC KR03 test material having 600 cm2 total surface area was
extracted for 24 hours at 70°C in 100 ml of HPLC grade water for all tests in Tables 6 through 12.
Evaluation for Nonvolatile Residue
A 50 ml sample of the extract and a 50 ml sample of HPLC grade water were transferred to individual, previously weighed crucibles which were then heated on top of a steam bath (100°C) for two hours, dried in an oven at 105°C for 1 hour and placed in a desiccator. The crucibles were weighed again. K-Resin® KR03 passed if the difference between the before and after weights was below the test limit of 15 mg.
Evaluation for Residue on Ignition Since the nonvolatile residue was < 5 mg, this test did not need to be performed.
Evaluation for Heavy Metals
A 20 ml sample of the extract was pipetted into a flask and the pH was adjusted to 3.78 with 1 N acetic acid. A 2 ml of Standard Lead Solution and 20 ml of the Blank were pipetted into a
second flask and adjusted with 1 N acetic acid to a pH of 3.97. Both samples were then diluted with HPLC grade water to 35 ml and mixed. Ten ml of freshly prepared hydrogen sulfide were added to each color comparison tube and diluted to 50 ml with HPLC grade water and mixed. Both flasks were placed on a white sheet of paper and observed 10 minutes later. KR03 passed if the color of the extract did not exceed the color of the Standard Lead Solution.
Evaluation for Buffering Capacity
A 20 ml sample of the extract and 20 ml of HPLC grade water were placed in separate beakers. The sample extract was titrated to a pH of 7.0 using 0.010 N hydrochloric acid and the blank was treated similarly with 0.010 N sodium hydroxide. K-Resin SBC KR03 passed if the difference in volumes between the two samples was below 10.0 ml.
D.2. Infrared Analysis
37.2 mg of K-Resin SBC KR03 pellets were dissolved in chloroform. A few drops of the
resulting solution were applied to the surface of a clean KBr (potassium bromide) disc and the
solvent was allowed to evaporate completely. An FTIR scan was performed on the resulting film.The infrared analysis indicated that the K-Resin® SBC sample was a styrene-butadiene
D.3. High Performance Liquid
Chromatography (HPLC) Characterization
Three separate K-Resin KR03 samples were weighed into 100 ml flasks. Each of the samples was brought to volume with tetrahydrofuran. Four 10μl aliquots were removed from each sample and analyzed by HPLC. Each of the resulting chromatograms was then analyzed. KR03 passed the test if the retention times of the main elution peaks agreed within ± 5% relative and the peak heights agreed within ± 10% relative.
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