Most pharmaceutical companies and contract manufacturers know exactly what is needed when outsourcing their chemical testing to meet regulatory and pharmacopoeial requirements. But when it comes to Biosafety Testing, requirements become somewhat less clear. What is Biosafety Testing as it applies to outsourced bioanaly-tical testing? Biosafety Testing in the pharmaceutical manufacturing/packaging arena can range from preclinical to clinical studies and involve such disciplines as microbiology, toxicology, and pharmacology. Biosafety Testing focuses mainly on the safety of the material in question and on anything from raw materials to intermediate and final product. Because Biosafety Testing is such a large area, the focus of this article will be mainly in the area of toxicology and the biocompatibility testing of drug containers and closures and medical devices.
One discipline of Biosafety Testing which has changed greatly over the years is the area of toxicology and, more specifically, biocompatibility testing. Early versions of the United States Pharmacopoeias (USP) included only one paragraph to explain these requirements. Now this important area of testing has grown, especially as it relates to pharmaceutical drug containers, closures and medical devices. Although there are many other toxicological requirements especially for medical devices, such as sensitization subchronic toxicity, genotoxicity, hemocompatibility, chronic toxicity, carcinogenicity, reproductive or developmental toxicity, these are beyond the scope of this article.
Drug Containers and Closures
Pharmaceutical manufacturers are required to demonstrate that the plastic containers and the associated closures do not exhibit toxicity (biological reactivity) when tested either in vitro or in vivo. The drug containers are filled with liquid components that can possibly extract compounds out of the plastic drug containers or closures that are toxic to animal cells. Also included are elastomeric closures (those which are pierced by a syringe). Elastomeric closures are composed of several chemical compounds such as plasticizers, fillers, accelerators, stabilizers, vulcanizing agents, pigments and natural or synthetic polymers which can possibly be extracted and have toxic effects. It is usually the elastomeric closures that most often exhibit toxicity during testing. Biocompatibility testing requirements can be found in the current USP under Injections <1>, Containers <661>, and Elastomeric Closures for Injections <381>.
The biocompatibility testing requirement of medical devices depends upon the type of contact it will make with the patient (i.e., direct or indirect). Examples of such medical devices are intravenous solution administration sets, intravenous cath-eters, blood administration sets, dialyzers and dialysis tubing, transfusion and infusion assemblies and intramuscular drug delivery catheters. The testing requirements can be found in the USP under Transfusion and Infusion Assemblies and Similar Medical Devices <161>.
The biological testing referenced in these requirements concentrate mainly on two USP chapters, Biological Reactivity Tests, In Vitro <87> and Biological Reactiv-ity Tests, In Vivo <88>. The testing is designed to determine if there is any toxicity to mammalian cell cultures or biological reactivity to animals.
In Vitro Testing
Since most containers and medical devices are too big to analyze in vitro testing is usually done using a representative sample of the material. Testing conducted according to USP Biological Reactivity Tests, In Vitro <87>, consists of three types of in vitro tests: MEM Elution, Direct Contact or Agar Diffusion. The factors affecting the choice of the test are the material, the final product and its intended use. Test sample preparation involves taking a specific surface area (usually 60 or 120 cm2 based on the thickness of the test sample) of a drug container or a per gram weight of an elastomeric closure (refer to USP 24 <88> for surface area or gram weight to extract). The test sample is then extracted either in serum-supplemented cell culture media or sodium chloride injection (0.9% NaCl). The extraction temperature and solvent for extraction is chosen based on the final temperature that will most closely mimic "in-use" conditions of the finished and sterilized product. For example, the most typically used extraction temperatures are 121°, 70°, 50° and 37°C for time intervals of 1, 24 or 72 hours. It should be noted that if cell culture medium with serum is used, then 37°C is the highest temperature that can be used or the serum in the medium will be destroyed. The extracts are then exposed to L-929 mammalian fibroblast cell line (a well-characterized cell line) at 37°C for 48 hours utilizing the in vitro tests mentioned previously. Other cell lines can be used, but the testing facility will have to scientifically justify the use of the alternate test system.
Three in vitro tests are used in the evaluations. The Elution test was designed for high-density polymeric material and for dose-response evaluations. At the same time, the cells are also exposed to a USP reference standard (a non-reactive high-density polyethylene) as a negative control and a USP Positive Bioreaction (the USP convention has not developed the Positive control thus a sterile non-lubricated latex condom is typically used as a positive control). The cells are evaluated microscopically to determine if there is any reactivity. The reactivity is graded from 0 (no reactivity) to a grade 4 (severe reactivity with nearly complete destruction of the cell layers). The sample meets the requirements for biological reactivity if the reaction is not greater than grade 2 (mildly reactive) and the controls showed a suitable response.
The Direct Contact Test was designed for materials in a variety of shapes and allows for simultaneous extraction and testing of leachable chemicals from a test sample. Very low- or high-density materials cannot be used in this test because the materials can cause mechanical damage to the cells. Not less than 100 mm2 portions of the test material are placed in direct contact with the L-929 cells in the presence of serum-supplemented cell culture medium. After 24 hours, the cells are examined. The sample meets the requirements for biological reactivity if the reaction is not greater than grade 2 (mildly reactive) and the controls showed a suitable response.
The Agar Diffusion Test is used for elastomeric closures that come in a variety of shapes. There is an agar overlay placed on the cells which protects the cells from mechanical damage but allows diffusion of leachable chemicals from the polymeric materials to reach the cells. Extracts of materials can also be applied to filter paper and tested on the agar overlay. Again a reactivity grade no greater than 2 (mildly reactive) is required to meet the biological reactivity requirements.
A plastic or polymer which does not meet the requirements of the in vitro testing just discussed is considered unsuitable material for drug containers or medical devices. If the requirements are met, no further testing is required. However, sometimes a class designation is desired, therefore, in vivo testing will need to be conducted in order to determine the class (I-VI).
The exception is for elastomeric materials. If the elastomeric materials fail the in vitro testing, the materials still can be classified as biocompatible materials for drug containers and for use in medical devices if they pass the in vivo testing. No class designation is given to elastomeric materials.
In Vivo Testing
If the materials do not pass the in vitro testing or require class designation, then in vivo testing is required. Testing is conducted according to USP Biological Reactivity Tests, In Vivo <88>. Three tests are used: 1) the Systemic Injection Test, 2) the Intracutaneous Test and 3) the Implantation Test. As with the in vitro tests, an extract is made of the plastic or polymers and the extracts are then injected systemically and intracutaneously into mice and rabbits. If the biological reactivity is no greater than the blank material (solvent used for extraction without test sample) which was used, the polymer is graded as class I, II, III or V (depending on the type of extract used). The third test is implantation and classifications of IV or VI can be given to the plastic or polymer based on these test results.
Classification of Plastics
Plastics consist of six classes and the classification is based on responses to a series of in vivo tests. The choice of the test is based on the final end use of the plastic or polymer and the choice of the material used for extraction will depend on the vehicle that the plastic will contact in normal use. Temperatures chosen for the test will depend on the heat resistance of the plastic. The class designation of the plastic will be followed by the temperature of extraction (e.g. IV-121°). The plastic classification does not apply to plastics that are intended for as containers for oral or topical products, or that may be used as an integral part of a drug formulation.
The Systemic Injection Test is designed to evaluate systemic responses of extracted material following injection into mice. Five mice in a test group are injected intravenously with the extracts and five in a test group with a blank. The animals are observed for a response at 4, 24, 48 and 72 hours after injection. To meet the requirements of this test, none of the animals in the sample extract group should show a greater biological reactivity than the blank group. Requirements of the test would not be met if any of the following conditions occur during the observation period: 1) two or more mice die, 2) two or more mice exhibit abnormal behavior such as convulsions or prostration, or 3) three or more mice show body weight loss greater than 2 grams occurs. A repeat test can be done using 10 mice per group if the animals treated with the sample shows only slight signs of biological reactivity and no more than one animal shows gross symptoms or dies. However, if the repeat is done, all 10 mice must meet the criteria previously discussed (i.e., no greater biological reactivity than the blank sample).
The Intracuta-neous Test evaluates local responses to extracts of materials after intracutaneous injection into rabbits. The fur is removed and the extracts are injected intracutaneously in two animals. Five sites are chosen on one side of the animal for the sample extract and five sites are chosen on the other side of the animal for the blank. The injection sites are observed for erythema, edema and necrosis at 24, 48 and 72 hours after injection. The average skin reactions are scored using a scoring system ranging from 0 (no erythema/edema) to 4 (severe erythema/edema). After the last scoring is done, all erythema and edema scores for the sample and blank sites are totaled separately and divided by 12 to obtain a mean score. The requirements of the test are met if the difference between the sample and blank mean score is 1.0 or less. A repeat test using three additional rabbits is done if the average reaction of the sample is much greater than the blank. The requirements of the three additional animals must be met as mentioned above.
The Implantation Test is for determining the reaction of plastic and other polymeric materials in direct contact with living tissue. Eight strips of 10 x 1 mm samples strips and 4 strips of USP High-density Polyethylene Reference Standard as a control are aseptically implanted into rabbits using a hypodermic needle. Four strips are implanted into the paravertebral muscle on one side of the spine of each of 2 rabbits, 2.5 to 5 cm from the midline and parallel to the spinal column and 2.5 cm2. The USP high density polyethylene RS is implanted on the other muscle using two strips. The implantation period is for no less than 120 hours. The tissue is examined macroscopically using a magnifying glass for necrosis, hemorrhage, discoloration and infections. The sites are also evaluted for encapsulation of the samples. The differences between average scores from the sample and control sites is calculated. The test requirements are met if 1) the difference between the sample and controls does not exceed 1.0 and 2) if the difference between the sample and control mean scores for more than one of the four implant sites does not exceed 1 for any implanted animal. A board certified pathologist will generally read the processed tissue slides to confirm the macroscopic observation.
Choosing a Testing Facility For Biocompatibility Testing
The first step on the road to outsourcing is determining what tests are required for the product to meet regulatory requirements. The next step is to choose the proper testing facility to carry out the testing. What does the manufacturer need to know when choosing a testing facility to perform these toxicology tests? Questions which should be asked are:
1) Does the laboratory perform the test and what are their credentials and expertise in this area?
2) Do they routinely test pharmaceutical drug containers, closures and medical devices?
3) Is the laboratory routinely inspected by the FDA and USDA?
4) Have they received any FDA 483s or warning letters?
5) Do they have an in vitro cell culture lab?
6) Do they have well maintained and monitored animal facilities and is there an animal care committee and veterinarian to insure the proper care and handling of the animals?
7) Can they handle large amounts of samples if required?
8) Can the testing facility meet the required timeline for testing?
9) Do they have a good working knowledge of the regulatory requirements for this type of testing?
10) Can work be scheduled easily and quickly?
11) Do they have an internal Quality Assurance Unit to evaluate the progress of the placed study?
12) Are they customer service oriented?
13) Committed to on-time delivery of testing?
14) Are client audits allowed?
Once all the above questions have been answered to the satisfaction of the manufacturer/packager, samples can be submitted to the appropriate contact in the test facility and testing initiated. It is clear that pharmaceutical biosafety testing encompasses a large variety of biological testing which was not in the scope of this article. It can be a difficult decision for the manufacturer to determine the testing that is required and where to outsource the work.