Staci Horme

Why Your Products Need APE Testing? An antimicrobial or preservative is defined as a chemical substance that will kill microorganisms or inhibit the growth of microorganisms, usually added to nonsterile products to protect them from microbiological contamination. If your product will be on the market, it is advised that it has been validated to be free from microbiological contaminants through Antimicrobial Preservative Effectiveness (APE) Testing. Antimicrobial Preservative Effectiveness (APE) Testing , also know as Antimicrobial Effectiveness Testing (AET) or Preservative Efficacy Testing (PET) is a microbial challenge methodology performed to evaluate if the chosen preservative is appropriate for a product formulation. This test measures the effectiveness of antimicrobial preservatives to inhibit the growth of microorganisms during the manufacturing process or product use. Antimicrobial effectiveness must be illustrated on all injections packaged in multiple-dose containers, multiple-dose topical and oral forms, and other dosage forms that contain antimicrobial preservatives. This test should be performed on all aqueous-based products including ophthalmic, otic, nasal, oral, irrigation, and dialysis fluids. How To Perform APE Testing? Both the EP 5.1.3 and the USP <51> give out guidance for conducting validation of the Recovery Method to determine the ability of the routine testing procedure to identify challenge microorganisms in the presence of a product. The potential of the media used in this process is accepted to promote microbial growth if the product appears antimicrobial properties. To evaluate the antimicrobial effectiveness of a product, the product is inoculated with a prescribed quantity of specified microorganisms. The common five Antimicrobial Preservative Efficacy test organisms are Candida Albicans, Staphylococcus Aureus, Escherichia Coli, Pseudomonas aeruginosa, and Aspergillus brasiliensis. The effectiveness of the preservative is evaluated by comparing the initial level of microorganisms to the test material at various time intervals over a period of 28 days at a specified temperature. The reduction of microorganisms is calculated logarithmically. STEMart offers multiple methods for Antimicrobial Preservative Effectiveness Testing following the USP General Chapter 51 on Antimicrobial Effectiveness Testing, as well as European Pharmacopoeia and other international standards. Our scientific testing will provide you with reliable and replicate testing results to help you determine whether or not your product is effective in this area, and you can submit data that is required by the appropriate regulatory agency. If you have any questions about Antimicrobial Preservative Effectiveness Tests or would like to find out more about our services, please feel free to contact us.

What is Antibiotic Potency Test? A microbiological potency assay measures the bioactivity or potency of an antibiotic by the degree of growth repression on susceptible microorganisms at different concentrations. All antibiotic products must go through potency testing prior to market release. Both the EP and the USP require testing of antibiotic potency using microbiological assays for some products. Compared with measuring the potency of an antibiotic chemically, assessing the potency of an antibiotic microbiologically verify the ability of the antibiotic to destroy the selected organisms and the concentration at which the antibiotic will work effectively. And one major advantage of microbiological potency testing is that it can measure the effectiveness of the antibiotic directly on a living microorganism. Testing Methods Typically, antibiotic products have their potency determined by comparison with a standard substance or a biological reference substance. Such potency is expressed as the ratio of the dose that inhibits the growth of a sensitive microorganism, relative to the standard substance dose that produces similar inhibition. In the pharmacopeia, there are two methods defined to perform a microbiological potency assay: the Diffusion (Cylinder Plate) Method and the Turbidimetric Method. Diffusion (Cylinder-Plate) method The Diffusion (Cylinder-Plate) method is constructed on the diffusion of the antibiotic throughout solid culture media. A metal cylinder or paper disk containing the antibiotic is placed on an agar media with a target microorganism. This test can be performed at multi-levels of concentrations to determine the minimum effective concentration. After incubation, the growth of the target organism is measured, the growth of the target organism will be inhibited by an antibiotic. Turbidimetric Method The turbidimetric method is constructed upon the effectiveness of the antibiotic to inhibit the growth of a target microorganism. Adding a series known concentrations of antibiotics into a liquid culture media which contains a target microorganism. Subsequently, incubating and measuring the turbidity of the mixture solution by spectrophotometric method. The turbidity of the solution positively correlates with microbial growth and therefore, it's a measure of the effectiveness of the antibiotic. STEMart offers Antibiotic Potency Tests following the Antibiotics-Microbial Assays in USP General Chapter 81. Our scientific and reliable testing with strict control can provide you with a high level of confidence in the testing results. If you have additional questions about Antibiotic Potency Tests or would like to find out more about our services, please feel free to contact us. Reference  "Pharmaceutical Microbiology Manual", FDA, 2014.

Biocompatibility evaluation of breathing gas pathways in medical devices is a powerful tool for the evaluation of contaminants from the air emitted from the device for Volatile Organic Compounds (VOCs) and particulate matter when the gas pathway is intended to contact a patients’ respiratory tract. Standard for Biocompatibility Evaluation of Breathing Gas Pathways in Medical Device "Chemical Characterization of medical device materials", part eighteen of the Biological evaluation of medical device standards(ISO 10993-18), gives out the general guidance for E/L testing including polar, semi-polar, and nonpolar solvents measure leaching of organic compounds. It also outlines that VOCs can be determined either by an analysis of aqueous extracts or direct analysis of test articles by heated headspace. ISO 18562, the standard for biocompatibility evaluation of material or medical device with a breathing gas pathway, presents instructions for E/L extraction with purified water to simulate humidified vapor condensate, VOC analysis for VOCs emitted in a gas stream under simulated use conditions(temperature, flow rate, duration) with GC/MS method, and particulates analysis for emissions of PM2.5 and PM10. It’s noteworthy that the flow rate and temperature for sampling chosen should be clinically relevant. Our Testing of Breathing Gas Pathways Services Medical devices with breathing gas pathways covered by our service as follows: Anesthesia workstations Ventilators Breathing system Oxygen conservation equipment Oxygen concentrators Mouthpieces Nebulisers Incubators Resuscitators Low-pressure hoses Breathing tubes Humidifiers Heat and moisture exchanges Breathing system filters Respiratory gas monitors Masks Y-pieces Accessories Particulate matter measurement for those emitted from the medical device. Flowing clean and dried air through the test material and collecting particulates matter from the output. Calculating the particulate matter levels of PM2.5 and PM10 and recording the minimum, maximum, and average particulate concentration. VOC analysis with Gas Chromatography Mass Spectrometry (GC/MS). We have two options for collecting and sampling VOCs: Thermal Desorption Tubes/Canisters Method and Stainless Steel SUMMA Canister Method. VOCs are collected by leading clean and dry air through the test material in a controlled environment. Subsequently, sampling particulates matter from the output. Gas samples are analyzed for VOCs by GC/MS. The concentration of VOCs in µg/m3 is reported. CO, CO2, and Ozone may also be analyzed. Leachables analysis in condensate with Gas Chromatography Mass Spectrometry (GC/MS) and Inductively Coupled Plasma (ICP) method. After collection of leachables, we provide determination of the metal ion concentrations with pharmacopeia methods, and identification and quantification of organic impurities with GC/MS. Interpretation of inhalation toxicity data relevant to breathing gas pathways for the medical device. STEMart offers biocompatibility evaluation of material or medical device with a breathing gas pathway utilized modern analytical techniques and instrumentation, following the biocompatibility guidelines modified for medical devices and using a solid scientific rationale. If you have additional questions about the Testing of Breathing Gas Pathways Services or would like to find out more about our services, please feel free to contact us.

The biocompatibility systemic toxicity tests are in vivo systemic tests used to evaluate the impairment or activation of a system, rather than the impairment of individual cells or organs. Acute systemic toxicity tests and pyrogenicity tests are included in the systemic toxicity tests. Acute Systemic Toxicity Testing Acute toxicity is considered to be the adverse effects that occur after administration of a single dose or repeated doses of a test article given within 24 hours. Acute systemic toxicity test determines the systemic toxic effect followed by a single acute exposure of medical biomaterials to the body. The acute systemic test can provide extensive instruction on health hazards which may be induced by acute exposure from a medical device or chemical material. The animals are administrated with test material extracts or the liquid chemical; subsequently, observed at 24 ± 2, 48 ± 2, and 72 ± 2 hours for signs of toxicity. Pyrogenicity Testing Pyrogens are some chemical or bacterial-sources compounds which can cause a fever when present in the body at sufficiently high doses. Pyrogenicity test is used to detect the material-mediated fever-causing reactions caused by extracts of medical devices or chemical materials. The material-mediated pyrogen evaluates the potential of a chemical substance in/on a medical device to cause a pyrogenic response. Remarkably, no single test can differentiate pyrogenic reactions material-mediated from those caused by endotoxin contamination. The animals are dosed with the test article extract and observed for 3 hours. Standard for Acute Systemic Toxicity and Pyrogenicity "Tests for systemic toxicity", part eleven of the Biological evaluation of medical devices standards(ISO 10993-11), gives out the general considerations that should be taken into account when evaluating the potential of inducing acute systemic toxicity and pyrogenicity of a medical device. According to the study procedures mentioned in ISO 10993-11 for the assessing of the potential of medical devices and their constituent materials to induce systemic toxicity, STEMart offers the following systemic toxicity testing service: Test Capabilities For Acute Systemic Toxicity Test Test Methods 1) Acute oral toxicity test; 2) Acute dermal toxicity test; 3) Acute inhalation toxicity test; 4) Acute intravenous toxicity test; 5) Acute intraperitoneal toxicity test. Test Animals Mice, rats, or rabbits Test Items 1) Visual observations of animals; 2) Routine hematology and serum chemistry tests with blood samples; 3) Microscopic examination with representative tissue samples. For Pyrogenicity Test Test Method Limulus amebocyte lysate (LAL) reagent test STEMart offers Acute System Toxicity and Pyrogenicity testing following the biocompatibility guidelines modified for medical devices. If you have additional questions about systemic toxicity tests or would like to find out more about our services, please feel free to contact us.   Reference Wallin, Richard F., et al. "A Practical Guide to IS0 10993-11: Systemic Effects." MEDICAL DEVICE AND DIAGNOSTIC INDUSTRY 20: 98-101 (1998).

The Biocompatibility Subacute and Subchronic Toxicity test is used to evaluate the toxicity effects likely to arise from repeated exposures would have on a patient, including any compound toxicity effects. This test determines the systemic effect of repeated doses of materials or their extracts for no less than 24 hours and no greater than 10% of the total lifespan of the test animal. Subacute toxicity (repeat-dose toxicity) focuses on adverse effects occurring after a single or repeated exposures to a test material per day during a period of 14 to 28 days. Subchronic toxicity indicates the adverse effects of a substance resulting from repeated exposure to a toxic agent over a period of several weeks or months. The subacute and subchronic toxicity studies include full clinical pathology (clinical chemistry, hematology, and coagulation), necropsy and organ weights, as well as histopathology. These tests can either be conducted with full histopathology or limited tissue evaluation. Standard for Subacute & Subchronic Toxicity Testing "Tests for systemic toxicity", part eleven of the Biological evaluation of medical devices standards(ISO 10993-11), gives out the general considerations that should be taken into account when evaluating the potential of inducing subacute/ subchronic toxicity of a medical device. It outlines the procedure of studies to assess the potential of medical devices and their constituent materials to induce systemic toxicity. Fig. 1 Subacute & subchronic toxicity tests during drug development. (Denny, 2017) The usual animals of choice for the subacute and subchronic toxicity tests are mice, rats, or rabbits, and oral, dermal, inhalation, intravenous, intraperitoneal, or subcutaneous application of the test substance may be used based on the considered application of the biomaterial. General Procedures of Subacute & Subchronic Toxicity Testing Administering mice or rats with a dose of 0.9% normal saline or cottonseed extract of the test biomaterial over a 14-day period; Observing the test animals once daily for signs of toxicity; Recording animal weights on Day 0, Day 7, and Day 14; On day 14, collecting blood samples for hematology and clinical chemistry analysis; Conducting a gross necropsy and collecting lesions. STEMart offers biocompatibility Subacute & Subchronic Toxicity testing following the biocompatibility guidelines modified for medical devices. The testing is designed and performed based on the route of exposure to the body including the oral, dermal, and inhalation exposures. If you have additional questions about Subacute & Subchronic Toxicity testing or would like to find out more about our services, please feel free to contact us.   Reference Denny, Kevin H., et al. "Acute, subacute, subchronic, and chronic general toxicity testing for preclinical drug development." A Comprehensive Guide to Toxicology in Nonclinical Drug Development 109-127(2017).

Cytotoxicity testing is a sensitive method to assess the biocompatibility of a material extracted through a specific cell culture media following exposure of the extracted fluid to L929 cells. This test is performed on raw materials in the manufacturing process, all medical devices contacting patients, and devices undergoing a sterilization validation. STEMart provides a wide range of in vitro cytotoxicity assays to qualitatively and quantitatively evaluate the ability of medical device to damage cells or tissues. Qualitative Methods of Cytotoxicity Testing Fig. 1 Qualitative cytotoxicity test with MEM elution. Extraction Method/ MEM Elution In Minimum Essential Medium (MEM), extract the leachable of a medical device for 24 hours. An extract is then placed over the L929 cells. The cells are examined microscopically for morphological changes, degeneration, and lysis of the cells following incubation. Agar Diffusion/ Agarose Overlay Assay In this method, a thin layer of nutrient-supplemented agar is placed over the L929 cells. The test material is placed on top of the agar layer, and the cells are incubated for 24 hours. Cytotoxic leachable diffuses into the cell layer via the agar layer, and a zone of malformed, degenerative, or lysed cells will be used to assess cytotoxicity. Direct Contact The test material is placed directly onto cells growing on culture medium without the agar layer, cell cultures are grown to a standard monolayer, the cells are then incubated for 24 hours at 37 ?. During the incubation, leachable chemicals in the test material can diffuse into the culture medium and contact the cell layer. Examining the monolayers for the presence of morphological changes, reduction in cell density, or lysis of cells to indicate the cytotoxicity. Fig. 2 Qualitative cytotoxicity test with direct contact method. Quantitative Methods of Cytotoxicity Testing MTT Assay MTT assay is used often in determining cell viability (detects viable cells). The MTT can be used to evaluate the cytotoxicity of toxic compounds and toxins and environmental pollutants. Calcein Assay The calcein-AM is used in the testing of cell viability as it can pass through the cellular membrane into live cells. After being transported into the cells, the acetomethoxy group is removed from intracellular esterases, the molecule gets trapped inside and gives out strong green fluorescence. Only live cells are labeled and counted by flow cytometry as dead cells lack active esterases. Neutral Red (NR) Assay The neutral red (NR) assay is a cell survival chemosensitivity assay. This assay is based on the incorporation of NR into the lysosomes of viable cells after being incubated with test agents. When a reduced cytotoxic effect was determined by the NR-test, which shows the activity of lysosomal enzymes, microscopically multiplication or enlargement of lysosomes was observed. Lactate Dehydrogenase (LDH) Leakage Assay Lactate dehydrogenase is a soluble cytosolic enzyme present in most eukaryotic cells, and it will release into culture medium upon cell death due to damage to the plasma membrane. The increase of the LDH activity in culture supernatant is proportional to the member of lysed cells. Subsequently, LDH activity can indicate cell membrane integrity and assess cytotoxicity resulting from chemical compounds or environmental toxic factors. STEMart offers medical device cytotoxicity testing service to advance your medical device development and facilitate the manufacturers submitting their medical device for regulatory approval. If you have additional questions about cytotoxicity or would like to find out more about our services, please feel free to contact us.    

Chemical Characterization is a powerful methodology for addressing biocompatibility endpoints, providing a risk assessment of biocompatibility using chemical, biocompatibility, and patient exposure data. Chemical Characterization involves the extraction of the device with various solvents, chemical analysis of compounds extracted from the device, and an evaluation of the toxicological risk associated with the patient exposure to the extracted compounds. Standard for Chemical Characterization "Chemical Characterization of materials", part eighteen of the Biological evaluation of medical devices standards (ISO 10993-18), gives out the general considerations for the analytical approach including the intended use of device contact time (blood, mucosal, skin contact), material composition, and surface area calculations. It also outlines for the extractables, the study design, solvents, temperature, and duration should be taken into account; while for the leachables, the drug formulation, storage, and handling of drug and delivery devices should be taken into account. STEMart offers Chemical Characterization service utilized modern analytical techniques and instrumentation, following the biocompatibility guidelines modified for medical devices and using a solid scientific rationale. The testing is designed and performed based on the route of exposure to the body including the skin, mucosal, or ocular. Our Chemical Characterization Services Extractables & Leachables STEMart offers Extractable & Leachables testing service utilized modern analytical techniques and instrumentation, following the ISO 10993 standard for medical devices. Medical device manufacturers can use it to show that materials of concern do not migrate from the device into the patient. Degradation Products According to ISO 10993 standard, STEMart provides comprehensive degradation product testing based on the material chemistry and end-use biological environment for polymeric, ceramic and metallic medical devices. Residual Ethylene Oxide According to ISO 10993 standard, STEMart offers residual ethylene oxide testing service to identify and quantify the content of ethylene oxide, ethylene chlorohydrin, and ethylene glycol in medical device with gas chromatography techniques. Raw Material Testing STEMart provides raw material identification and characterization services to the medical device to help manufacturer select their suppliers, avoid costly production problems and delays, and ensure consistence of their products. Techniques for Chemical Characterization Gas Chromatography (GC) for volatile organics GC-Mass Spectrometry (MS) for analyte identification of solvents, plasticizers, and degradation products High Performance Liquid Chromatography (HPLC) with MS detection for non-volatile/ semi-volatile analyte identification Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for elemental analysis (i.e., heavy metals and other process impurities) HPLC with laser light scattering or LC/MS can monitor the polymers/lubricants from the device or manufacturing process. STEMart provides comprehensive chemical characterization service to help medical device manufacturer to evaluate hazards related to device and manufacture process, support process control and prove the equivalence of the proposed materials. If you have additional questions about Chemical Characterization or would like to find out more about our services, please feel free to contact us.

Sensitization testing is used to assess the potential of chemicals and medical devices to cause a delayed hypersensitivity reaction following a single or repeated exposure to the body. Sensitization is one of the three most common biocompatibility tests required to ensure the safety of medical devices. "Tests for irritation and skin sensitization", part ten of the Biological evaluation of medical devices standards (ISO 10993-10), gives out the general considerations that should be taken into account when evaluating the potential of inducing sensitizing of a medical device. It outlines the procedure of studies to assess the potential of medical devices and their constituent materials to induce irritation and skin sensitization. General Procedures of the Sensitization Test Exposing the animals to the test materials or extracts across multiple induction phases and a single challenge phase; Observing the challenge sites for about 24 and 48 hours after the removal of the test materials or extracts and evaluating skin sensitization reactions. Sensitization reactions are noted by observing erythema and/or edema as it interacts with the body's immune system. STEMart offers several methods as in vivo and in vitro assays for sensitization biocompatibility testing following the biocompatibility guidelines modified for medical devices. The testing is designed and performed based on the route of exposure to the body and acceptance of the notified body. Sensitization Testing Capabilities Two types of tests have been developed: an adjuvant test in which sensitization is potentiated by the injection of Freund's Complete Adjuvant (FCA), and non-adjuvant tests. Local Lymph Node Assay (LLNA) LLNA studies the induction period of skin sensitization and delivers quantitative data for dose-response assessment. LLNA is an in vivo method that will not eliminate the use of animals but have the potential to reduce the number of animals required for this purpose and cause less pain and distress. LLNA does not require that challenge-induced dermal hypersensitivity reactions be elicited or the use of an adjuvant.   Fig. 1 The local lymph node assay method. (Gerberick, 2000) Guinea Pig Maximization Test (GPMT) The GPMT is an in vivo test to screen for substances that cause human skin sensitization. The test animals are put together with an adjuvant, exposed intradermally to the test articles to enhance the immune reaction.   Buehler Assay (BA) The Buehler Assay is a non-adjuvant in vivo test to screen for substances that cause human skin sensitization (i.e. allergens). In the test, Guinea pigs are exposed to a high dose of the substance, followed by a challenge dose, which is the highest dose that does not cause irritation.   In Vitro Sensitization We offer three-tiered in vitro skin sensitization tests: Direct Peptide Reactivity Assay (DPRA); ARE-Nrf2 Luciferase Test (KeratinoSens); Human Cell Line Activation Test (hCLAT)   If you have additional questions about Sensitization Testing or would like to find out more about our services, please feel free to contact us. Reference Gerberick, G. Frank, et al. "Local lymph node assay: validation assessment for regulatory purposes." American Journal of Contact Dermatitis 11.1 (2000): 3-18.

Irritation represents local tissue inflammation response to chemicals, without a systemic immunological component, and irritation is characterized by inflammation, redness, swelling, heat, or pain. Irritation testing is used to assess the potential of a medical device to cause an immediate irritation reaction in the skin, mucosal, or ocular tissues following a single or repeated exposure to the body. Irritation is one of the three most common biocompatibility tests required to ensure the safety of medical devices. Standard for Irritation Testing Irritation testing is instructed by FDA regulations and international standards. “Assessing the Irritation and Sensitization Potential of Transdermal and Topical Delivery Systems for ANDAs”, introduced by the FDA as guidance for industry, provides guidance for the design and carry out of studies to assess the potential of in vivo skin irritation and sensitization induced by a medical device. “Tests for irritation and skin sensitization”, part ten of the Biological evaluation of medical devices standards (ISO 10993-10), gives out the general considerations that should be taken into account when evaluating the potential of inducing irritation of a medical device. It outlines the procedure of studies to assess the potential of medical devices and their constituent materials to induce irritation and skin sensitization. STEMart offers several methods as in vivo and in vitro assays for irritation biocompatibility testing following the biocompatibility guidelines modified for medical devices. The testing is designed and performed based on the route of exposure to the body including the skin, mucosal, or ocular. Irritation Testing Capabilities Topical Irritation Testing In the Topical Irritation Testing, the erythema and edema (redness and swelling) on the injection site are assessed after injection of extracts of the test material. It is suitable for the detection of the potential for local irritation caused by chemicals extracted from a biomaterial. Skin Irritation Testing Since the skin irritation is always caused by chemical-induced cell damage and subsequent inflammatory cascade, our skin irritation testing measures cell viability in reconstructed human epidermis (RhE) tissues after a single chemical exposure. Fig. 1 Procedures of skin irritation test (SIT). (Lin, 2019) Ocular Irritation Testing We provide several in vitro test methods for the identification of ocular irritation to satisfy the specific medical device requirements, including bovine corneal opacity and permeability (BCOP), the isolated chicken eye (ICE) test methods, Cytosensor microphysiometer (CM) test method, chorioallantoic membrane (HET-CAM) test method and isolated rabbit eye (IRE) test method. In vivo test is also provided for weak irritants. Oral Irritation Vaginal Irritation Nasal Irritation Penile Irritation Rectal Irritation Intracutaneous Reactivity Testing In the Intracutaneous Reactivity Testing, the erythema and edema (redness and swelling) on the injection site are assessed after injection of extracts of the test material. It is suitable for the detection of the potential for local irritation caused by chemicals extracted from a medical device. If you have additional questions about Irritation Testing or would like to find out more about our services, please feel free to contact us. Reference Lin, Yu-Chun, et al. “Testing Method Development and Validation for in Vitro Skin Irritation Testing (SIT) by Using Reconstructed Human Epidermis (RhE) Skin Equivalent-EPiTRI®.” Alternatives to Animal Testing 8-19 (2019).