Sterilization of health care products - Microbiological methods - Part 3: Bacterial endotoxin testing (ISO 11737-3: 2023).
Tests not accredited in our laboratory.
Tests with the Certificate of Good Laboratory Practices (GLPs).
The ISO 11737-3:2023 Standard specifies the general criteria to be applied in the determination of bacterial endotoxins on or in health care products, components or raw materials using bacterial endotoxins test methods using amebocytes lysate reagents. In our laboratory, we perform the chromogenic end point method that is based on the linear relationship between endotoxin concentration and formation of a colour (chromogenic) measured by optical density at a given wavelength, which is assessed over a standard curve. The endotoxin level in an unknown is calculated by measuring the optical density of the sample and interpolating the endotoxin concentration from the standard curve.
According to the ISO 11737-3 there are two types of sampling plans: batch testing and alternatives to batch testing. For batch testing, non-pyrogenicity is confirmed through the testing of end-products in each production batch. Alternatives to batch testing may be used if it has been demonstrated that the manufacturing process and materials are suitably controlled.
For batch testing, the number of product units chosen for routine testing is dependent on the size of the defined batch, level of control, statistical considerations, and historical performance. In most cases, each batch of a product shall be tested using an appropriate number of samples, no more than 10, taken at random to represent the quality of the batch. A sampling plan based on the batch size has been commonly used and is generally considered to be acceptable: 2 samples for a batch size lower than 30 units, 3 samples for a batch size between 30 to 100 units, and 3% of the batch up to maximum of 10 for a batch size higher than 101 units. Alternate sampling plans that use small sample sizes or that do not test each batch of product shall be clearly defined and supported/justified by a risk assessment. On the other hand, other sampling plans requiring a greater number of samples, can be necessary for validation or investigational purposes. If sampling plans are used that require selecting more than 10 samples from a batch, no more than 10 samples should be pooled within a test.
Regarding the selection of samples, it must be taken into account that samples selected for testing shall include all factors that can affect or contribute to the levels of endotoxin. All parts of the health care products shall be included in the testing process, and the exclusion of any part of the product shall be justified and documented. For example, if a health care product has portions of the product that are sealed and as such do not come into contact with the patient, and such portions may be excluded from endotoxin testing. Moreover, for products for which a claim of non-pyrogenicity applies only to a portion of the product, endotoxin testing does not apply to the portions of product not intended to be non-pyrogenic. Besides, regarding the testing of multi-component kits (procedure packs) or sets of individual products within the same sterile barrier system, depending upon how the product is used, there are instances where each component may be evaluated individually and other instances where the entire contents may be considered as a single entity. It should also be taken into account that samples may be obtained prior to sterilization (pre-sterilization) or after sterilization (post- sterilization). Post-sterilization samples encompass all the factors that can affect the product or the endotoxin test and of pre-sterilization samples are selected for testing, the acceptability of the samples in representing the endotoxin level on sterilized product shall be justified and documented.
Depending on the type of the product, the analysis of an extract of the medical device may be necessary. Solid health care products must be analysed by extraction and can be tested either individually or pooled (no more than 10 samples should be pooled within a test). The medical devices may be cut or disassembled for the extraction. On the other hand, aqueous health care products, biological components in aqueous form, or other aqueous samples may be tested without extraction. Powders, gels, and pastes shall be in suitable form for testing and may require extraction for analysis if they do not dissolve correctly and homogeneously in water. For products that need extraction, the maximum volume of extraction fluid (MVD) that may be used for the pooled extract for a given endotoxin limit must be calculated. Nevertheless, the volume of extraction fluid used to extract the product units may be adjusted to facilitate extraction, depending on the size of the device, without exceeding the calculated MVD. In our laboratory we follow the ISO 10993-12 standard to calculate the fluid extraction volume taking into account the surface or mass of the test product. To calculate the MVD the client must specify the endotoxin limit (EU/device) established by the competent authorities.
The limit set by the US Pharmacopeia (USP) <161> and the FDA (Food and Drug Administration) is 20 EU/device in contact with the circulatory system or lymphatic system (200 EU in pools extracts of 10 devices), and 2.15 EU/device for devices in contact with cerebrospinal fluid. The FDA also includes the limits per mL of extract, using 40 mL of extraction fluid, stating the limit in 0.5 EU/mL for products that directly or indirectly contact the cardiovascular system and lymphatic system, and 0.06 EU/mL for devices in contact with cerebrospinal fluid. For other types of medical devices, endotoxin limits higher than 20 EU/device can be justified and can require regulatory acceptance. For example, a limit of 35 EU/device or higher can be appropriate for medical devices without systemic exposure such as subcutaneous implantable health care products. The FDA also states that that for devices that are in direct or indirect contact with the intraocular environment, a lower endotoxins limit may apply.