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Endotoxin Limits

How are endotoxin limits calculated? How do my endotoxin test results relate to those limits?
 
Endotoxin limits for drug products specify the amount of endotoxin that can safely be present. Limits are based on human tolerance and generally specified in the USP monograph or if no monograph exists, calculated via USP <85> Bacterial Endotoxin Test. The testing limit calculation considers:
Route of administration
Dose of the product per kilogram of body weight (which should include the maximum bolus dose and the minimum patient weight)
Duration (time) of administration
Once a limit is determined, USP <85> allows a testing facility flexibility to determine the appropriate test dilution to reduce drug sample properties that interfere with the test. Using the endotoxin limit and the assay's sensitivity, a maximum valid dilution (MVD) is calculated. The MVD provides the maximum dilution possible to obtain a result and maintain the sensitivity necessary to determine a pass or fail. For example, if a sample is submitted with an endotoxin test limit of NMT 14 EU/mL and the MVD is determined to be 1400, then the product concentration can be diluted no further than 1:1400 and still generate a result with the appropriate sensitivity.
 
The test result is calculated by the assay software using the test dilution and assay sensitivity. In a case where no endotoxins are present, for example, a 1:1000 dilution was used and no presence of endotoxin was detected, a result of <10 EU/mL is reported. A result which includes a "less than" value does not indicate that the actual endotoxin value is near the number reported, only that the dilution factor used allowed for that level of sensitivity in results calculation. This means that results of <10 EU/mL and <4 EU/mL both show no presence of endotoxin at the dilution with which they were tested; and neither result offers any indication of the exact level of endotoxin present below the values reported.
 
If you would like to always receive the same result when endotoxins are not detected, a validation of the endotoxin test method is required.
 
Contact ARL at info@arlok.com or 800-393-1595 to schedule your endotoxin test method validation.

Hand Sanitizer Guidance...

Hand sanitizers are regulated as over the counter (non-prescription) drugs by the U.S. Food and Drug Administration. The FDA has issued warnings to consumers about hand sanitizer products containing methanol. According to the CDC, Alcohol-based hand sanitizers should only contain ethanol or isopropanol, but some products have been found to contain methanol. Ethanol is a valid active ingredient, but methanol, even in small amounts, can lead to patient adverse events.

USP Hand Sanitizer Tool Kit

Have you downloaded USP's Hand Sanitizer Tool Kit? September 1, 2020, is the official date for USP Alcohol and Dehydrated Alcohol monographs. These monographs are revised to include the Limit of Methanol test in the Identification (ID) section. https://www.usp.org/covid-19/hand-sanitizer-information

 

 USP Hand Sanitizer Tool Kit

Register for the Rapid Sterility Strategies Webinar

Register today for the Rapid Sterility Strategies Webinar! Discover real-world applications for rapid sterility testing for compounded sterile preparations in the hospital pharmacy. Moses H. Cone Memorial Hospital will provide a case study on applying rapid sterility testing in sterile compounding. https://bit.ly/RapidStrategies

What Factors Influence Stability?

 

Stability measures the extent to which a product retains, within specified limits, and throughout its period of storage and use, the same properties and characteristics it possessed at the time of compounding. According to USP <1191>, pharmacists should establish and maintain compounding conditions that ensure drug stability to help prevent therapeutic failure and adverse responses.

FDA Includes Methanol Testing...

Today, FDA issued updated guidances to provide additional clarification on testing of alcohol used in hand sanitizers manufactured under FDA’s temporary policies to help ensure that harmful levels of methanol are not present in these products. This will help ensure widespread access to alcohol-based hand sanitizers that are free of contamination.

FDA has updated their guidances to provide clarification that companies test each lot of the active ingredient (ethanol or isopropyl alcohol (IPA)) for methanol if the ethanol or IPA is obtained from another source. FDA recommends using the test methods described in the USP monograph for alcohol (ethanol) and conducting the testing in a laboratory that has been previously inspected by FDA and is compliant with current good manufacturing practice (CGMP).

Additionally, any alcohol (ethanol) or IPA found to contain more than 630 ppm methanol does not fall within the policies described in the temporary guidances and may be considered evidence of substitution and/or contamination. Alcohol-based hand sanitizers that are contaminated with methanol are subject to adulteration charges under the FD&C Act. The alcohol (ethanol) or IPA should be destroyed following guidelines for hazardous waste and the manufacturer or compounder should contact FDA regarding the test results and the alcohol’s source.

Expiration Dates and Retesting...

Pharmaceutical ingredients are controlled by cGMP regulations. Users of pharmaceutical materials should be informed regarding when the material can be re-tested for continued use.

ICH Q1A(R2)/FDA Definition:

Expiration date: The date placed on the container/labels of an API designating the time during which the API is expected to remain within established shelf-life specifications if stored under defined conditions and after which it should not be used.

Retest date: The date when a material should be reexamined to ensure that it is still suitable for use.

Retest period: The period of time during which the drug substance is expected to remain within its specification and, therefore, can be used in the manufacture of a given drug product, provided that the drug substance has been stored under the defined conditions. After this period, a batch of drug substance destined for use in the manufacture of a drug product should be retested for compliance with the specification and then used immediately. A batch of drug substance can be retested multiple times and a different portion of the batch used after each retest, as long as it continues to comply with the specification. For biotechnological/biological substances known to be labile, it is more appropriate to establish a shelf-life than a retest period. The same may be true for certain antibiotics.

USP <85> Bacterial Endotoxin Validation

ARL Bio Pharma utilizes the kinetic turbidimetric method described in USP <85> Bacterial Endotoxins Test to provide results. An endotoxin test detects toxins that are released from the cell wall of disrupted gram-negative bacteria. This is a reaction over time test that quantitates the amount of endotoxin in a sample compared to a standard curve. This article discusses USP <85> challenges and why method validation is important to provide consistent results.

The most common challenge encountered while performing endotoxin testing is that many drug products cause the assay reaction to slow down (inhibition) or speed up (enhancement) during the testing process, which does not allow for an accurate result to be obtained. Those properties which cause the inhibition or enhancement must be overcome to obtain a reliable and repeatable test result. Microbiologists performing testing must determine the degree in which inhibition or enhancement is occurring. This is done by adding known amounts of endotoxin to test samples and comparing the results to those known values. If there is no inhibition or enhancement, results from the test samples spiked with a known amount of endotoxin will be the same as the expected results. Inhibition would artificially lower results for the test sample, and enhancement would increase results for the test sample, relative to the expected spike concentration. 

Once inhibition or enhancement is understood, microbiologists can develop a plan of action to overcome those sample properties.

Growth Promotion Testing

Growth Promotion demonstrates that media used in environmental monitoring programs, media fills, or personnel qualification are capable of supporting microorganism growth. 

Benefits of growth promotion testing:  

  • Reduces risk of false negative results if media does not support growth
  • Reduces risk of false positive results if media is contaminated (negative control sample required)
  • Ensures media used in environmental monitoring provides an accurate representation of environmental control in compounding areas
  • Meets FDA and USP microbiological growth media requirements
  • Verifies media vendor's certificate of analysis

ARL has a new submission form specific for "Growth Promotion" tests. This new form is a quick selection form for media types, organism tests, and incubation time and temperature.

Container-Closure Systems...

The container closure system for a drug product provides critical protection for stability and sterility. It consists of all components intended to seal and protect the package/delivery system and its contents. Container-closure integrity (CCI) is the ability of the system to keep contents in, and to keep harmful environmental contaminants out.

CCI testing (CCIT) detects leaks in the container closure system. A passing test result indicates the packaging system can maintain sterility and relevant physicochemical specifications. Three types of leaks that can be detected during CCIT include:

  • entry of microorganisms
  • escape of the product dosage form or entry of liquids or solids
  • escape of nitrogen gas or entry of oxygen, water vapor, or air gases

In February 2008, the Food and Drug Administration (FDA) introduced CCIT in lieu of sterility testing as a component of a stability written testing program for sterile products. A stability study measures the extent to which a product retains, within specified limits, and throughout its period of storage and use, the same properties and characteristics it possessed at the time of compounding. Sterility is a stability characteristic that should be conducted throughout a stability study to confirm a product's shelf life.