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What does compliance of a product with USP-NF standards entail? |
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What is the role of USP-NF standards, in terms of U.S. law (i.e., the Federal Food, Drug, and Cosmetic Act, FDCA)? |
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How much can I modify a chromatographic procedure and still be in compliance? Can column length, internal diameter, mobile phase composition be modified? |
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What brand of HPLC/GC column was used in the development and/or validation of a particular test? Is there an alternative chromatographic column for a particular test? |
| |
How can I obtain copies of monographs or general chapters proposed in Pharmacopeial Forum, or monographs or general chapters that have appeared in an edition of the USP-NF? |
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How do I convert the potency of a vitamin expressed in International Units (I.U.) to g or mg (and vice versa)? |
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How can I verify that the balances in our laboratory are suitable for weighing the amounts specified in a USP monograph? |
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How does a plastic material receive a "Class VI" classification? |
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How are microbial limits and objectionable microorganisms determined? |
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Can I use an alternative, noncompendial analytical procedure for a test specified in a USP-NF monograph to demonstrate compliance? |
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Do I have to perform all tests specified in a monograph to ensure compliance? |
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Do I have to use the quantities and concentrations specified in the monograph or may I make some adjustments? |
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How do I contact the USP about questions on a specific monograph or general chapter? |
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Do I have to meet the specifications described in the Description and Solubility section of the USP-NF? |
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What is the status of my revision proposal? What is the status of the revision proposal that appeared in Pharmacopeial Forum? |
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Do
I have to meWhere can I find specifications for reagents? Who are the suppliers for a particular reagent mentioned in a USP-NF monograph? |
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How do I find out if a particular Reference Standard is available? |
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What is the expiration date of a Reference Standard? |
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Can I obtain a Certificate of Analysis for a particular USP Reference Standard? |
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What is the "purity" of a USP Reference Standard where no value is stated on the label? |
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What is the chemical name of a related compound specified in a test for impurities in a monograph? |
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How do I propose new monographs and revisions to official standards in the USP-NF or to revisions already proposed in Pharmacopeial Forum? |
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How do I inform USP that there may be an error in the text of the USP-NF or proposed in Pharmacopeial Forum? |
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What is the total organic carbon (TOC) limit for Purified Water and Water for Injection? |
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Why are there no microbial requirements included in the monographs for Purified Water and Water for Injection? |
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How often do I perform the system suitability test in Total Organic Carbon <643>? |
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How long can I store and reuse reference standard solutions prepared for the Total Organic Carbon <643> system suitability test? |
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What is the main reason for KCl addition in the Water Conductivity <645> test for pH measurement? |
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Can I use other strains than those that are cited in the USP? |
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Is there a method that provides verification that there is 100 CFU in the inoculum? |
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When are you actually supposed to do the negative control: when testing the suitability of the method, or when testing the product, or in both situations? |
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What is the purpose of the negative control? |
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Does it have to be done every time the product is tested or during the method validation or is it possible to do it periodically? |
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Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation? |
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Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch? |
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Why do growth promotion tests have to be performed on sabouraud-dextrose agar (SDA) and casein soya bean digest agar (CSA) for C. albican and A. niger? |
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As bacteria growing on SDA are also counted as part of TYMC, why aren't the growth promotion tests required to be performed on SDA with bacterial strains? |
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What does the factor of 2 mean and how is it calculated? |
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What is the sufficient volume of the microbial suspension of not more than 100 CFU? What does 100 CFU refer to? |
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What are the specifications when we compare a fresh batch with a previous batch for growth promotion properties? Do we need to take a factor of 2 into account? |
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You should not incubate more than the --shortest incubation time prescribed--. How exactly are the given times (e.g. 18 - 24 h) to be followed? |
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In the growth promotion test of Rappaport Vassiliadis Salmonella enrichment broth there is no visible growth after the incubation time, but after subculturing on selective agar there is typical growth. Is this the case only in our laboratory?
Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch? |
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Does it mean that for each test strain, individual suitability tests have to be performed, or is it possible to use a mixed inoculum of all 4 strains? |
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Test strains must be inoculated individually using a number of micro-organisms equivalent to not more than 100 CFU, could you clarify if this means that only the specific micro-organism under detection in the test method is inoculated into the growth medium or if each of the 4 micro-organisms are added individually to the growth medium for each of the specific test methods. |
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Which test micro-organisms should one use? Just the same micro-organisms as used for testing the growth promoting properties of the respective media, or also the micro-organisms used for testing inhibitory properties of the media? |
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Must all products be tested? |
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What is meant by "at the time of mixing"? Bile-tolerant gram-negative bacteria: At the time of sample preparation, or at the time of addition to the resuscitation broth, or at the time of inoculation of the Mossel Broth? E.coli: At the time of sample preparation, or at the time of addition to pre-broth, or at the time of inoculation of the MacConkey broth etc.? |
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What does "100 cfu in the inoculated test preparation" mean? How should this be done? |
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Should the test organism (<100 cfu) be added to the 1:10 diluted test solution (with 10g Product) and then the inoculated test solution be inoculated into the selective medium? If the inactivators (see Table 2, USP<61>) cannot inactivate the antimicrobial activity of the product, can proceed as in USP<61> to add the test organisms to a higher dilution of the product (<100cfu in 1g product) or at a later time in the test? |
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Why shall I use the shortest incubation period? |
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What does "The specified micro-organisms must be detected with the indication reactions" as described under "Testing of Products" mean? |
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What do I have to show to be able to proceed as stated: "If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralised, then it is to be assumed that the inhibited micro-organism will not be present in the product." |
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What are "the Bile-tolerant Gram-negative bacteria"? |
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In the sub-section Selection and Subculture under Escherichia coli, what is the purpose of the elevated temperature 42 - 44° C?
Can one utilize an alternative temperature range, e.g. 35 - 37° C provided that one demonstrates the recovery of E. coli during qualification? |
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With accumulation of the minimal incubation (3 x 18h) for pre-culture, enrichment culture, and sub-culture, it is unavoidable to work at night-hours to perform the validation. How can we avoid these awkward working hours for our personnel? |
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If 10 g of sample is added to the initial broth for a test such as E. coli (4.2), but an amount is immediately sub-cultured into a second broth that is only representative of 1g of actual product and this broth is incubated. At the end of testing, can this test be classified, for a negative result, as "none detected per 10 g" or as "none detected per g". |
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It is observed that on selective media of S. aureus, yellow colonies of gram-positive cocci in chains are seen, but the yellow colonies are without clear zones in the test sample. Whereas positive culture shows yellow colonies of gram-positive cocci in clusters surrounded by yellow zones? |
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For which pharmaceutical products or raw materials is it prescribed to search for Clostridia? |
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Are there alternatives media that are acceptable? |
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How can we do the pH measurement for culture media? It is written to measure the pH at 25° C. At this temperature, agar is solid. Therefore, how can we adjust the pH? |
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If we have growth problems of S. aureus and inhibitory problems of E. coli with mannitol salt agar medium that is recommended in the harmonised method, what is the cause? |
|
| Q. |
What does compliance of a product with USP-NF standards entail? |
| A. |
An article of commerce that is recognized in the USP-NF complies with the USP-NF standards when the article meets all of the requirements stated in the article's monograph, applicable General Chapters, and the General Notices (with monograph requirements superseding those of the General Chapters and General Notices, in any cases where requirements differ). Applicable standards apply at all times in the life of an article, from production to expiration. Thus, any official article is expected to meet the compendial standards if tested, and any official article actually tested as directed in the relevant monograph must meet such standards to demonstrate compliance. Frequency of testing and sampling are left to the preferences or direction of those performing compliance testing, including manufacturers, buyers, or regulatory authorities.
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| Q. |
What is the role of USP-NF standards, in terms of U.S. law (i.e., the Federal Food, Drug, and Cosmetic Act, FDCA)? |
| A. |
In the United States under the FDCA, both USP and NF are recognized as "official compendia." A drug with a name recognized in USP-NF must comply with compendial identity standards, or be deemed adulterated, misbranded, or both. To a void being deemed adulterated, such drugs must also comply with compendial standards for strength, quality, and purity, unless labeled to show all respects in which the drug differs. See, e.g., FDCA Sections 501(b) and 502(e)(3)(b); see also FDA regulations, 21 CFR 299.5. In addition, to avoid being deemed misbranded, drugs recognized in USP-NF must also be packaged and labeled in compliance with compendial standards. See FDCA Section 502(g). With regard to dietary supplements (regulated separately under the Dietary Supplement Health and Education Act, DSHEA), a dietary supplement represented as conforming to specifications in USP will be deemed a misbranded food if it fails to so conform. See FDCA Section 403(s)(2)(D).
Enforcement of USP standards is the responsibility of FDA and other government authorities, in the U.S. and elsewhere. USP has no role in enforcement.
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| Q. |
How much can I modify a
chromatographic procedure and still be in compliance? Can
column length, internal diameter, mobile phase composition be
modified? |
| A. |
Chromatography <621> contains a list of allowed adjustments to chromatographic systems. However, the user should verify the suitability of the method under the new conditions by assessing the relevant analytical performance characteristics potentially affected by the change (see section System Suitability under Chromatography <621>)
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| Q. |
What brand of HPLC/GC column was
used in the development and/or validation of a particular
test? Is there an alternative chromatographic column for a
particular test? |
| A. |
The information about the brand of HPLC columns, used for the developing of USP-NF methods, is available in the electronic version of the USP-NF under Chromatographic Column. The link to the information is at the end of each monograph. The complete database, called Chromatographic Columns could also be searched through a Quick search, or through "advanced search" under "General".
Print copies of the publication Chromatographic Columns are available through the USP store online at http://store.usp.org or through USP Customer Services: phone 301-881-0666 or custsvc@usp.org.
Please note that the identification of chromatographic column reagents by brand name is provided solely for informational purposes as a matter of convenience, without implication of approval, endorsement, or certification. In addition, the omission of a particular brand or product does not indicate that the article was judged to be unsatisfactory or inadequate.
This response has been provided for informational purposes only, and should not be construed as an official interpretation of USP text, or be relied on to demonstrate compliance with USP standards or requirements.
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| Q. |
How can I obtain copies of monographs or general chapters proposed in Pharmacopeial Forum, or monographs or general chapters that have appeared in an edition of the USP-NF? |
| A. |
Due to copyright requirements, for nonsubscribers, single copies of monographs and general chapters published either in USP-NF or in Pharmacopeial Forum (both of which are copyrighted) can be obtained for a fee by contacting USP Customer Service at custsvc@usp.org. Please note that for compliance with current USP requirements for a particular article, one must comply not only with the current monograph but also with all applicable General Chapters and with the USP General Notices.
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| Q. |
How do I convert the potency of a vitamin expressed in International Units (I.U.) to g or mg (and vice versa)? |
| A. |
The USP has discontinued the use of
International Units and now specifies the potency of vitamins
in terms of metric mass units such as μg or mg. The following
table shows the conversions.
| Vitamin A, 1 I.U. = |
0.3 μg all-trans retinol |
| " |
0.344 μg all-trans retinyl acetate |
| " |
0.55 μg all-trans retinyl palmitate |
|
|
| Vitamin D, 1 I.U. = |
0.025 μg ergocalciferol or cholecalciferol |
|
|
| Vitamin E, 1 I.U. = |
0.91 mg d,l-alpha tocopherol |
| " |
1.0 mg d,l-alpha tocopheryl acetate |
| " |
1.12 mg d,l-alpha tocopheryl acid
succinate |
| " |
0.67 mg d-alpha tocopherol |
| " |
0.74 mg d-alpha tocopheryl acetate |
| " |
0.83 mg d-alpha tocopheryl acid
succinate |
|
|
| Beta carotene, 1 I.U. = 0.6 μg of all-trans beta carotene |
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| Q. |
How can I verify that the balances in our laboratory are suitable for weighing the amounts specified in a USP monograph? |
| A. |
Refer to general chapters Weights and Balances <41> and Weighing on an Analytical Balance <1251> in the USP-NF. The procedure used to determine the suitability of balances is indicated in Weights and Balances <41>. Note that under Weighing on an Analytical Balance <1251>, it is stated that "...it is important for each balance to be serviced and calibrated regularly by a specially trained internal or external service person." Please check the index of the most recent issue of the Pharmacopeial Forum for proposed updates of these general chapters.
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| Q. |
How does a plastic material receive a "Class VI" classification? |
| A. |
The following is stated under general tests chapter Biological Reactivity Tests,
In Vivo <88>: "This classification is based on responses to a series of in vivo tests for which extracts, materials,
and routes of administration are specified." For example, for a material to be classified as Class VI, the material
would need to produce satisfactory results for all of the tests listed in Table 1 of Biological Reactivity Tests, In Vivo <88>.
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| Q. |
How are microbial limits and objectionable microorganisms determined? |
| A. |
In determining the appropriate microbial limit, the USP Analytical Microbiology Expert Committee
considers such matters as the route of administration, the form of the product, and the source material. For example, it is
never appropriate to have any microorganisms in a product intended for injection intravenously. Therefore, you will never see
a microbial limit listed for such products. Instead, those products must meet the test for Sterility <71>. Other products may
be in a form possessing extremely low water activity, such that microbial growth could not occur. You may not see microbial
limits provided for some of those products. Many other products, such as those intended for oral administration, will have
limits provided. Those limits are selected such that the risk of harm to the consumer is extremely low, while being reasonable
from a manufacturing and quality control perspective. Products from botanical sources may have higher limits due to the larger
bioburden associated with the raw materials. Again, the limits are set such that consumer risk is minimized without creating an
untenable situation for the manufacturer. Consideration is also given to the likelihood of product spoilage. Since some products
are more prone to spoilage due to microbial contamination, limits may be set lower for these. The issues discussed above also
influence which, if any, specific organisms must be tested.
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| Q. |
Can I use an alternative, noncompendial analytical procedure for a test specified in a USP-NF monograph to demonstrate compliance? |
| A. |
Yes, you may use an alternative, validated method to demonstrate compliance with a monograph. In the current USP-NF General Notices, the following is stated: "Compliance may be determined also by the use of alternative methods, chosen for advantages in accuracy, sensitivity, precision, selectivity or adaptability to automation or computerized data reduction or in other special circumstances. Such alternative or automated procedures shall be validated." However, there is a caveat to the use of alternative methods. "Pharmacopeial standards and procedures are interrelated; therefore, where a difference appears or in the event of a dispute, only the results obtained by the procedure given in this Pharmacopeia is conclusive."
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| Q. |
Do I have to perform all tests specified in a monograph to ensure compliance? |
| A. |
Applicable standards apply at all times in the life of an article, from production to expiration.
Thus, any official article is expected to meet the compendial standards if tested, and any official article actually tested
as directed in the relevant monograph must meet such standards to demonstrate compliance. A manufacturer's specifications
that have been approved by FDA, and related current good manufacturing practices (GMPs) required by FDA regulations
(21 CFR Parts 210, 211), all can help assure that an article will comply with compendial standards. As noted in the
General Notices, repeats, replicates, statistical rejection of outliers, or extrapolation of results to larger populations,
as well as the necessity and appropriate frequency of batch testing, are neither specified nor proscribed by the compendia.
Frequency of testing and sampling are left to the preferences or direction of those performing compliance testing, including
manufacturers, buyers, or (as again exemplified by FDA's GMP regulations) regulatory authorities.
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| Q. |
Do I have to use the quantities and concentrations specified in the monograph or may I make some adjustments? |
| A. |
You may make some adjustments. In the current USP-NF General Notices, the following is stated: "In the performance of assays and test procedures, not less than the specified number of dosage units should be taken for analysis." Furthermore, "[p]roportionately larger or smaller quantities than the specified weights and volumes of assay or test substances and Reference Standards may be taken, provided the measurement is made with at least equivalent accuracy and provided that any subsequent steps, such as dilutions, are adjusted accordingly to yield concentrations equivalent to those specified and are made in such a manner as to provide at least equivalent accuracy."
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| Q. |
How do I contact the USP about questions on a specific monograph or general chapter? |
| A. |
Staff members, their assignments, phone numbers and E-mail addresses can be found at http://www.usp.org/aboutUSP/contactUs. Staff members, their assignments, phone numbers and E-mail addresses are also located in the Staff Directory (one of the front sections of every Pharmacopeial Forum). In addition, contact information can be found in all electronic versions of the USP-NF. Finally, you can contact the USP Science Support at 1-301-816-8151 or mailto:stdsmonographs@usp.org.
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| Q. |
Do I have to meet the specifications described in the Description and Solubility section of the USP-NF? |
| A. |
The specifications in the Description and Solubility section are not considered official requirements by the USP. Within this section it states "The "description" and "solubility" statements pertaining to an article (formerly included in the individual monograph) are general in nature. The information is provided for those who use, prepare, and dispense drugs, solely to indicate descriptive and solubility properties of an article complying with monograph standards. The properties are not in themselves standards or tests for purity even though they may indirectly assist in the preliminary evaluation of the integrity of an article".
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| Q. |
What is the status of my revision proposal? What is the status of the revision proposal that appeared in Pharmacopeial Forum? |
| A. |
Contact the USP staff member responsible for the revision as indicated in Pharmacopeial Forum. Staff members, their assignments, phone numbers and E-mail addresses can be found at http://www.usp.org/aboutUSP/contactUs. Staff members, their assignments, phone numbers and E-mail addresses are also located in the Staff Directory (one of the front sections of every Pharmacopeial Forum). In addition, contact information can be found in all electronic versions of the USP-NF Finally, you can contact the USP Science Support at 1-301-816-8151 or mailto:stdsmonographs@usp.org.
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| Q. |
Where can I find specifications for reagents? Who are the suppliers for a particular reagent mentioned in a USP-NF monograph? |
| A. |
The specifications for the reagents cited in USP-NF are generally found in the section Reagents Specifications of the USP-NF. When there is only one supplier for a particular reagent or a particular reagent grade, a footnote will cross-reference to the supplier information in the section Reagent Footnotes of the USP-NF. If the reagent you are interested in is not listed in the Reagents Specifications section, please contact USP by email to mailto:stdsmonographs@usp.org.
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| Q. |
How do I find out if a particular Reference Standard is available? |
| A. |
Check the USP Catalog or on the USP Home Page: www.usp.org. If a particular USP Reference Standard is not listed, it is not available. Interested parties may sign up for an email notification program which informs about new releases of USP Reference Standards. To sign up, go to http://www.usp.org/referenceStandards/notificationProgram.html.
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| Q. |
What is the expiration date of a Reference Standard? |
| A. |
The only USP Reference Standards labeled with expiration dates are Certified Reference Materials. Otherwise, USP Reference Standards are not labeled with predetermined expiration dates and are official until otherwise stated in the USP Reference Standard Catalog. The new lot is indicated in the column "Curr. Lot." The column "Previous Lot/Valid Use Date" indicates the date on which the previous lot will no longer be official.
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| Q. |
Can I obtain a Certificate of Analysis for a particular USP Reference Standard? |
| A. |
Generally no. USP provides any needed information about the proper use of a Reference Standard on the label and in associated documents. A full Certificate of Analysis is not provided, except for USP Certified Reference Materials. If a special circumstance arises, interested parties may submit a Document Disclosure Request for consideration by the Secretary of the USP Convention. For more information, see the current USP Document Disclosure Policy at www.usp.org.
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| Q. |
What is the "purity" of a USP Reference Standard where no value is stated on the label? |
| A. |
Where no value is stated on the label, the calculation value is considered to be 100.0% when handled as described on the label.
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| Q. |
What is the chemical name of a related compound specified in a test for impurities in a monograph? |
| A. |
If there is a USP Reference Standard for the particular related compound, the chemical name may be found in general chapter <11> USP Reference Standards in the USP-NF.
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| Q. |
How do I propose new monographs and revisions to official standards in the USP-NF or to revisions already proposed in Pharmacopeial Forum? |
| A. |
Contact the USP in writing with any change you wish to make to the USP-NF. USP contact information can be found at http://www.usp.org/aboutUSP/contactUs, in the front pages of the USP-NF or the Staff Directory, printed in one of the front sections of every Pharmacopeial Forum. You can also contact USP Science Support at 1-301-816-8151 or stdsmonographs@usp.org. As described in the USP general chapter Validation of Compendial Methods <1225>, all proposals must be supported by relevant data including scientific rationale, complete analytical procedures, and appropriate validation data. Revision proposals will be considered only after receipt of supporting data and justification. Proprietary data should be labeled "Confidential".
All proposals approved by the USP Expert Committees are published in the Pharmacopeial Forum for public comment, with the eventual goal of official adoption in USP-NF.
Detailed information on how to submit monographs and revisions is available online at http://www.usp.org/USPNF/submitMonograph/. Detailed Request for Revision guidance is also available online; note in particular the section on "General Information for All Submissions." http://www.usp.org/USPNF/submitMonograph/subGuide.html
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| Q. |
How do I inform USP that there
may be an error in the text of the USP-NF or proposed
in Pharmacopeial Forum? |
| A. |
Please contact the USP in writing or by email with the nature of the error found in official text of the USP-NF or text proposed in Pharmacopeial Forum. USP contact information can be found at http://www.usp.org/aboutUSP/contactUs, in the front pages of the USP-NF or the Staff Directory, printed in one of the front sections of every Pharmacopeial Forum. You can also contact the USP Science Support at 1-301-816-8151 or stdsmonographs@usp.org.
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| Q. |
What is the total organic carbon
(TOC) limit for Purified Water and Water for
Injection? |
| A. |
There is a "target limit response" of 500 ppb carbon The true limit is the response of the TOC measurement system to a 500 ppb sucrose solution, Rs, corrected for the response to reagent water, Rw. This is equal to Rs-Rw.
The actual number will vary based upon your reference standard solution, your equipment, background carbon etc. You will determine compliance using the USP Reference Standards.
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| Q. |
Why are there no microbial requirements included in the monographs for Purified Water and Water for Injection? |
| A. |
Because of the various uses of these waters, microbial requirements are not included in these monographs since this would require some uses to adhere to meaningless and/or inappropriate requirements. Microbial guidelines are provided under the non-mandatory informational chapter Water for Pharmaceutical Purposes <1231>. We recommend that you implement Alert and Action Levels no higher than, and preferably lower than, those listed in Water for Pharmaceutical Purposes <1231> based on the normal microbial performance trends of your water system. The purpose of Alert and Action Levels is to trigger additional, rather than routine, microbial control measures. These additional control measures should avoid objectionable levels and types of microorganisms for the water's use.
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| Q. |
How often do I perform the system suitability test in Total Organic Carbon <643>? |
| A. |
USP Chapter <643> intentionally says nothing about how often the system suitability test (SST) should be run. The reasoning is that this frequency depends on the stability of the Total Organic Carbon (TOC) instrument response and other factors associated with the water quality and risk. If the TOC of a quality water system is very low, say <20 ppb, then many opt to reduce the frequency of testing due to less risk. The stability of different TOC measurement technologies may vary over extended periods of time. The instrument manufacturer can advise the user on this matter and user experience can also be valuable in determining a suitable frequency. Another factor is the risk of a non-conforming system suitability test result since the Rs-Rw result used in this calculation is the limit response for the instrument, the crucial pass/fail value for the TOC test. If a non-conforming system suitability result is obtained, it implicates the accuracy of all TOC test results since the previous successful system suitability test. For this reason, many users choose to perform the system suitability test more frequently than the stability of the TOC instrument response might suggest, just to minimize the impact of a possibly non-conforming result. This is why a typically low TOC water system is at less risk, even with a failed SST. If the SST fails, some remediation is necessary to re-adjust the instrument, replace a lamp, or some other means of instrument improvement. But even a 50% error will have little impact on the past TOC readings (since the readings, even with this error, are so low relative to the Limit). On a high TOC water system, the failure of the SST is possibly more critical. This is up to the risk the user is willing to assume, knowing the historic stability of their instrument and other factors. Therefore, Total Organic Carbon <643> is silent on the frequency of performing the system suitability test because it is up to the user to decide what is appropriate.
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| Q. |
How long can I store and reuse reference standard solutions prepared for the Total Organic Carbon <643> system suitability test? |
| A. |
Where USP is silent on storage conditions and the stability of prepared reference standard solutions, the implication is that the solutions should be prepared fresh with every use, starting with the solid material provided by USP. Only with suitable stability studies performed over various periods using controlled storage conditions can a user's storage conditions be justified as suitable. It should be noted that a number of factors can influence the stability of the reference standard solutions used for the system suitability test in the <643> Total Organic Carbon (TOC) test. These include concentration, microbial decomposition, adsorption to the container surface, and the degradative effects facilitated by oxygen, light, and temperature. The development of turbidity, additional color, or performance variability relative to freshly prepared solutions are indicators of instability. If the user wishes to store and re-use these prepared reference standard solutions or solution concentrates, then the user must have stability data for these solutions under their own storage conditions. If working standard solutions are purchased from a third party supplier, then that supplier should certify the suitability of their materials based on stability studies they have performed. Otherwise, the user should prepare fresh solutions each time the system suitability test is performed. Most of the suppliers specify expiry dates. But as a practical matter, concentrated reference standard solutions of Sucrose last 3-6 months, and analogous solutions of 1,4 Benzoquinone (pBQ) last about 2 months, assuming they are stored at appropriate temperatures in appropriate containers and protected from light (for pBQ). It is recommended to use refrigeration since this slows down all degradation.
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| Q. |
What is the main reason for KCl addition in the Water Conductivity <645> test for pH measurement? |
| A. |
In Stage 3, a neutral electrolyte (KCl) is added to accurately measure the pH of the solution. If the ionic strength of the solution is not increased, the pH measurement will be highly unstable and inaccurate. So KCl is added so that there is minimal concentration gradient across the pH electrode's membrane between the water and sensor's internal electrolyte. As a neutral salt, KCl does not impact the pH. It impacts the conductivity of the water, but you do not measure the conductivity again at this stage of the test, only a pH measurement. The method says to use this pH result and the conductivity result from Step 4 (Stage 2), and go to the Stage 3 table. If the measured conductivity from Stage 2 is less than the value in the Stage 3 table (at the measured pH), then you pass.
The <645> Stage 1-3 must be done sequentially, otherwise the user will reach wrong conclusions.
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USP Chapter <61>:
Microbiological Examination of Nonsterile Products:
Microbial Enumeration Tests
|
| Q. |
Can I use other strains than those that are cited in the USP? |
| A. |
You should use the strains that are cited in this chapter, or equivalent strains from other culture collections.
For example, if Pseudomonas aeruginosa ATCC 9027 is indicated, you should use this strain or strains from other culture collections claiming equivalence to ATCC 9027. Other strains such as ATCC 14149 are not appropriate.
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| Q. |
Is there a method that provides verification that there is 100 CFU in the inoculum? |
| A. |
You may establish a turbidimetric calibration curve or use another suitable method and then you will be able to get an estimate of the concentration of your inoculum. This can be later confirmed by standard plating methods, such as described in USP <51>. You can also use ready-to use certified strains.
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| Q. |
When are you actually supposed to do the negative control: when testing the suitability of the method, or when testing the product, or in both situations? |
| A. |
You are supposed to do the negative control at the same time as when you are testing the product.
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| Q. |
What is the purpose of the negative control? |
| A. |
The purpose of the negative control is to show that there is no contamination during the testing of the product. If a positive result is obtained with a negative control, the test can be regarded as invalid and may be repeated.
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| Q. |
Does it have to be done every time the product is tested or during the method validation or is it possible to do it periodically? |
| A. |
Negative controls should be included every time that the product is tested.
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| Q. |
Is it necessary to test the growth promotion on all received batches or does it serve just for microbiological validation? |
| A. |
Growth promotion must be checked for each new batch of medium. Growth promotion must be checked on agar media and nutritive broth but not on diluted broth.
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| Q. |
Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch? |
| A. |
You do not have to test a previous batch in parallel. You can do the comparison 'on paper' if growth was clearly described.
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| Q. |
Why do growth promotion tests have to be performed on sabouraud-dextrose agar (SDA) and casein soya bean digest agar (CSA) for C. albican and A. niger? |
| A. |
This is a matter of definition. TAMC by definition includes yeast and moulds. Therefore the media have to be checked with these micro-organisms.
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| Q. |
As bacteria growing on SDA are also counted as part of TYMC, why aren't the growth promotion tests required to be performed on SDA with bacterial strains? |
| A. |
TYMC is by definition yeasts and moulds count so growth promotion with bacteria is not essential. SDA with antibiotics may be used as an alternative when the TYMC is expected to exceed the acceptance criterion due to the bacterial growth.
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| Q. |
What does the factor of 2 mean and how is it calculated? |
| A. |
It means that the result can be twice that of the innoculum. For example, with an inoculum of 100 CFU, acceptable counts are: 100/2 = 50 CFU to 100 x 2 = 200 CFU. The factor is introduced to take account of the variability of the method.
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| Q. |
What is the sufficient volume of the microbial suspension of not more than 100 CFU? What does 100 CFU refer to? |
| A. |
The micro-organisms are to be added to the diluted / suspended product at the end of the preparation (usually a 1 in 10 dilution is prepared) or after the neutralization (in the last fraction of the rinsing fluid in the case of filtration or simultaneously with the preparation in/on the Petri dish in the case of the plate count method) if inhibition of growth by the sample cannot otherwise be avoided. The 100 CFU refers to the innoculum.
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USP Chapter <62>:
Microbiological Examination of Nonsterile Products:
Tests for Specified Microorganisms
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| Q. |
What are the specifications when we compare a fresh batch with a previous batch for growth promotion properties? Do we need to take a factor of 2 into account? |
| A. |
The factor of 2, as described in USP <61> can be used. No strict requirement was deliberately given in this chapter because the test is qualitative, not quantitative. You can define the comparability criterion yourself. For example, colony size at the shortest incubation time prescribed.
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| Q. |
You should not incubate more than the --shortest incubation time prescribed--. How exactly are the given times (e.g. 18 - 24 h) to be followed? |
| A. |
For growth promoting properties of media you must incubate not more than 18 h (worst case conditions).
For inhibitory properties of media you must incubate not less than 24 h (worst case conditions).
For indicative properties of the media you could incubate within the specified range (here, from 18 h to 24 h).
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| Q. |
In the growth promotion test of Rappaport Vassiliadis Salmonella enrichment broth there is no visible growth after the incubation time, but after subculturing on selective agar there is typical growth. Is this the case only in our laboratory?
Do we have to test systematically in parallel a previous and approved batch in order to compare with the new batch?
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| A. |
This can be observed, since for Rappaport Vassiliadis Salmonella enrichment broth, we inoculate low numbers of Salmonella sp (usually the inoculum is around 20 CFU per 10 mL Rappaport Vassiliadis Salmonella enrichment broth).
Even if the enrichment broth seems clear, you must confirm recovery of Salmonella by sub-culturing the Rappaport Vassiliadis Salmonella enrichment broth to solid agar.
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| Q. |
Does it mean that for each test strain, individual suitability tests have to be performed, or is it possible to use a mixed inoculum of all 4 strains?
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| A. |
The method states that the strains are inoculated individually. No mixed inoculum is permitted.
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| Q. |
Test strains must be inoculated individually using a number of micro-organisms equivalent to not more than 100 CFU, could you clarify if this means that only the specific micro-organism under detection in the test method is inoculated into the growth medium or if each of the 4 micro-organisms are added individually to the growth medium for each of the specific test methods.
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| A. |
It is only the specified micro-organism under detection which is inoculated.
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| Q. |
Which test micro-organisms should one use? Just the same micro-organisms as used for testing the growth promoting properties of the respective media, or also the micro-organisms used for testing inhibitory properties of the media?
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| A. |
You do not have to use an inhibitory strain in order to test the suitability of the method.
For example if you test the suitability of the method for E. coli, you should use only E. coli as test micro-organism for growth promotion.
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| Q. |
Must all products be tested?
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| A. |
Not always. For products differing only in amount of active ingredient a bracketing approach may be applied.
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| Q. |
What is meant by "at the time of mixing"? Bile-tolerant gram-negative bacteria: At the time of sample preparation, or at the time of addition to the resuscitation broth, or at the time of inoculation of the Mossel Broth? E.coli: At the time of sample preparation, or at the time of addition to pre-broth, or at the time of inoculation of the MacConkey broth etc.?
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| A. |
In both cases, the micro-organisms should be added at the time of mixing with the pre- incubation or resuscitation broth. If Bile-tolerant gram-negative bacteria are taken as an example it refers to the sub-section "Sample Preparation and Pre-Incubation". The micro-organisms are added to the casein soy bean digest broth (SCDB) immediately before or after the product to be examined is added. The micro-organisms are therefore present during the whole resuscitation period of 2-5 hours.
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| Q. |
What does "100 cfu in the inoculated test preparation" mean? How should this be done?
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| A. |
This is to simulate the situation that 100 cfu's are present in the sample to be examined, usually 1 g of the product but 10 g for Salmonella (cf. section Salmonella), resp.
Example: Suppose 10 g of product is diluted in 100 ml of CSDB. Then less than 100 cfu's are added to this suspension.
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| Q. |
Should the test organism (<100 cfu) be added to the 1:10 diluted test solution (with 10g Product) and then the inoculated test solution be inoculated into the selective medium? If the inactivators (see Table 2, USP<61>) cannot inactivate the antimicrobial activity of the product, can proceed as in USP<61> to add the test organisms to a higher dilution of the product (<100cfu in 1g product) or at a later time in the test?
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| A. |
The test organisms should be added at the time of mixing with the medium for pre-incubation. Inactivation of antimicrobial activity should be attempted as far as possible. Addition at a later time of the test is not a reasonable measure. If inactivation cannot be achieved, the test can be abandoned for the product because it is assumed that the specified micro organism will not be able to survive in the product.
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| Q. |
Why shall I use the shortest incubation period?
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| A. |
You have to show that the worst conditions work. Moreover you are working with healthy cells; these should give the required response in the shortest time.
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| Q. |
What does "The specified micro-organisms must be detected with the indication reactions" as described under "Testing of Products" mean?
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| A. |
Characteristic colonies are observed on the selective agar, and no such colonies are observed with a non-inoculated product, examined simultaneously as a negative blank.
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| Q. |
What do I have to show to be able to proceed as stated: "If for a given product the antimicrobial activity with respect to a micro-organism for which testing is prescribed cannot be neutralised, then it is to be assumed that the inhibited micro-organism will not be present in the product."
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| A. |
Once you demonstrate that you have tried all possible approaches, then you can refer to the escape clause.
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| Q. |
What are "the Bile-tolerant Gram-negative bacteria"?
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| A. |
There is no strict definition of this group of micro-organisms. They are defined operationally as those micro-organisms that show growth in the stated conditions on Violet Red Bile Glucose Agar medium. They include, Gram negative bacteria that grow in the presence of bile salts, non-lactose fermenting but able to utilize glucose, e.g., some Bile Tolerant Gram Negative Bacteria includes members of the family Enterobacteriaceae, Pseudomonads and Aeromonas.
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| Q. |
In the sub-section Selection and Subculture under Escherichia coli, what is the purpose of the elevated temperature 42 - 44° C?
Can one utilize an alternative temperature range, e.g. 35 - 37° C provided that one demonstrates the recovery of E. coli during qualification?
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| A. |
The purpose of the elevated temperature, 42 - 44° C is to allow for selective conditions for faecal E. coli. The selected temperature is usually a compromise between sensitivity and specificity as not all strains of E. coli will grow, or grow and produce gas, at these higher incubation temperatures.
An alternative temperature range would depart from the USP method, but you can always use alternatives methods as described in the General Notices of the USP and USP<1223>.
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| Q. |
With accumulation of the minimal incubation (3 x 18h) for pre-culture, enrichment culture, and sub-culture, it is unavoidable to work at night-hours to perform the validation. How can we avoid these awkward working hours for our personnel?
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| A. |
You have to confirm that the test works for the minimum time for routine testing.
In fact, should a company find during suitability testing, that the minimum incubation time is not sufficient for a given product but a longer incubation time is needed, prolongation would be a necessary variation of the test.
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| Q. |
If 10 g of sample is added to the initial broth for a test such as E. coli (4.2), but an amount is immediately sub-cultured into a second broth that is only representative of 1g of actual product and this broth is incubated. At the end of testing, can this test be classified, for a negative result, as "none detected per 10 g" or as "none detected per g".
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| A. |
The quantity that is pre-incubated is 1 g therefore the outcome of the test is "absence in 1 g". For Salmonella, you will note that an "absence in 10 g" test has been implemented.
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| Q. |
It is observed that on selective media of S. aureus, yellow colonies of gram-positive cocci in chains are seen, but the yellow colonies are without clear zones in the test sample. Whereas positive culture shows yellow colonies of gram-positive cocci in clusters surrounded by yellow zones?
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| A. |
Your product can be contaminated, maybe not by the species described in the USP but by another micro-organism. Good laboratory practice should make you think that there is a problem and that you should investigate (e.g. identify the species and find out where it comes from). Probably the product cannot be released, but it is up to the QC laboratory manager to decide.
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| Q. |
For which pharmaceutical products or raw materials is it prescribed to search for Clostridia?
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| A. |
For powdered animal organs, products with risk of faecal contamination by Clostridia and possible proliferation, natural raw materials, possible telluric contamination. However it has not been introduced in any monograph yet. The test is particularly relevant where a preparation is exposed to anaerobic or low-oxygen conditions during use.
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| Q. |
Are there alternatives media that are acceptable?
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| A. |
Methods using alternative media are considered as alternative methods, which may be used when consistent with General Notices 6.30.
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| Q. |
How can we do the pH measurement for culture media? It is written to measure the pH at 25° C. At this temperature, agar is solid. Therefore, how can we adjust the pH?
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| A. |
You may use a robust electrode. There are electrodes for measurement in semisolid samples such as meat, cheese and fruit. These electrodes are certainly suitable for measurements in solid agar. Adjustment of pH must be made during preparation of the medium for ensuring that the criterion for pH is met in the final medium.
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| Q. |
If we have growth problems of S. aureus and inhibitory problems of E. coli with mannitol salt agar medium that is recommended in the harmonised method, what is the cause?
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| A. |
The composition of mannitol salt agar has been optimised to recover S. aureus and inhibit E. coli (pH, nutritive qualities...). You should verify that the temperature of incubation is correct. Moreover there could be a problem of stability of the medium and you should therefore verify that the medium has been stored in adequate conditions.
Lastly, you could try to use different media suppliers, which may give better results.
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