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The environmental control program is one of the essential elements for controlling the quality of pharmaceutical production environments, so it is essential to use monitoring solutions that can detect any drift that may lead to the loss of this control. .
Specifically, the control of the environment relies mainly on the use of agar culture media which must make it possible to recover each possible living microorganism but also neutralize the presence of residues of disinfectants that can inhibit the growth of certain strains while promoting sufficient growth to to be seen by an operator.
The quality of test results depends on the performance of these media as very clearly does the pertinence of the resultant trend analyses. But validating culture media that are able to recover stressed bacteria from the environment in a cleanroom is not so simple! To gain a better understanding of the interaction between culture media and the disinfectants dedicated to environmental control, we have studied the properties of the biocides most commonly used in the pharmaceutical industry and have suggested a methodology to evaluate their impact on the performance of these media.
The manufacture of medicines in an aseptic environment remains a complex task. The maintenance of the quality of production areas is achieved by observing numerous procedures, including those related to cleaning and decontamination, and very clearly by using different biocides. The choice of the right disinfectant therefore proves crucial to ensure proper bioburden control. All this is necessary but it is not enough. The task is then to make sure of the level of cleanliness of surfaces, and agar culture media is widely used among pharmaceutical manufacturers for this purpose. These media, generally in the format of agar contact plates, are used to grow the microorganisms which may be present and to assess surface contamination. They will be in direct contact with these disinfectants. But how to estimate the compatibility between disinfectant and culture medium without running complicated validations for this purpose?
We will begin by taking a look at the different biocides used in the pharmaceutical industry.
|Aldehydes||Sporicidal agent||2% Glutaraldehyde|
|Alcohols||General purpose disinfectant, antiseptic, antiviral agent||70% Isopropyl alcohol, 70% alcohol|
|Chlorine and sodium hypochlorite||Sporicidal agent||Sodium hypochlorite 0.5%|
|Phenolics||General purpose disinfectant||Micrograms per g Chlorocresol 500, 500 micrograms per g chloroxylenol|
|Ozone||Sporicidal agent||8% by weight Gas|
|Hydrogen peroxide||Vapor Phase Sterilant, liquid Sporicidal agent, antiseptic||4 mcg per g H2O2 vapor, 10% -25% solution, 3% solution|
|Substituted diguanides||Antiseptic agent||0.5% chlorhexidine gluconate|
|Peracetic acid||Sterilant Liquid, vapor-phase Sterilant||0.2% Peracetic acid, 1 micrograms per g Peracetic acid|
|Ethylene oxide||Vapor-Phase Sterilant||600 mcg per g Ethylene oxide|
|Quaternary ammonium compounds||General purpose disinfectant, antiseptic||200 micrograms per g benzalkonium chloride|
|ß-propiolactone||Sporicidal agent||100 μg per g -. propiolactone|
General Classification of Antiseptics, Disinfectants, and Sporicidal Agents - USP <1072> Disinfectants and Antiseptics
The choice of disinfectant is a complex task. There are different texts which can guide manufacturers in their choice. U.S.P. <1072>1 offers a useful classification of molecules with antimicrobial properties while indicating the intended target(s) and gives examples of effective concentrations.
ASTM (2) also directs the pharmacy industry towards the selection of one or two disinfectants and a sporicide but especially towards the use of formulations incorporating more than one type of chemical agents in order to obtain the best balance between the disinfection performance and limited aggression of the surfaces to be treated. ASTM points out that since the products used in disinfectants have activity spectra which can vary, the mixtures make it possible to guarantee the greatest possible efficiency. As a result, the majority of disinfectants on the market is a mixture of several active molecules.
We see that finding the right product is not a simple thing and this difficulty is added the new European REACH Directive which will greatly limit the introduction of new molecules or even to question the use of certain of biguanides them as such.
We must therefore consider increasing complexity disinfectant formulations and obviously increased neutralization challenge for culture media.
What is the impact of disinfectants on environmental testing?
The suppliers of biocides offer a wide variety of formulations with the different agents cited previously. But depending on the family of the chemical compound or its working concentration, the amount of product residue can vary considerably. The chemical compounds can be classified in 2 major categories: compounds which leave residues and those which do not.
|H2O2 / Peracetic acid||High||Means||No|
Source: Shield Medicare - 10 / 2005 Cleanroom Technology
To develop our model, we selected different disinfectants commonly used by the pharmaceutical industry, and relying on their formulation, constructed a classification based on their propensity to leave residues on surfaces.
Four categories of residue scores (SR) were created based on the likelihood of the disinfectant leaving residues on the surfaces (of SR = 0,5 low to SR = 2 very high, see table).
We then evaluated their antimicrobial performance on a diverse panel of microorganisms incorporating 3 strains of the Pharmacopoeia (Pseudomonas aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538) and 4 susceptible strains from industry cleanroom isolates. pharmaceutical (Staphylococcus epidermidis, Bacillus pumilus, Corynebacterium striatum, Common Penicillium). For this purpose, a volume of 500 μl of disinfectant (at the selected concentration) was deposited on standard agars without neutralizer. After drying, these media were inoculated with the panel strains at a concentration between 10 and 100 CFU and the incubated media. Recovery rates are finally calculated.
|Disinfectants ready to use||Residue score (SR)||Chosen concentration|
|A||Very high (2)||Pure|
|B||Very high (2)||Phone 1|
|Hydrogen peroxide||Low (0,5)||Phone 1|
|70% isopropyl alcohol||Low (0,5)||Pure|
The chosen concentration represents the concentration which allows weak growth of at least one panel strain as a minimum.
From the levels obtained for all the strains, a first score on the antimicrobial performance (SA) can be given.
SA = 1 - (Average panel recovery rate)
if the chosen concentration is pure
SA = 2 - (Average panel recovery rate)
if the chosen concentration is 1 / 10
SA is a value between 0 and 2.
The final impact score of the disinfectant (SI) on the middle is then calculated by multiplying SR and SA. This impact score is an estimate of the risk that a disinfectant represents in terms of neutralization and thus to induce poor recovery rates of microorganisms in the environment and a trend analysis that is not representative of reality.
|70% isopropyl alcohol||0,03||0,5||0,02|
A graphic representation of SI allows a rapid visualization of the potential impact of the disinfectant on the growth performance of a culture medium.
It is interesting to note that isopropyl alcohol comes out as a disinfectant which will not show a major impact. Conversely, disinfectant B will need to be the subject of a serious assessment with the neutralizers present in the agar used for environmental testing. In effect there is a substantial risk of having a high concentration of residues with strong antimicrobial potency, and the neutralizing capacity of the culture medium used will therefore have to be tailored to these levels to permit growth of potential microorganisms that are still viable but which are inhibited by these residues.
The results for hydrogen peroxide and disinfectant A demonstrate that there is no correlation between the amount of residues and the risk on the culture medium since 4 disinfectants with very different residual levels are all found in the high risk category. The H2O2 remaining the test compound in the panel with the highest antimicrobial potency but which also has the least residues.
Control of bioburden cleanrooms requires validation of disinfectants and culture media. Knowledge of interactions and validation of compatibility between the two products depend on the results of environmental tests. However there is no concordance between the guide biocide suppliers and culture media. We then sought to develop a simple model to measure, beyond the performance of disinfection, the impact of disinfectant formulations of culture media. This model does not replace a validation but is an effective tool to measure risk.
This methodology can also help aid in the selection of culture media that will be used. Different forms of culture media are commercially available with varying levels of neutralization. Some agars are proposed with 2 neutralizing (lecithin and polysorbate 80) or 4 (lecithin, thiosulfate, histidine and polysorbate 80) and there are also new agar ranges with reinforced neutralizing.
The application of the methodology can provide a first orientation on the expected performance of neutralization based on the disinfectant risk scale. Today, most of the formulations suggested by the media providers include neutralizing 4, which seems to correspond to the majority of neutralizing needs.
However we see that new biocide formulations offer increasingly complex, as for example disinfectant B, they represent a higher risk of poor neutralization.
In consequence, the search for media with a wider neutralizing spectrum will become inevitable to ensure that the results of environmental testing are pertinent.
Laurent LEBLANC - BIOMÉRIEUX
ASTM: : American Society for Testing Materials
IPA : Isopropylic alcohol
REACH : Registration, Evaluation and Authorization of Chemicals
USP : United States Pharmacopoeia