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Development of the antimicrobial effectiveness test as USP Chapter 〈51〉
Abstract
The antimicrobial effectiveness test first appeared as a USP General Chapter in the 18th revision, official September 1, 1970. This chapter, at the beginning, was designed to evaluate the performance of antimicrobials added to inhibit the growth of microorganisms that might be introduced during or subsequent to the manufacturing process. As Good Manufacturing Practices (GMPs) became a governing principal in pharmaceutical manufacturing, the purpose of the test was refined to focus on activity of the preservative system as a protection against inadvertent contamination during storage and usage of the product. This article will review the history of the antimicrobial test; its function, technique, and the background discussions that resulted in the changes from the test that appeared in USP XVIII to that of the current USP 25.
... The purpose of this analysis was to determine the effect of exposure to Fibergraft BG Morsels on the viability of a range of microorganisms based on the Antimicrobial Effectiveness Test. Throughout history, this particular test has evolved to study a system's ability to protect against microbial contamination during storage and usage of a product [8]. ...
... Fibergraft ® Bone Graft Substitute Material was used for one viability study on five different microorganisms that are familiar to today's practitioners [8]. The antimicrobial effectiveness was assessed by a third-party commercial laboratory, Biotest Laboratories, Inc., by using the USP <51> Antimicrobial Effectiveness Test (AET). ...
... The antimicrobial effectiveness was assessed by a third-party commercial laboratory, Biotest Laboratories, Inc., by using the USP <51> Antimicrobial Effectiveness Test (AET). The AET is designed to demonstrate the ability of a pharmaceutical product to inhibit the growth of a contaminant in the product, commonly referred to as its preservative system [8]. It is important for practitioners to keep in mind that the AET is a laboratory test performed under careful controls and is not intended to be a simulation of real-world clinical situations [8]. ...
... To confirm the antimicrobial activity of APs, a simplified preservative efficacy test was performed (Sutton and Porter, 2002;Hutchings et al., 2013). A primary culture of E. coli BL21(DE3) cells was incubated overnight at 37 C in a shaker. ...
... Table 1 lists the concentrations of these APs used in formulations. Fig. 2A shows the antimicrobial efficacy of these APs, measured using a simplified test (Sutton and Porter, 2002;Hutchings et al., 2013). We tested their effect on the growth of E. coli bacteria. ...
... Preservative efficacy test of APs on BL21(DE3) E. coli cells(Sutton and Porter, 2002;Hutchings et al., 2013). The cell count was monitored by measuring the relative optical density at 600 nm as a function of the growth time.Addition of APs inhibited bacterial growth. ...
... To confirm the antimicrobial activity of APs, a simplified preservative efficacy test was performed 41 . A primary culture of Escherichia coli DH5α cells was incubated overnight at 37°C in a shaker. ...
... To demonstrate the antimicrobial efficacy of the five APs, we tested their effect on the growth of Escherichia coli bacteria 41 . For this purpose, we used DH5α cells in LB media and monitored the cell count by measuring changes in the optical density at 600 nm as a function of the growth time. ...
... Our results presented here show that a similar strategy of stabilizing the weakest links in a protein might also decrease its AP-induced aggregation. Preservative efficacy test of APs on DH5α E. coli cells 41 . The circles represent the data untreated with APs, while the other symbols represent the data treated with various APs: 1% v/v benzyl alcohol (BA), 0.3% v/v m-cresol (CR), 0.5% v/v phenol (PH), 0.5% v/v phenoxyethanol (PE), and 0.5% v/v chlorobutanol (CB), respectively. ...
One-third of protein formulations are multidose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation "hot-spot" reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol (CB). All these enhanced the partial protein unfolding that includes a local region, which was predicted to be the aggregation "hot-spot." The extent of destabilization correlated with the extent of aggregation. Further, we showed that stabilizing the "hot-spot" reduced aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas CB causes the least protein aggregation. The same protein region acts as the "hot-spot" for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
... Marketed product stability testing of preservatives may be conducted using the chemical assay and not by microbiological challenge tests [4]. Preservatives are selected during formulation development [3] by preservative effectiveness test that was first described in the 18 th edition of General Chapter of USP on September 1, 1970 [5]. Preservative effectiveness test is now described in major official compendia, such as United States Pharmacopoeia (USP) [6], European Pharmacopoeia (Ph.Eur.) ...
... (4) NR stands for no recovery. (5) NI stands for no increase. It is defined as not more than 0.5 log 10 higher than the value measured at previous time point [6,8]. ...
... Marketed product stability testing of preservatives may be conducted using the chemical assay and not by microbiological challenge tests [4]. Preservatives are selected during formulation development [3] by preservative effectiveness test that was first described in the 18 th edition of General Chapter of USP on September 1, 1970 [5]. Preservative effectiveness test is now described in major official compendia, such as United States Pharmacopoeia (USP) [6], European Pharmacopoeia (Ph.Eur.) ...
... (4) NR stands for no recovery. (5) NI stands for no increase. It is defined as not more than 0.5 log 10 higher than the value measured at previous time point [6,8]. ...
Preservatives are some synthetic or natural antimicrobial chemicals incorporated in pharmaceutical formulations to prevent the proliferation of microorganisms that may cause product destabilization, leading to degradation, or potential life-threatening contamination with harmful microbes. They are effectively used for the production of sterile parenteral multi-dose administrations. The current methods for determination of preservative effectiveness are according to compendial methods. These methods are by simulation of the independent single challenge of bacteria, yeast, and mold into the product formulation followed by comparing their calculated recovery over a 28-day period to the established acceptance criteria. Compendial methods are not harmonized in many aspects for determination of preservative effectiveness. For example, recommended panel of microorganisms, incubation period, plating times as well as a method of recovery and acceptance criteria are not the same in these methods. Under special circumstances, World Health Organization requires that preservative effectiveness test to be performed by simulation of multiple challenges of bacteria over a 14-day period at two different incubation temperatures, followed by calculation of the recovery over a 28-day, without providing any acceptance criteria. This review article attempts to provide a unified method for determination of preservative effectiveness test in multi-dose parenteral products. It is a revised hybrid of the WHO multi-challenge method with the selection of the microorganisms listed in United States Pharmacopoeia or Japanese Pharmacopoeia and recommended European Pharmacopoeia acceptance criteria. The ultimate goal is to achieve harmonization of the preservative effectiveness test that is globally acceptable by all regulatory agencies.
... A concentração inibitória mínima pode ser utilizada para a avaliação quantitativa de diferentes formulações, porém este método não fornece informações referentes ao comportamento do produto durante o período de uso, sendo mais interessante a utilização de procedimentos analíticos que permitam avaliar o perfil de morte microbiana, a exemplo dos métodos de avaliação da eficácia de sistemas conservantes. Orth [12][13] propôs a utilização do método de regressão linear para avaliar sistemas conservantes em cosméticos, que permite calcular o tempo de redução decimal (valor D), equivalente ao tempo necessário para reduzir 90% da população microbiana. Protocolos analíticos como os métodos para avaliação da atividade germicida de sanitizantes, publicado pela AOAC 14 e para avaliação da atividade antimicrobiana de desinfetantes e antisépticos da AFNOR 15-16 também avaliam a capacidade de reduzir populações microbianas utilizadas em testes de desafio, considerando como eficazes os produtos capazes de reduzir 99,999% da população microbiana em 30 segundos de contato, no caso do método AOAC e no tempo estabelecido de uso do produto, no caso da AFNOR. ...
... O terceiro subcultivo foi quantificado de acordo com os compêndios farmacopêicos 17 , em ágar Caseína de soja (DIFCO ® ), utilizando as mesmas condições de incubação descritas anteriormente. Após a determinação da carga microbiana, a suspensão, mantida sob refrigeração, foi ajustada com solução salina estéril para conter entre 7,5 x 10 7 e 1,2 x 10 8 UFC/mL [13][14][15][16] . ...
... Preserved products must also be tested in accordance with the United States Pharmacopeia (USP) preservative efficacy test, [16] the USP antimicrobial effectiveness test, [17,18] and the International Standards Organization protocol 14730 for preservative effectiveness test standards [19,20]. ...
... Five types of microorganisms, i.e., Staphylococcus aureus TISTR 1466, Pseudomonas aeruginosa ATCC 25783, Escherichia coli ATCC 25922, Candida albicans ATCC 10231, and Aspergillus niger, were added to the anti-cellulite emgel formulation. The criteria of acceptance and the consideration of preservative stability were measured according to USP 29 Chapter 51 Antimicrobial Effectiveness [35]. ...
Recently, the herbal compress was successfully developed and applied for cellulite treatment. The aim of this study was to formulate a more convenient dosage form of herbal application from the original formula. In addition, we aimed to characterize and evaluate the stability of the developed dosage form. A gelled emulsion, or an “emgel,” incorporated with 0.1 wt% tea and coffee extracts (1:1 ratio) plus 5 wt% essential oils (mixed oil) was prepared. The caffeine content in the finished product obtained from tea and coffee extracts analyzed by HPLC was 48.1 ± 2.3 µg/g. The bio-active marker monoterpenes of mixed oil characterized by headspace GCMS were camphene 50.8 ± 1.8 µg/mg, camphor 251.0 ± 3.2 µg/mg, 3-carene 46.7 ± 1.8 µg/mg, α-citral 75.0 ± 2.1 µg/mg, β-citral 65.6 ± 1.3 µg/mg, limonene 36.8 ± 6.7 µg/mg, myrcene 53.3 ± 4.5 µg/mg, α-pinene 85.2 ± 0.6 µg/mg, β-pinene 88.4 ± 1.1 µg/mg, and terpinene-4-ol 104.3 ± 2.6 µg/mg. The stability study was carried out over a period of 3 months at 4, 25, and 50 °C. The caffeine content showed no significant changes and passed the acceptance criteria of ≥80% at all tested temperatures. However, monoterpenes showed their stability for only 2 months at 50 °C. Therefore, the shelf-life of the emgel was, consequently, calculated to be 31 months using the Q10 method. Thus, the anti-cellulite emgel was successfully formulated. The characterization methods and stability evaluation for caffeine and monoterpenes in an emgel matrix were also successfully developed and validated.
... All the glassware and media were sterilized by autoclaving at 121 • C for 2 h before the antibacterial tests. The antibacterial study of GO was evaluated with modified the United States Pharmacopoeia Antimicrobial Effectiveness Test (AET) (Sutton and Porter, 2002). The model bacterium, S. aureus, was selected to investigate the effects of concentration and contact time of GO on its antibacterial property. ...
Graphene oxide (GO) has high-efficient antibacterial activity to diverse pathogenic bacteria. However, the detailed antibacterial mechanism of GO is not fully clear. Herein the antibacterial properties of GO against model Gram-positive (Gram+) (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Gram−) bacteria (Pseudomonas aeruginosa and Escherichia coli) were compared by plate count methods. Results showed that 4 mg/L of GO induced the mortality of Gram+ and Gram− bacteria by >99% and <25%, respectively. GO had greater adsorption affinity to teichoic acids, the unique components existing in the cell wall of Gram+ bacteria, mainly via π−π interaction. The adsorption efficiency of teichoic acids was 27 times higher than that of peptidoglycan when they were simultaneously exposed to 100 mg/L GO. The superior adsorption of teichoic acids onto GO increased one order of magnitude of atlA expression, the autolysin related gene. As a result, these accelerated bacterial death by hydrolyzing peptidoglycan in cell walls. Exogenous addition of 50 mg/L teichoic acids could impair 4~5 fold of antibacterial activity of GO against S. aureus. These new findings illuminate the antibacterial mechanism of GO against Gram+ bacteria, which paves the way for the further application of graphene-based materials in water disinfection and pathogen control.
... 20 However, as preservative ingredients in the TJM technology drug delivery system, the preservative antibacterial effectiveness test is done as per USP chapter 51. 21 A pharmacokinetic study conducted in rabbits (study no: BRP17038NG, unpublished data) has shown that maximum concentration and the area under the curve achieved with TJM technology bimatoprost 0.01% is equivalent to low strength bimatoprost 0.01% marketed formulation. Therefore, to further our understanding of this new technology, the present study was undertaken to compare the efficacy and safety of the new formulation TJM bimatoprost 0.01% (TJM-bimatoprost) and marketed bimatoprost 0.01% (BKC-bimatoprost) in healthy beagle dogs. ...
Background: This study was undertaken to compare the efficacy and safety of the new technology tight junction modulation (TJM) bimatoprost 0.01% (TJM-bimatoprost), containing polyhexamethylene biguanide hydrochloride as a preservative, and marketed bimatoprost 0.01% (BKC-bimatoprost) in healthy beagle dogs.Methods: This was a cross-over study and all animals in the study were assigned to one of two treatment arms to receive either TJM-bimatoprost (n=6) or BKC-bimatoprost (n=6) ophthalmic solution. Dosing for period 1 was started on day 3 (8 AM everyday) and it continued till day 12. Assessments were carried out every day at 8 AM, 9 AM, 2 PM and 8 PM throughout the study period till day 17.Results: For the pooled analysis (n=12 in each group) of period 1 and 2, there was a significant decrease (p<0.001) in mean intra-ocular (IOP) 1-hour post administration as compared to the baseline and this trend continued all throughout the study in both treatment arms. Twenty fours after last dose, on day 12, IOP measurements were 14.20±1.59 mmHg and 13.89±1.5 mmHg in the TJM-bimatoprost and the BKC-bimatoprost group respectively. The analysis of the primary end point revealed that 95% confidence interval for the between group differences in mean IOP values were well within the pre-defined equivalence margin of ±1.5 mmHg. In terms of safety, there was no difference in mean pupillary diameter in the TJM-bimatoprost and BKC-bimatoprost group.Conclusions: The results of this study enhance our understanding of the proprietary TJM technology by establishing efficacy and safety of TJM-bimatoprost in animal models.
... Summer and Winter resins were evaluated for impact on bacterial populations for all seven bacteria listed in Table 2. Activity of resins was classified based on antimicrobial effectiveness tests [14]. Activity was categorized into three levels based on the degree of difference (x) between the response of the no-resin control and that of the 50 μL dose of resin suspension: ...
Sciadopitys verticillata (Sv) produces a white, sticky, latex-like resin with antimicrobial properties. The aims of this research were to evaluate the effects of this resin (Sv resin) on bacterial populations and to determine the impact of its primary volatile components on bioactivity. The impact of sample treatment on chemical composition of Sv resin was analyzed using Fourier transform infrared spectroscopy (FTIR) coupled with principal component analysis. The presence and concentration of volatiles in lyophilized resin were determined using gas chromatography/mass spectrometry (GC/MS). Changes in bacterial population counts due to treatment with resin or its primary volatile components were monitored. Autoclaving of the samples did not affect the FTIR spectra of Sv resin; however, lyophilization altered spectra, mainly in the CH and C=O regions. Three primary bioactive compounds that constituted >90% of volatiles (1R-α-pinene, tricyclene, and β-pinene) were identified in Sv resin. Autoclaved resin impacted bacterial growth. The resin was stimulatory for some plant and foodborne pathogens (Pseudomonas fluorescens, P. syringae, and Xanthomonas perforans) and antimicrobial for others (Escherichia coli, Bacillus cereus, Agrobacterium tumefaciens, and Erwinia amylovora). Treatment with either 1R-α-pinene or β-pinene reduced B. cereus population growth less than did autoclaved resin. The complex resin likely contains additional antimicrobial compounds that act synergistically to inhibit bacterial growth.
... The antimicrobial efficacy of benzyl alcohol in JJAV diluent was evaluated using the United States Pharmacopoeia Antimicrobial Effectiveness Test (AET) [19]. For the noninferiority analysis, we performed an independent t-test on the log transformed IgG data, and assessed whether the upper limit of the 95% confidence interval for the difference between treatments (low dose adjuvanted JJAV treatment minus standard high dose JJAV alone treatment) was below a predefined margin of non-inferiority of 0.5. ...
A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.
... godine) i bilo je predviđeno za procenu efikasnosti konzervanasa u slučaju kontaminacije proizvoda u toku procesa proizvodnje. Primenom principa Dobre proizvođačke prakse u farmaceutskoj i kozmetičkoj industriji smanjena je mogućnost ovakve kontaminacije proizvoda, a svrha ispitivanja je procena efikasnosti konzervisanja odnosno smanjenje kontaminacije proizvoda u toku čuvanja i upotrebe (49,50). Test se sastoji od veštačke kontaminacije finalnog proizvoda, nakon koje sledi procena da li je nivo smanjenja obezbedio mikrobiološke granice za proizvode kategorije 1 i 2. ...
The European regulatory framework in the field of cosmetic products requires of manufacturers to provide an assessment of microbial limits tests and results of preservation challenge test, as well as microbiological stability data which should be documented in the cosmetic product safety report. During the development of a cosmetic product, it is necessary to consider the risk of microbiological contamination in the selection of starting cosmetic ingredients and packaging, manufacturing process, alongside with conditions of storage and use. The deviation of the microbiological quality of the cosmetic product from the manufacturer's specification presents a risk for consumers, especially if it is used around the eyes, on mucous membranes in general, on damaged skin or it is intented for use in specific groups of consumers (children under the age of 3, elderly people and immunocompromised persons).
... Disadvantages of the pharmacopeia monographs are missing consideration of potential resistance of the microorganisms which increases variation and that the tests are not fully validated [63][64][65][66]. ...
Antimicrobial testing is a time consuming and cost-intensive but essential method for evaluation of newly developed pharmaceutical formulations for topical use. In this study the correlation between free preservative concentration in emulsion gels measured by equilibrium dialysis and the successful preservative effectiveness testing for Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis (analyzed according to Ph. Eur. and USP) was investigated. The higher the lipophilicity of the oil phase and the lower the content of the aqueous phase with regard to dissolved ingredients the more preferably distributed is phenoxyethanol to the water phase and, consequently, the higher was the efficacy against the microbes. Increased emulsifier concentrations reduced the free amount of the preservative due to micellar interactions. Aspergillus brasiliensis was the most resistant and Staphylococcus aureus the most sensitive germ towards phenoxyethanol in o/w-emulsion gels.
... The preservative inactivation is considered successful when the number of the microorganisms inoculated at zero time deviates by no more than 1 log10 from the one theoretically predicted. The survival rate can be either qualitatively or quantitatively evaluated [110]. Several independent researchers have applied other microorganism counting methods in the efficacy test of preservatives, including impedance, direct epifluorescence (DEF), and ATP bioluminescence (ATP-B). ...
Cosmetics, like any product containing water and organic/inorganic compounds, require preservation against microbial contamination to guarantee consumer’s safety and to increase their shelf-life. The microbiological safety has as main goal of consumer protection against potentially pathogenic microorganisms, together with the product’s preservation resulting from biological and physicochemical deterioration. This is ensured by chemical, physical, or physicochemical strategies. The most common strategy is based on the application of antimicrobial agents, either by using synthetic or natural compounds, or even multifunctional ingredients. Current validation of a preservation system follow the application of good manufacturing practices (GMPs), the control of the raw material, and the verification of the preservative effect by suitable methodologies, including the challenge test. Among the preservatives described in the positive lists of regulations, there are parabens, isothiasolinone, organic acids, formaldehyde releasers, triclosan, and chlorhexidine. These chemical agents have different mechanisms of antimicrobial action, depending on their chemical structure and functional group’s reactivity. Preservatives act on several cell targets; however, they might present toxic effects to the consumer. Indeed, their use at high concentrations is more effective from the preservation viewpoint being, however, toxic for the consumer, whereas at low concentrations microbial resistance can develop.
... The Ag/AgCl and Ag NPs produced using the same synthesis conditions employed for those used for the anti-proliferative tests were used in the assessment of antibacterial activities against Grampositive S. aureus (ATCC 25923) and B. cereus (ATCC 11778), and Gram-negative E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853). The antimicrobial effectiveness test was adopted for this assessment (Sutton & Porter, 2002). The initial concentrations of the bacterial stocks were determined based on the apparent turbidity compared with McFarland No. 0.5. ...
A facile phytosynthesis of Ag/AgCl and Ag nanoparticles (NPs) was developed using a crude water extract of Citrus hystrix DC (kaffir lime) leaves. The phytochemical and chloride contents of the extract, and various synthesis parameters were studied. The obtained Ag/AgCl and Ag NPs were of the cubic phase and mostly spherical in shape. Particle sizes of the yielded NPs were distributed in a narrow range with an average size of 20 and 38. nm for the Ag/AgCl and Ag NPs, respectively. The reducing function of the phytochemicals and the stabilizing ability of gelatin were demonstrated. The NPs showed significant anti-proliferative activities against the carcinoma HCT 116 and adenocarcinoma Caco-2 colorectal cell lines, but had a negative effect toward human fibroblasts. The NPs exhibited excellent antibacterial activities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus cereus. The inactivation kinetics obtained in the presence of the NPs showed a biphasic behavior that can be explained by the Cerf model. © 2018 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences.
... According to the USP 39 (General Chapter 51), BMicrobiological Attributes of Nonsterile Pharmaceutical Products,m icrobiological evaluation should be performed by considering the nature of product's use and its potential for causing hazard. This pharmacopeial specification considers the relation between the source of materials, the processing conditions, and the final products; and it recommends testing for the presence of USP-specified indicator organisms for a finished pharmaceutical product (30). Injections and other parenterals, including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles 2 ...
This review gives a brief overview about microbial contamination in pharmaceutical products. We discuss the distribution and potential sources of microorganisms in different areas, ranging from manufacturing sites, pharmacy stores, hospitals, to the post-market phase. We also discuss the factors that affect microbial contamination in popular dosage forms (e.g., tablets, sterile products, cosmetics). When these products are contaminated, the microorganisms can cause changes. The effects range from mild changes (e.g., discoloration, texture alteration) to severe effects (e.g., changes in activities, toxicity). The most common method for countering microbial contamination is the use of preservatives. We review some frequently used preservatives, and we describe the mechanisms by which microorganisms develop resistance to these preservatives. Finally, because preservatives are inherently toxic, we review the efforts of researchers to utilize water activity and other non-preservative approaches to combat microbial contamination.
... The purity of standard strains and the identification of waterborne bacterial isolate was conducted using diminutive biochemical identifications kits BBL™ Crystal™ enteric/non fermenter (E/NF) and Gram-positive (GP) Identification System Identification System and Gramstain reagents purchased from BD (Becton Dickinson Microbiology Systems, Cockeysville, Md.). PET study was conducted based on the method and criteria of pharmacopeial guide [15]. Neutralization procedure was done similar to that described be Eissa et al., 2014 [16]. ...
Protection of medicinal dosage forms from microbial spoilage is a mandatory requirement that must be appropriately demonstrated by the pharmaceutical firms to ensure microbiological safety of the products before reaching the market. Special attention must be brought to those multidose packaged products with significant water activity where product spoilage may easily occur due to microbial contamination from the manufacturing sites and/or during consumption. In the current study, an oral liquid medication at two strength
–
based on Prednisolone Sodium Phosphate equivalent to 5 and 15 mg/5 ml of the active pharmaceutical ingredient (API) base - was tested using graded ratio of Methyl to Propyl Parabens
–
due to precipitation of Methyl Paraben from the formula - after applying validated neutralization procedure for the antimicrobial properties of the product. Product neutralization at dilution ratio1:10 (v/v) proved to be statistically optimum for the preservative efficacy test (PET). All tested preservative-modified products formulae met antimicrobial efficacy test of United States Pharmacopeia (USP). None of the tested standard strains were recovered after 28 days testing with all results reported as <10 CFU/ml. Interestingly, inhibition of
Aspergillus niger
(
brasiliensis
) increased with the increasing concentration of Methyl Paraben with maximum activity between 1.9 and 3.0 mg/5 ml.
On the other hand,
Pseudomonas aeruginosa
and
Candida albicans
were the most sensitive along the whole concentration range. While
Staphylococcus aureus
followed by
Escherichia coli
showed relatively greater tolerance especially at the lowest API concentration formulae. The mold spores showed the greatest resistance to the product if compared with other microbes.
... According to the EP requirements (EP 2002), an oral preparation is defined to be effectively preserved if the number of bacteria and fungi recovered per gram is reduced by a factor of 10 3 , respectively 10 1 , within 14 days of challenge, with no subsequent increase at the 28 th day. The USP requirements are currently less stringent (Sutton and Porter 2002), although ICH efforts are on-going to eliminate these pharmacopoeial differences. Only formulations 9, 10, 11 and 12 did comply with all pharmacopoeial requirements, whereas the other batches complied with the USP criteria but not with the EP criteria. ...
... Microorganism selected were based on the possibility of their growth in ophthalmic solutions, which may possess maximum probability of causing eye infection (as per U.S.P, B.P) such as S. aureus, P. aeruginosa, E. Coli, C. Albicans and A. Niger. Acceptance criteria for bacteria: not less than 1.0 log reduction from the initial calculated count at 7 days, not less than 3.0 log reductions from the initial microbial count at 14 days, and no increase from the 14 days count at 28 days and for Yeast and Moulds: No increase from the initial calculated count at 7, 14 and 28 days [9][10][11]. ...
The present study was to find the role of prostaglandin (PG) analogue (Latanoprost) and an effort to develop its anti-glaucoma ophthalmic solution (using solubility agents via safe preservative system). To formulate the eye drops of Latanoprost wherein benzalkonium chloride (BKC; cause external disturbance of the corneal epithelium) was replaced with sodium perborate as the preservative using optimize concentration of surfactant's for better aqueous solubility and evaluated its effectiveness in ocular drug delivery system. The results obtained during the optimization of process in conjunction with process parameters are showed maximum solubility and stability with Tween-80 (Polysorbate-80) in water (0.25% w/v) in phosphate buffer at pH 6.8. In the presence of phosphonic acid, sodium perborate (0.3 mg) has maximum stability at pH ranging from six to seven which gave optimum preservative action during the preservative efficacy test (PET). Due to the results of the assay of the drug and preservative content, and minimizes leaching of impurities Eto sterilized vials were used for storage as compared to gamma sterilized vials. Also, the stability study (short term excursion, freeze thaw and thermal cycling studies), there is no significant change has been observed. The formulation was stable enough with better shelf-life and potency, withstand in extreme temperature condition during shipment.
... The harmonization involved almost all aspects related to the test methodology including size of the inoculum, frequency of sampling, recovery media, neutralization procedure and the assessment of results 6 . It was also pointed out that a single challenge with one level of microbial count using reference microorganisms should be employed in the evaluation studies 7 . These details are valuable as they eliminate bias in results that could arise due to the use of different test procedures Sutton 8 indicated that work done by the compendia in harmonization, assumes the equivalence of test strains from the various culture collections while, in reality this claim of equivalence might not be true beyond the culture collection catalogues. ...
The microbial challenge test used for the evaluation of preservatives efficacy in none sterile liquid pharmaceutical preparations has been recently harmonized between the United States, Europeans and Japanese pharmacopoeias. This investigation reports on the assessment of this test using 2 sets of microorganisms. The first was composed of recommended strains derived from the American Type Culture Collection and the second was of clinical isolates with multi drug resistance. Testing was carried out on a prototype antacid preparation in accordance with documented methodology. It is shown that although similar results were achieved by using either sets of cultures, many of the clinical isolates persisted in the challenged product for a longer period of time. In all cases three log reductions were obtained for the challenge organisms within one week of inoculation and remained with no increase till the end of the experiment which lasted for 28 days. It is concluded that clinical isolates with multi drug resistance can be used effectively in the test as the recommended strains provided their adaptability and potentials to grow in the un-preserved product is established. The impact of this investigation on the pharmaceutical industries of the developing countries in regard to registration of new products is discussed.
... The analytical procedures that enable the evaluation of kinetic microbial death are based on those used in the preservative efficacy testing. Orth and Sutton et al. (18,25) proposed the use of the linear regression method to evaluate the preservative efficacy of cosmetics, with calculation of D-value, which is the time required for decreasing the number of viable microorganisms in one logarithmic unit. This value is used as a predictor for responses beyond the data to estimate the time required for reaching a level of established activity. ...
The antimicrobial activity of Curcuma zedoaria (Christm) Roscoe extract against some oral microorganisms was compared with the antimicrobial activity of five commercial mouthrinses in order to evaluate the potential of the plant extract to be incorporated into formulas for improving or creating antiseptic activity. The in vitro antimicrobial efficacy of plant extracts and commercial products were evaluated against Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus and Candida albicans using a linear regression method to evaluate the microbial reduction obtained in function of the exposure time, considering as effectiveness a 99.999% reduction in count of standardized microbial populations within 60 seconds. The results showed that the antimicrobial efficacy of Curcuma zedoaria ( Christm) Roscoe extract was similar to that of commercial products, and its incorporation into a mouthrinse could be an alternative for improving the antimicrobial efficacy of the oral product.
... 10 Despite these efforts, a unique, primary risk factor for the sudden increase in cases remains undiscovered, suggesting that multiple risk factors may be involved. 5,11 Ophthalmic formulations, specifically multidose commercial preparations, are required to resist contamination as demonstrated by a standard test regimen against a small number of stock bacteria and fungi, 12 usually achieved by the addition of a preservative compound with a non-specific antimicrobial spectrum extending far beyond the test organisms. Benzalkonium chloride (BAK), the predominant ophthalmic drug preservative for the last several decades, is a quaternary ammonium surfactant with this broad range of antimicrobial activity. ...
Importance The significant antiacanthamoebal effect of benzalkonium chloride, at or below concentrations used for preservation of common ophthalmic preparations, should be understood both when choosing empiric antibiotic therapy for infectious keratitis and when assessing the persistent rise in Acanthamoeba cases in the United States since 2003.
Objective To characterize the antiacanthamoebal efficacy of low concentrations of benzalkonium chloride (BAK) for drug preservation and therapeutic effect against Acanthamoeba.
Design Experimental study with a review of the literature.
Setting Laboratory.
Exposures A concentration of 104 trophozoites of 3 well-characterized clinical strains of Acanthamoeba were exposed at 0.5, 2.0, 3.5, 5.0, and 6.5 hours to BAK (0.001%, 0.002%, and 0.003%), moxifloxacin hydrochloride (0.5%), and moxifloxacin (0.5%) + BAK (0.001% and 0.003%) with hydrogen peroxide (3%) and amoeba saline controls.
Main Outcomes and Measures Amoeba survival was calculated using the most probable number method recorded as log kill values. The relationship of BAK concentration and exposure time as well as the relative effect of BAK and moxifloxacin on acanthamoebal survival were analyzed.
Results Amoebicidal activity of BAK is both time dependent and concentration dependent in pooled and strain-stratified analyses (P < .001). Moxifloxacin demonstrated no significant independent inhibitory effect or additive effect to BAK efficacy on acanthamoebal survival. The profound antiacanthamoebal effect of BAK, 0.003%, was similar to that of hydrogen peroxide for certain strains.
Conclusions and Relevance Low concentrations of BAK, previously demonstrated to concentrate and persist in ocular surface epithelium, exhibit significant antiacanthamoebal activity in vitro at or below concentrations found in commercially available ophthalmic anti-infectives. The unexplained persistence of the Acanthamoeba keratitis outbreak in the United States, clusters abroad, and clinical studies reporting resolution or modification of Acanthamoeba keratitis without specific antiacanthamoebal therapy suggests that other contributing factors should be considered, including changes in the formulations used for empirical therapy of presumed infectious keratitis occurring in the same period.
... Likewise, propylene glycol when used at concentrations around 20% is fully effective as a preservative for molds and yeast. When DEGEE was first selected as the solvent system for a topical dapsone gel, the potential preservative properties were thoroughly evaluated using the USP preservative efficacy test 28 in which gram-positive, gram-negative, molds, and yeasts are inoculated into the product to assure that bacterial colony-forming units are quickly reduced, while molds and yeasts are not allowed to propagate. DEGEE repeatedly showed inertness with regard to microbial growth. ...
The solvent diethylene glycol monoethyl ether (DEGEE) is currently used in over 500 cosmetic products and has enabled the formulation of a topical 5% dapsone gel for the treatment of acne. It is anticipated that this common cosmetic ingredient will be a component in numerous future prescription topical products approved for the US market. Dermatologists are already treating patients that apply products containing 5-40% of this solvent multiple times each day.
To provide dermatologists a review of this solvent's safety and tolerance in addition to describing how it interacts with the stratum corneum, sebum, and resident microflora.
To critically review technical and patent literature that provides insight into this novel solvent.
Diethylene glycol monoethyl ether when used in a 99.9+% pure pharmaceutical grade is safe and well tolerated. Up to half of the applied solvent crosses the skin's barrier and becomes systemic. For certain drug actives, this solvent provides for an intracutaneous depot. This solvent has not demonstrated any inherent antimicrobial properties but was found to be mildly inhibitory toward Propionibacterium acnes.
This safe, well-tolerated solvent is already used in many cosmetics and will become an ingredient in an increasing number of prescription products. Its ability to modify the skin delivery of actives it is formulated with (or formulation components that are applied just shortly before or after) make it important for dermatologists to have an understanding of this emerging solvent.
... According to the EP requirements (EP 2002), an oral preparation is defined to be effectively preserved if the number of bacteria and fungi recovered per gram is reduced by a factor of 10 3 , respectively 10 1 , within 14 days of challenge, with no subsequent increase at the 28 th day. The USP requirements are currently less stringent (Sutton and Porter 2002), although ICH efforts are on-going to eliminate these pharmacopoeial differences. Only formulations 9, 10, 11 and 12 did comply with all pharmacopoeial requirements, whereas the other batches complied with the USP criteria but not with the EP criteria. ...
The influence of three variables, i.e. the concentrations of benzyl alcohol (BA), butylated hydroxytoluene (BHT) and tert-butyl-4-hydroxyanisol (BHA), on the preservative efficacy and antioxidant activity of an oily veterinary formulation was investigated using quantitative experimental designs and applying pharmacopoeial methods as part of the robustness-evaluation. Preservative Efficacy Tests (PETs) were performed using the validated European Pharmacpoeia (EP) methodology with 7 test-organisms over one month on lab-scale test-formulations. These were independently prepared according to a Box-Behnken experimental design with a triplicate central point at 0.75% m/V BA, 0.05% mN BHT and 0.05% m/V BHA, and with an additional control-point outside the Box-Behnken cube containing no preservative ingredient. The preservative efficacies were evaluated against the USP and EP criteria for formulations for oral use, as well as by the statistical comparison of the slopes obtained by linear regression of the log of CFU/g versus time. The peroxide values were determined after two months storage at 50 degrees C, using the EP titrimetric method. No interactions between the preservatives were observed for any of the seven tested micro-organisms in the PETs. BA had a very significant preservative effect against several of the tested microorganisms, while no antimicrobial effect for BHT and BHA was observed. Aspergillus niger was the most preservative-resistant micro-organism, while Staphylococcus aureus was the most sensitive test-germ. Compliance with USP-PET criteria was found for all formulations tested, even those without preservatives, while the EP-PET criteria showed compliance for those formulations with the highest BA concentration only. Stored in glass vials, a statistically significant antioxidant effect was demonstrated for BA only, although all tested formulations showed acceptable anti-oxidative properties. No significant antioxidant effects were shown for BHT or BHA.
... According to the PhEur requirements, [14] an oral preparation is effectively preserved if the number of bacteria, respectively fungi, recovered per gram is reduced by a factor of 10 3 , respectively 10 1 , within 14 days of challenge, with no subsequent increase at the twenty-eighth day. The USP requirements are currently less stringent, [16] although International Conference on Harmonization (ICH) efforts are ongoing to eliminate these pharmacopoeial differences. ...
The aim of this study was to statistically evaluate the influence of the concentration of the co-solvent propylene glycol on the preservative efficacy of a complex pharmaceutical suspension-emulsion formulation containing methyl- and propylparaben. Preservative Efficacy Tests (PETs) were performed using the validated pharmacopoeial methodology with five test organisms over 1 month on lab-scale test formulations. These were independently prepared according to a Box-Behnken experimental design with a triplicate central point at 0.22% m/m methylparaben, 0.22% m/m propylparaben, and 2.75% m/m propylene glycol, and with an additional corner point of the Box-Behnken cube. We evaluated the preservative efficacies against the criteria of the United States Pharmacopeia (USP) and European Pharmacopoeias (PhEur) for formulations for oral use, as well as by the statistical comparison of the slopes obtained by linear regression of log (CFU/g) vs. time. With an initial bacterial challenge of 10(6) CFU/g for each of the three bacterial strains, no survivals were detected after 7 days. For the two fungal strains, box plots and analysis of variance showed significant, concentration-dependent, main effects: the three variables significantly influenced the kill-rate of C. albicans, while A. niger was predominantly influenced by the cosolvent propylene glycol, and only to a minor extent by methylparaben and not at all by propylparaben. These findings were confirmed by taking the pharmacopoeial criteria as the evaluation basis, where the dominant influence of propylene glycol concentration is apparent. It was concluded that the cosolvent propylene glycol is at least of equal preservative importance than both parabens.
Eye drops are sterile preparations intended for instillation into the eye. All major pharmacopoeias require these products to pass the antimicrobial effectiveness test (AET). This test is similar to that used for oral dosage form despite the fact that both product categories differ in their microbiological specifications. The eye drops might pass the official requirements of the AET but in practice, contaminants introduced into the preparation might not be killed prior to its next use by the patient and this may compromise ocular health. The objective of this work was to investigate the possible application of a limited sterility testing in a multi-challenge test that mimics more closely actual life use of eye drops. The AET was performed on 12 brands of eye drops and results were compared with the suggested pass criteria of various pharmacopoeias. The multi-challenge test was designed and used to demonstrate the ability of each tested product to kill the entire challenge organism population within a few hours. The results demonstrated that all products investigated were in compliance with the AET acceptance requirements of the United States Pharmacopoeia <51> and the "B" criteria of the European Pharmacopoeia <5.1.3>. Only 2 of the tested products did not comply with the no recovery term of Ph Eur <5.1.3> 'A' criteria. Products repeatedly challenged with Pseudomonas aeruginosa ATCC 9027 (103 CFU ml-1) were found to be self-sterilizing within 2 h of each inoculation. In conclusion, all tested products passed the acceptance criteria of the USP <51>, class B of the Ph Eur <5.1.3> and the multi-challenge test. The size of the challenge organism population in the AET seems to be severe for eye drops and the pass criteria of the BP Appendix XVI is the most stringent. The no recovery term given in the Ph Eur <5.1.3> should be defined to a specified range.
Modern antimicrobial preservatives authorized for use in dosage form technology are reviewed. The nomenclature and various classifications of preservatives according to chemical nature, mechanism and spectrum of antimicrobial action, optimum effective concentrations for antimicrobial activity, and separate factors affecting the activity of antimicrobial preservatives in various dosage forms, e.g., optimum solution pH values and specific adsorbents reducing preservative activity, are presented. Antimicrobial preservatives used widely in pharmaceutical technology, i.e., parabens, sorbic acid and its salts, benzoic acid and its salts, and benzalkonium chloride, are discussed in detail. Ascience-based approach to selecting antimicrobial preservatives is shown to produce the most stable and safest medicines.
In recent years, the use of natural preservatives for protection of vegetable oils against microbial and chemical deterioration is one of the interesting issues. The purpose of this study was to evaluate the preservative activity of Satureja khuzistanica essential oil (SKEO) against microbial and chemical deterioration in sesame and flaxseed vegetable oils. Chemical composition of SKEO, chemical profiles, antioxidant and preservative potencies of inoculated vegetable oils with different concentration of SKEO were determined. Carvacrol was the main component of SKEO. The chemical profile of vegetable oils in presence of SKEO had no changes. Sesame and flaxseed vegetable oils had the IC50 equal to 26 and 22 µg/ml, respectively. Inoculation the SKEO (1%v/v) in vegetable oils decreased the IC50 for vegetable oils. SKEO showed promised antimicrobial activity against food microorganisms. Inoculation the SKEO (0.75%v/v) in sesame oil inhibited completely the bacteria and fungi after 14 days. Flaxseed oil inoculated with SKEO (1% v/v and lower concentrations) decreased the bacteria and fungi populations after 28 days. Therefore, the use of SKEO as natural preservative can protect vegetable oils from deterioration; also it gives the vegetable oils the other pharmacological effects such as anti-inflammatory and analgesic effects with applications in different industries.
Based on our previous results, which proved that each of G. glabra and R. officinalis extracts potentiate the antibacterial effect of the other aganist MRSA when combined together at their sub-MIC. We thought to investigate the capacity of Glycyrrhiza glabra(Licorice) and Rosmarinus officinalis (Rosemary) extracts to act as preservatives for topical formulations, which in our knowledge is the first to be used together. Two Oil/Water(O/W )cream were formulated : using methyl paraben, a common used preservative (MP) and the combination between Licorice and Rosemary (LR) as preservatives, were tested for their Primary skin irritation on Laboratory experimental animals which proved that they were devoid of any primary skin irritation, erythema, or edema even after 48 h of application, and by challenging them with microbial indicators: Bacterial; (Escherichia Coli 25922, Pseudomonas aeruginosa 27853 and Staphylococcus aureus 29737), Yeast; (Candida albicans10231), and fungi; (Aspergillus niger 1015), revealed that the concentration of each test microorganisms decreased during the test period.
This chapter will discuss microbiology as it pertains to Cleaning Validation. In practice, the primary focus of Cleaning Validation is the removal of chemical residues, either from active ingredients or cleaning agents, and microbiological issues are of an incidental nature. It should be understood that the purpose of cleaning procedures should never be seen as being used to reduce microbial residues to acceptable levels.
Background: In this study antimicrobial effectiveness test was performed on eye-drops which had high microbial contaminations in hospital practice to find out whether their antimicrobial efficacies affect the magnitude of microbial contamination during their uses. Materials and Methods: Artificial tear, atropine sulfate, betamethasone, homatropine hydrobromide, phenylephrine hydrochloride, phenylephrine zinc, pilocarpine hydrochloride, tetracaine hydrochloride and tropicamide eye-drops were subjected to the United States Pharmacopeia (USP) and British Pharmacopeia (BP) antimicrobial preservative effectiveness tests. Results: The results of this study showed that eight out of the nine products met the BP 'B' and USP criteria. The preservative employed in phenylephrine zinc eye-drop did not possess adequate antimicrobial activity against P. aeruginosa. Other eye-drops showed appropriate reductions in bacterial viability after 6 hrs, 24 hrs and 7 days, but showed a very low bacterial recovery after 28 days which didn't comply with the no recovery (NR) term of BP 'A' criteria. Since viable microbial counts were usually determined by plate count method, it seems that the term of NR should define an acceptable range. Conclusion: The results indicated that there is not a clear correlation between antimicrobial efficacy testing of eye-drops and the rate of their microbial contamination while are being used. Other factors such as hygienic practices of eye-drops, proper bottle design and training of patients could influence their microbial contaminations. Regulation of in-use efficacy testing of eye-drops which is influenced by the environment, the frequency and technique of use, might be essential.
Background: In this study antimicrobial effectiveness test was performed on eye-drops which had high microbial contaminations in hospital practice to find out whether their antimicrobial efficacies affect the magnitude of microbial contamination during their uses. Materials and Methods: Artificial tear, atropine sulfate, betamethasone, homatropine hydrobromide, phenylephrine hydrochloride, phenylephrine zinc, pilocarpine hydrochloride, tetracaine hydrochloride and tropicamide eye-drops were subjected to the United States Pharmacopeia (USP) and British Pharmacopeia (BP) antimicrobial preservative effectiveness tests. Results: The results of this study showed that eight out of the nine products met the BP 'B' and USP criteria. The preservative employed in phenylephrine zinc eye-drop did not possess adequate antimicrobial activity against P. aeruginosa. Other eye-drops showed appropriate reductions in bacterial viability after 6 hrs, 24 hrs and 7 days, but showed a very low bacterial recovery after 28 days which didn't comply with the no recovery (NR) term of BP 'A' criteria. Since viable microbial counts were usually determined by plate count method, it seems that the term of NR should define an acceptable range. Conclusion: The results indicated that there is not a clear correlation between antimicrobial efficacy testing of eye-drops and the rate of their microbial contamination while are being used. Other factors such as hygienic practices of eye-drops, proper bottle design and training of patients could influence their microbial contaminations. Regulation of in-use efficacy testing of eye-drops which is influenced by the environment, the frequency and technique of use, might be essential. INTRODUCTION Ophthalmic drops are sterile preparations which are usually packed in multi-dose containers. In their uses, microbial contamination may lead to product degradation or result in ocular infection (1-4). Protection of these multiple dose products against microbial contamination is usually achieved by addition of a suitable preservative system (5-7). The antimicrobial effectiveness test is designed to provide a laboratory test that gauges the level of antimicrobial activity by a pharmaceutical product and to evaluate how well a product withstands microbial contamination while being used (8, 9). The method is similar in both British Pharmacopeia (BP) (10) and United States Pharmacopeia (USP) (11), but sampling times and logarithmic (log) reduction performance criteria of the BP are more stringent than those in the USP. It has been reported that there is a correlation between the performance of eye-drops according to the BP antimicrobial efficacy test and magnitude of microbial contamination during their uses (12), suggesting other investigators to extend similar studies on other multi-dose products. The aim of this study was to determine the antimicrobial efficacy of eye-drops produced by Iranian manufacturers according to the both United States and British Pharmacopeia to assess the correlation of antimicrobial performance of the eye-drops with magnitude of microbial contamination during their uses.
The objective of this study was to investigate the application of established D-value calculations to survival curves for various bacteria using the following antimicrobials: acidified sodium chlorite, triclosan, octanoic acid, and sodium hydroxide. D-values can be calculated in 3 ways, a linear regression, an endpoint calculation, or an average of multiple endpoint calculations. The assumption made in calculating a D-value is that the rate of kill follows 1st-order kinetics under specified treatment conditions. Each antimicrobial solution was challenged with approximately 108 CFU/mL of Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica subsp. enterica, and Escherichia coli independently and in triplicate. Test systems were sampled at each of the 10 time points over a period of 7 min, neutralized, pour plated then incubated at 35 °C for 48 h (AOAC official method 960.09). Survival curves using the log-transformed data were calculated using regression analysis. Correlations coefficients for all linear regression analyses ranged between 0.291 and 0.982, with 6 of the 16 different treatment systems having an R2 value below 0.7. Methods used for calculating D-values should lead to the same result if the survival curve in a given condition is linear. The calculated D-values were different using endpoint analysis (Stumbo method), linear regression, and average of multiple endpoints. This study demonstrates the nonlinearity of inactivation curves of antimicrobials. D-value estimations cannot be reliably used to illustrate biocidal activity in antimicrobial test systems.
Long-term intravenous infusion of epoprostenol and treprostinil for treatment of pulmonary arterial hypertension (PAH) via a central venous catheter is associated with the risk of bloodstream infection (BSI). While several potential explanations exist for possible differences in BSI incidence among intravenous prostanoids, one hypothesis suggests that the alkaline pH of epoprostenol in Sterile Diluent for Flolan (SDF) has greater antimicrobial activity compared with the neutral pH of other common diluents such as sterile saline or water, which have been used for treprostinil.
The chemical stability and antimicrobial activity of 4 microg/ml and 130 microg/ml treprostinil in SDF were assessed according to United States and European Pharmacopeia.
At both concentrations, treprostinil in SDF remained stable after incubation at 40 degrees C and ambient relative humidity for up to 52 h. Solution pH also remained stable (range 10.4-10.6), and the solutions were essentially free of particulate at all time points examined. Antimicrobial activity was measured using an antimicrobial effectiveness test after inoculation with five species of bacteria, yeast and mould. The antimicrobial activity of both concentrations of treprostinil met United States Pharmacopeia requirements. Further, the antimicrobial activity of treprostinil in SDF against gram-negative bacteria (> 4 log(10) reductions) exceeded that previously described for treprostinil in sterile saline.
These results suggest that dilution of treprostinil with the alkaline solution SDF may reduce the risk of infection from inadvertent patient contamination compared with dilution of treprostinil in sterile saline.
This article is a comprehensive review of the published activities of the Analytical Microbiology Expert Committee (AMB) for the 2000-2005 revision cycle. The major thrust of the activities during this revision cycle were directed at international harmonization, and to provide guidance in the changing field of pharmaceutical microbiology. In addition to reviewing the changes accomplished, this article discusses the rationale for many of the changes and some background information regarding new initiatives underway. Where appropriate, changes in the USP that did not fall under the direct purview of the AMB Expert Committee (EC) but of interest to the microbiology community are also discussed.
It is difficult to predict accurately the ultimate effectiveness of a preservative in any but the simplest cosmetic or pharmaceutical formulation. It is thus necessary to obtain some assurance of its likely in-use performance in a formulated product. A challenge test is a procedure in which a product is challenged by exposure to specified types of bacteria and fungi to determine whether it is adequately preserved. Assessment of preservative efficacy is needed over the intended shelf-life of that product. Test organisms should be representative of those likely to occur as contaminants during use and should consist of Gram-positive and Gram-negative bacteria, mould and yeast. In-house factory isolates obtained as a result of contamination of earlier batches of a product may also be included. The organisms, as single or mixed inocula, are inoculated (usually as a single challenge, although some advocate multiple challenges) into samples of the product and aliquots removed at appropriate intervals for the determination of survivors. Interpretation of data is normally based on pharmacopoeial or other official protocols. Challenge testing should be undertaken at the beginning, during and at the end of the shelf-life of the product. An alternative, the D-value, approach is open to criticism and further studies are required that utilize rapid methods, e.g. impedance, for the detection of survivors before these can be considered to be a suitable replacement for the more traditional but very time-consuming, viable counting procedures.
Determination of a D value for specific test organisms is a component of the efficacy evaluation of new contact lens disinfecting solutions. This parameter is commonly defined as the time required for the number of surviving microorganisms to decrease 1 logarithmic unit. The assumption made in establishing a D value is that the rate of kill exhibits first-order kinetics under the specified conditions. Such exponential kill rates are seen with thermal contact lens disinfection system. A comparison of the death rate kinetics for a variety of chemical contact lens disinfecting solutions was undertaken to ascertain the suitability of D-value determination for these chemical disinfectants. The active agents of these different solutions included hydrogen peroxide, thimerosal, chlorhexidine, tris(2-hydroxyethyl)tallow ammonium chloride, thimerosal, polyaminopropyl biguanide, and polyquaternium-1. The solutions were challenged with 10(6) CFU of either Pseudomonas aeruginosa, Serratia marcescens, or Staphylococcus hominis per ml, and survival rate was determined. This study clearly demonstrates the nonlinear nature of the inactivation curves for most contact lens chemical disinfecting solutions for the challenge organisms. D-value determination is, therefore, an inappropriate method of reporting the biocidal activity of these solutions.
The dispensing closure used for containers plays an important role in protecting cosmetics from in-use microbial contamination. This hypothesis was tested by aseptically packing unpreserved shampoo and skin lotion into containers with three different closure types which provided various degrees of protection against consumer and environmental microbial insults. Shampoo was packed in containers with slit-cap (n = 25), flip-cap (n = 25), or screw-cap (n = 28) closures. Skin lotion was packed in containers with pump-top (n = 21), flip-cap (n = 18), or screw-cap (n = 21) closures. The products were then used by volunteers under actual in-use conditions for 3 (shampoo) or 2 (skin lotion) weeks. After use, the products were evaluated for microbial contamination by using standard methods for enumeration and identification. The standard screw-cap closure provided only minimal protection against microbial contamination of both the shampoo (29% contamination incidence) and the skin lotion (71%). The slit-cap closure on the shampoo container and the flip-cap closure on the skin lotion container provided slightly enhanced degrees of protection (21 and 39% contamination incidence, respectively). The greatest amount of protection (i.e., lowest contamination incidence) was provided by the flip-cap closure for the shampoo container (0%) and the pump-top closure for the skin lotion container (10%). As a result, closure type plays an important role in protecting poorly preserved products from in-use microbial contamination.
We initiated a comparative study of four methods of yeast inoculum preparation: a spectrophotometric method, the Wickerham card method, a hemacytometer method, and the Prompt inoculation system. The variability in inoculum size obtained when each method was applied to two strains each of Candida albicans, Candida tropicalis, Candida parapsilosis, Torulopsis glabrata, Cryptococcus neoformans, and Saccharomyces cerevisiae was analyzed in a single laboratory. Each method was performed in triplicate on the same day and on three separate days to provide estimates of within-day and between-day variations. Inoculum size was determined by viable colony counts. The greatest range of inoculum sizes was seen with the Wickerham card method. Viable counts ranged from 1.1 X 10(6) to 24.2 X 10(6) CFU/ml among the 12 yeast isolates. The greatest variation was observed with the Prompt system. Within-day coefficients of variation averaged 19% (range, 4 to 45%), and between-day coefficients of variation averaged 22% (range, 3 to 51%). Variation between laboratories was evaluated by comparing inoculum values obtained by each method in three different laboratories for two strains of C. albicans. The spectrophotometric method was the least variable and the Wickerham card and hemacytometer methods were the most variable methods between laboratories. The spectrophotometric method is recommended as the method of choice for preparation of a standardized inoculum suspension for susceptibility testing of yeasts.
The decimal reduction time (D-value) is the time required for inactivation of 90% of the population of test organisms subjected to a lethal agent. The D-value is calculated from the plot of the log number of surviving organisms/g as a function of the time after inoculation into the product. This value provides a quantitative expression of the rate of death of specific test organisms in a particular product. COSMETIC PRESERVATIVE EFFICACY is established by determining the D-VALUES for several test organisms used to challenge the cosmetic products, and seeing if these values meet the acceptance criteria of D-values _
Preservative efficacy tests were performed in triplicate on each of three batches of three formulated nasal spray preparations to assess the inter- and intra-batch variation in preservative performance which typically results from these procedures, and to assess the relative importance of factors influencing preservative performance in nasal products.
Tests were conducted using procedures conforming, as far as possible, to both the European and the US pharmacopoeias and the results interpreted using the performance criteria of both. Despite the adoption of practices designed to maximize reproducibility, a marked variation in the degree of microbial inactivation was observed, both within and between batches of product.
A preservative system comprising benzalkonium chloride and phenylethyl alcohol was found to be far superior to combinations of either benzalkonium chloride plus disodium edetate or potassium sorbate plus disodium edetate, both of which failed to satisfy the EP performance criteria on a number of occasions. Proposals are made for the adoption of inactivation criteria which incorporate realistic error limits reflecting the inherent problems of reproducibility of the viable counting procedures involved.
Spray application of cycloplegics and mydriatics is efficacious and frequently easier to use than a standard dropper bottle in the pediatric population. However, no documentation regarding the sterility of drugs dispensed from spray bottles is available. This study was conducted to determine whether contamination of ophthalmic drugs occurs with spray bottle use.
Fifteen milliliters of 1% cyclopentolate hydrochloride or 0.5% tetracaine hydrochloride were transferred to each of 15 disinfected spray bottles, stored at room temperature or refrigerated, and sprayed three times weekly for 12 weeks. Cultures were obtained from the spray bottles and drugs before transfer of the drug and from spray bottle contents at 0, 2, 4, and 6 to 12 weeks of storage.
No cultures showed significant bacterial growth. The bactericidal action of the preservative and sterility of the drugs were maintained.
Despite the transfer to and use of a spray bottle there appears to be minimal risk of instilling contaminated diagnostic drugs using the spray method when a single drug is stored in a spray bottle.
To evaluate and compare the microbial contamination arising from 1 and 2 weeks' use of eye drops by hospital inpatients and hence determine the validity of apportioning a 2 week in use expiry date for these preparations.
Eye drop residues were collected from inpatients of Worthing, Southlands, and Brighton General hospitals after 7 days' use (341 samples) and also after 14 days' use (295 samples). The contents of the containers were examined for the presence of contaminating bacteria and fungi.
The incidence of microbial contamination was shown to be not significantly different (p > 0.1 chi 2 test) between the 7 and 14 day samples. In addition, the contaminating micro-organisms were of a broadly similar pattern between the two sample groups and were mostly those normally associated with the skin. Less frequent contaminants were organisms of environmental origin. None of the micro-organisms isolated were considered to be of clinical significance and the mean number of cells found per sample was very low.
The evidence therefore suggests that increasing the period of use for eye drops in hospitals from 7 to 14 days would not present a clinically significant threat to patients' health and yet may lead to annual savings to the NHS of Pounds 0.5 million.
The abilities of nine antimicrobial systems to preserve an experimental water-based cosmetic formulation were evaluated by six microbiological challenge tests: the U.S. Pharmacopeia test; the British Pharmacopeia test; the Cosmetic, Toiletry, and Fragrance Association test; the rapid screen test; the sequential challenge test; and the post-use test. The antimicrobial systems contained various combinations and amounts of two parabens and a quaternary compound in order to provide a broad range of preservation. The results obtained were compared with the abilities of the formulations to support maintenance and growth of microorganisms in microfloras obtained from human axilla areas and finger skin during an 8-week simulated in-use test. Without statistical analysis all of the tests predicted the results obtained with well-preserved or poorly preserved formulations. The rapid screen test was the best test for predicting differences at intermediate levels of preservation. Statistically, all of the tests were equivalent predictors of preservation efficacy in the in-use test (P = 0.05). At the P = 0.10 level, only the U.S. Pharmaceopeia, British Pharmacopeia, rapid screen, Cosmetic, Toiletry, and Fragrance Association tests were significantly predictive. The results of prediction by a test, based on the preservative levels used, agreed well with the in-use test results (P = 0.01). A total of 20% of the formulations that contained excessive microbial levels contained human axilla microorganisms. The levels of preservation in failed products were similar to the levels of preservation in unused controls.
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Note for guidance on maximum shelf-life of sterile products for human use after first opening or following reconstitution
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CPMP/QWP/159/96, corr "Note for guidance on
maximum shelf-life of sterile products for human
use after first opening or following reconstitution,"
effective July 1998.
Note for guidance on in-use stability testing of veterinary medicinal products (excluding immunological veterinary medicinal products)
- Emea Cvmp
EMEA/CVMP/424/01, "Note for guidance on in-use
stability testing of veterinary medicinal products
(excluding immunological veterinary medicinal
products)," effective September 2002.
Ranitidine Oral Solution," First Supplement to The United States Pharmacopeia 22
The United States Pharmacopeial Convention, Inc.,
(effective January 1, 1990), "Ranitidine Oral Solution," First Supplement to The United States Pharmacopeia 22/National Formulary 17, Rockville,
MD., p 2167.
Linear regression method for rapid determination of cosmetic preservative efficacy
- D S Orth
Orth, D. S., (1979), "Linear regression method for
rapid determination of cosmetic preservative efficacy," J. Soc. Cosmet. Chem., 30, 321-332.
Note for guidance on in-use stability testing of human medicinal products
- Cpmp Qwp
CPMP/QWP/2934/99 "Note for guidance on in-use
stability testing of human medicinal products." Effective September 2001.
Eighth Supplement to The United States Pharmacopeia 23
The United States Pharmacopeial Convention, Inc.,
(effective May 15, 1998), Eighth Supplement to The
United States Pharmacopeia 23/National Formulary
18, Rockville, MD, p. 1681.
The United States Pharmacopeial Convention The United States Pharmacopeia 19 th Revision
The United States Pharmacopeial Convention, Inc.,
(effective July 1, 1975), The United States Pharmacopeia 19 th Revision, Rockville, MD., p. 587.
The United States Pharmacopeial Convention The United States Pharmacopeia 20 th Revision
The United States Pharmacopeial Convention, Inc.,
(effective July 1, 1980), The United States Pharmacopeia 20 th Revision, Rockville, MD., p. 873.
- M Leitz
Leitz, M., (1972), "Critique of U.S.P. microbiological test," Bull Parenter Drug Assoc., 26 (5), 212216.
Note for guidance: In-use stability testing of veterinary medicinal products
- Emea Cvmp
EMEA/CVMP/127/95. "Note for guidance: In-use
stability testing of veterinary medicinal products,"
effective September 1996.
The United States Pharmacopeial Convention
The United States Pharmacopeial Convention, Inc.,
(effective January 1, 1985), The United States Pharmacopeia 21 st Revision Rockville, MD. p. 1151.