Chemical Disinfectants, Antiseptics and Preservatives PDF

343 18 Chemical Disinfectants, Antiseptics and Preservatives Sean P. Gorman1 and Brendan F. Gilmore2 1 Emeritus Professor of Pharmaceutical Microbiology, Queen’s University Belfast, Belfast, UK 2 Professor of Pharmaceutical Microbiology, School of Pharmacy, Queen’s University Belfast, Belf...

343 18 Chemical Disinfectants, Antiseptics and Preservatives Sean P. Gorman1 and Brendan F. Gilmore2 1 Emeritus Professor of Pharmaceutical Microbiology, Queen’s University Belfast, Belfast, UK 2 Professor of Pharmaceutical Microbiology, School of Pharmacy, Queen’s University Belfast, Belfast, UK CONTENTS 18.1 Introduction, 344 18.1.1 European Union Regulation, 344 18.1.2 Definitions, 345 18.1.2.1 Disinfectant and Disinfection, 345 18.1.2.2 Antiseptic and Antisepsis, 345 18.1.2.3 Preservative and Preservation, 346 18.1.3 Economic Aspects, 346 18.2 Factors Affecting Choice of Antimicrobial Agent, 346 18.2.1 Properties of the Chemical Agent, 346 18.2.2 Microbiological Challenge, 346 18.2.2.1 Vegetative Bacteria, 347 18.2.2.2 Mycobacterium tuberculosis, 347 18.2.2.3 Bacterial Spores, 347 18.2.2.4 Fungi, 347 18.2.2.5 Viruses, 347 18.2.2.6 Protozoa, 349 18.2.2.7 Prions, 349 18.2.3 Intended Application, 350 18.2.4 Environmental Factors, 350 18.2.5 Toxicity of the Agent, 350 18.3 Types of Compound, 352 18.3.1 Acids and Esters, 352 18.3.1.1 Benzoic Acid, 352 18.3.1.2 Sorbic Acid, 352 18.3.1.3 Sulphur Dioxide, Sulphites and Metabisulphites, 352 18.3.1.4 Esters of p-­Hydroxybenzoic Acid (Parabens), 354 18.3.2 Alcohols, 354 18.3.2.1 Alcohols Used for Disinfection and Antisepsis, 354 18.3.2.2 Alcohols as Preservatives, 354 18.3.3 Aldehydes, 355 18.3.3.1 Glutaraldehyde, 355 18.3.3.2 Ortho-­phthalaldehyde, 355 18.3.3.3 Formaldehyde, 356 18.3.3.4 Formaldehyde-­releasing Agents, 356 18.3.4 Biguanides, 356 18.3.4.1 Chlorhexidine, 356 18.3.4.2 Polyhexamethylene Biguanides, 357 18.3.5 Halogens, 357 18.3.5.1 Chlorine, 357 18.3.5.2 Hypochlorites, 357 18.3.5.3 Organic Chlorine Compounds, 357 Hugo and Russell’s Pharmaceutical Microbiology, Ninth Edition. Edited by Brendan F. Gilmore and Stephen P. Denyer. © 2023 John Wiley & Sons Ltd. Published 2023 by John Wiley & Sons Ltd. Companion website: https://www.wiley.com/go/HugoandRussells9e 344 18 Chemical Disinfectants, Antiseptics and Preservatives 18.3.5.4 Chloroform, 358 18.3.5.5 Iodine, 358 18.3.5.6 Iodophors, 358 18.3.6 Heavy Metals, 359 18.3.6.1 Silver, 359 18.3.6.2 Copper, 359 18.3.6.3 Mercurials, 359 18.3.7 Hydrogen Peroxide and Peroxygen Compounds, 359 18.3.8 Phenols, 360 18.3.8.1 Phenol (Carbolic Acid), 361 18.3.8.2 Clear Soluble Fluids, Black Fluids and White Fluids, 361 18.3.8.3 Synthetic Phenols, 361 18.3.8.4 Bisphenols, 361 18.3.9 Surface-­active Agents, 361 18.3.9.1 Cationic Surface-­active Agents, 361 18.3.10 Other Antimicrobials, 362 18.3.10.1 Diamidines, 362 18.3.10.2 Dyes, 362 18.3.10.3 Quinoline Derivatives, 362 18.3.11 Antimicrobial Combinations and Systems, 363 18.4 Disinfection Policies, 363 References, 364 Further Reading, 364 18.1 ­Introduction ‘active substances and preparations containing one or more active substances, put up in the form in which they are sup- Disinfectants, antiseptics and preservatives are chemical plied to the user, intended to destroy, deter, render harm- agents that have the ability to destroy, inactivate or inhibit less, prevent the action of, or otherwise exert a controlling the growth of microorganisms. They play a major role in effect on any harmful organism by chemical or biological the control of infection and contamination in medical and means’. The BPD had been transposed into domestic UK health care, and also find widespread use in the law by the Biocidal Products Regulation (BPR) 2001 ([SI management of livestock, the environment, paints and 2001/880] as amended). The BPD was superseded by the coatings, plastics, pharmaceutical, food and beverage EU BPR (Regulation (EU) 528/2012), the text of which was manufacture, textiles, the catering industry and consumer adopted in May 2012, entered into force in July 2012 and products. The term biocide is typically used to describe took effect in member states from September 2013, repeal- chemical agents employed to control harmful organisms ing and replacing the BPD (Directive 98/8/EC). The EU more broadly; this term is used widely throughout BPR concerns the placing on the market, and the use, of European Union (EU) directives. Microbicides, agents biocidal products intended to protect humans, animals, specifically used to control microorganisms, fall under this materials or articles against harmful pests or bacteria, general definition of biocide. through the action of active substances contained in a bioc- idal product. The BPR aims to improve the functioning of 18.1.1 European Union Regulation the biocidal products market within the EU member states, and ensure high levels of protection of human and animal Regulation of biocides continues to develop in the health and the environment, and applies within the European Union and many other countries wherein European Economic Area (EEA), including Iceland, guidance is defined for both the manufacturers and the Liechtenstein and Norway alongside EU member states. users of these products. Between 2000 and 2012, the The EU BPR aims to harmonise the internal market for Biocidal Products Directive (BPD) 98/8/EC and the related biocidal products, introducing several important changes directives – Medicinal Products for Human Use Directive to the previous regulations which include: 2001/83/EC and the Veterinary Medicinal Products Directive 2001/82/EC – governed the production, extending the scope of the regime to cover treated arti- marketing and use of non-­agricultural products intended cles and materials containing biocides; for biocidal purposes. Additionally, ‘agricultural pesticides’ adopting a community authorisation scheme for certain were regulated by the Plant Protection Products Directive types of products; 91/414/EC. The BPD regulated biocides are defined as requiring mandatory data-­sharing;  18.1 ­Introductio 345 Table 18.1 Levels of disinfection attainable when products are used according to manufacturer’s instructions. Disinfection level Low Intermediate High Microorganisms killed Most vegetative bacteria Vegetative bacteria including All microorganisms unless an Some viruses M. tuberculosis extreme challenge or resistance Some fungi Most viruses including hepatitis exhibited B virus (HBV) Most fungi Microorganisms surviving M. tuberculosis Bacterial spores Extreme challenge of resistant Bacterial spores Prions bacterial spores Prions Prions reducing the burden of data collection requirements; microorganisms but reducing them to a level acceptable for harmonising fee structures across member states. a defined purpose, for example, a level that is harmful nei- ther to health nor to the quality of perishable goods. The EU BPR defines a treated article as ‘any substance, Chemical disinfectants are capable of different levels of mixture or article which has been treated with, or action (Table 18.1). The term high-­level disinfection intentionally incorporates, one or more biocidal products’. indicates destruction of all microorganisms, but not Following the end of the ‘transition period’ on 31 necessarily bacterial spores; intermediate-­level disinfection December 2020, Great Britain no longer participates in the indicates destruction of all vegetative bacteria including EU scheme for biocidal products regulation. Prior to the Mycobacterium tuberculosis, but may exclude some United Kingdom’s exit from the European Union, the UK resistant viruses and fungi and implies little or no sporicidal Government Health and Safety Executive (HSE) was activity; low-­level disinfection can destroy most vegetative engaged in negotiations with all EU member states, the bacteria, fungi and viruses, but this will not include spores European Commission and the European Parliament in and some of the more resistant microorganisms. Some the development of a new directly acting EU law, and con- high-­level disinfectants have good sporicidal activity and tributed to the drafting and approval of the EU Biocidal are described as liquid chemical sterilants or chemosterilants Products Regulation (EU BPR 528/2012). As a result, on to indicate that they can effect a complete kill of all leaving the European Union, the existing EU BPR was microorganisms, as in sterilisation. In defining each of copied into British law, as the GB Biocidal Products these disinfection levels, the activity and outcome are Regulation (GB BPR) 2020. The GB BPR controls biocidal determined by correct use of the disinfectant product in products in Great Britain (England, Scotland and Wales), regard to concentration, time of contact and prevailing while the EU BPR controls biocidal products in Northern environmental conditions as described in subsequent Ireland. A number of minor differences between GB BPR sections of this chapter. and the EU BPR exist, which essentially allow GB to make different decisions to the EU, EEA and Switzerland and 18.1.2.2 Antiseptic and Antisepsis develop its own programmes for review of existing active Antisepsis, a specific type of disinfection, is defined as substances; however, the core regulatory instruments are destruction or inhibition of microorganisms on living largely identical. tissues having the effect of limiting or preventing the harmful results of infection. It is not a synonym for disinfection. The chemicals used are applied to skin and 18.1.2 Definitions mucous membranes, so as well as having adequate Some key definitions relevant to chemical microbicides are antimicrobial activity they must not be toxic or irritating given below. Other terms used to describe antimicrobial for these tissues. Antiseptics are mostly used to reduce the activity are considered in Chapter 19. microbial population on the skin before surgery or on the hands to help prevent spread of infection by this route. 18.1.2.1 Disinfectant and Disinfection Sanitisation, a regulated term, refers to the use of Disinfection is the process of removing microorganisms, disinfectants for reduction, not necessarily elimination, of including pathogens, from the surfaces of inanimate microorganisms from surfaces to levels considered objects. The British Standards Institution (BSI) further acceptable or safe as determined by public health regula- defines disinfection as not necessarily killing all tions. Therefore, sanitisation has a specific meaning with 346 18 Chemical Disinfectants, Antiseptics and Preservatives respect to disinfection applications or antisepsis, namely, ammonium compounds [QACs] and amine compounds), the removal or inactivation of microorganisms that pose a organometallics, organosulphurs, chloroisocyanurates threat to public health. The use of alcohol-­based hand sani- and phenolics. There are around 250 chemicals that have tisers has become increasingly common globally as an been identified as active components of microbicidal infection control measure during the COVID-­19 pandemic. products in the EU. Antiseptics are sometimes formulated as products contain- The aim of this chapter is to introduce the range of ing significantly lower concentrations of agents used for chemicals in common use and to indicate their activities disinfection. and applications. 18.1.2.3 Preservative and Preservation Preservatives are included in pharmaceutical and many 18.2 ­Factors Affecting Choice other types of formulations, both to prevent microbial of Antimicrobial Agent spoilage of the product and to minimise the risk to the consumer of acquiring an infection when the preparation Choice of the most appropriate antimicrobial compound is administered (see Chapter 17). Preservatives must be for a particular purpose depends on many factors and the able to limit proliferation of microorganisms that may be key parameters are described below. introduced unavoidably into non-­sterile products such as oral and topical medications during their manufacture and use. In sterile products, where multiuse preparations 18.2.1 Properties of the Chemical Agent remain available, preservatives should kill any microbial The process of killing or inhibiting the growth of contaminants introduced inadvertently during use. It is microorganisms using an antimicrobial agent is basically essential that a preservative is not toxic in relation to the that of a chemical reaction and the rate and extent of this intended route of administration of the preserved reaction will be influenced by concentration of agent, preparation. Preservatives tend to be employed at very low temperature, pH and formulation. The significance of concentrations and consequently levels of antimicrobial these factors on activity is considered in Chapter 19, and is action also tend to be of a lower order than for disinfectants referred to when discussing the individual agents in or antiseptics. This is illustrated by the British, European Section 18.3. Tissue toxicity will influence whether a and US pharmacopoeial requirements for preservative chemical can be used as an antiseptic or preservative, and efficacy where a degree of bactericidal activity is necessary, this limits the range of agents for these applications or although this should be obtained within a few hours or necessitates the use of lower concentrations of that agent. over several days of microbial challenge depending on the This is discussed further in Section 18.2.5. type of product to be preserved. 18.2.2 Microbiological Challenge 18.1.3 Economic Aspects The types of microorganism present and the levels of The international antimicrobial chemical market, particu- microbial contamination (the bioburden) both have a larly in the case of disinfectants, is expected to grow sig- significant effect on the outcome of treatment. If the nificantly over the coming years, on the basis of concerns bioburden is high, long exposure times and/or higher about bacterial and other pathogenic threats and the concentrations of antimicrobial may be required. increasing emphasis on hygiene in the home and work- Microorganisms vary in their sensitivity to the action of place. In particular, the SARS-­CoV-­2 pandemic has signifi- chemical agents. Some organisms merit attention either cantly increased demand for disinfectant and antimicrobial because of their resistance to disinfection (for further chemicals; this is expected to continue to drive significant discussion, see Chapter 20) or because of their significance growth in the sector, even post pandemic, as consumer in cross-­infection or healthcare-­associated infections (see and workplace practices with respect to disinfection, sani- Chapter 16). Of particular concern is the significant tisation and hygiene are likely to be altered in the longer increase in resistance to disinfectants resulting from term. The global value of the antimicrobial and disinfect- microbial growth in the biofilm form rather than free ant chemical market was estimated at $9.1 billion in 2019 suspension (see Chapter 8). Microbial biofilms form and is projected to increase to $17.1 billion by 2027, repre- readily on available surfaces, posing a serious problem for senting a combined annual growth rate of 6.7% over hospital infection control committees in advising suitable this period. Key disinfectant products in use contain disinfectants for use in such situations. Recently, the ­aldehydes, iodophors, nitrogen compounds (quaternary description of dry surface biofilms (dehydrated biofilms  18.2 ­Factors Affecting Choice of Antimicrobial Agen 347 which have been shown to exist on dry inanimate sur- contaminated with mycobacteria if the patient is a carrier faces in healthcare, food processing and domiciliary envi- of this organism. It is important to be able to disinfect the ronments) with their demonstrable environmental equipment to a safe level to prevent transmission of persistence and significantly elevated tolerance to infection to other patients (Table 18.2). antimicrobial challenge (even compared with hydrated or 18.2.2.3 Bacterial Spores wet biofilms) highlights yet another potential challenge to selection of an appropriate disinfectant agent or Bacterial spores can exhibit significant resistance to even approach. Dry surface biofilms, as yet, lack a clear or the most active chemical disinfectant treatment. The agreed definition in the literature, or standardised test majority of disinfectants have no useful sporicidal action in methods, further complicating appropriate disinfectant a pharmaceutical context, which relates to disinfection of selection. materials, instruments and environments that are likely to The efficacy of an antimicrobial agent must be be contaminated by the spore-­forming genera Bacillus, investigated by appropriate capacity, challenge and in-­use Clostridioides and Clostridium. However, certain aldehydes, tests to ensure that a standard is obtained which is halogens and peroxygen compounds display very good appropriate to the intended use (Chapter 19). In practice, it activity under controlled conditions and are sometimes is not usually possible to know which organisms are used as an alternative to physical methods for sterilisation present on the articles being treated. Thus, it is necessary to of heat-­sensitive equipment. In these circumstances, categorise agents according to their antimicrobial activity correct usage of the agent is of paramount importance, as and for the user to be aware of the level of antimicrobial safety margins are lower in comparison with physical action required in a particular situation (see Table 18.1). methods of sterilisation (Chapter 21). Clostridioides difficile is a particularly problematic 18.2.2.1 Vegetative Bacteria contaminant in hospital environments, resulting in high At in-­use concentrations, chemicals used for disinfection levels of morbidity and mortality. In addition to stringent should be capable of killing bacteria and other organisms handwashing, meticulous environmental disinfection expected in that environment within a defined contact procedures must be in place, for example, using solutions period. This includes ‘problem’ organisms such as of 5.25–6.15% sodium hypochlorite for routine disinfection. methicillin-­resistant Staphylococcus aureus (MRSA), When high-­level disinfection of C. difficile is required, 2% vancomycin-­resistant enterococci (VRE) and species of glutaraldehyde, 0.55% o-­phthalaldehyde and 0.35% per- Listeria, Campylobacter and Legionella. Antiseptics and acetic acid are effective. preservatives are also expected to have a broad spectrum of The antibacterial activity of some disinfectants and antimicrobial activity, but at their in-­use concentrations, antiseptics is summarised in Table 18.2. after exerting an initial biocidal (killing) effect, their main function may be biostatic (inhibitory). Gram-­negative 18.2.2.4 Fungi bacilli, which are a major cause of healthcare-­associated The vegetative fungal form is often as sensitive as vegetative infections, are often more resistant than Gram-­positive bacteria to chemical antimicrobial agents. Fungal spores species. Pseudomonas aeruginosa, an opportunistic (conidia and chlamydospores; see Chapter 4) may be more pathogen (see also Chapter 7), has gained a reputation as resistant, but this resistance is of much lesser magnitude the most resistant of the Gram-­negative organisms. than that exhibited by bacterial spores. The ability to rapidly However, problems mainly arise when a number of destroy pathogenic fungi – such as the important nosocomial additional factors such as heavily soiled articles or diluted pathogen Candida albicans, filamentous fungi such as or degraded disinfectant solutions are employed. Trichophyton mentagrophytes and spores of common spoilage moulds such as Aspergillus niger – is put to advantage 18.2.2.2 Mycobacterium tuberculosis in many applications of use. Many disinfectants have good M. tuberculosis and other mycobacteria are resistant to activity against these fungi (Table 18.3). In addition, ethanol many bactericides. Resistance is either (i) intrinsic, mainly (70%) is rapidly and reliably active against Candida species. due to reduced cellular permeability, or (ii) acquired, due to mutation or the acquisition of plasmids (Chapter 13). 18.2.2.5 Viruses Tuberculosis remains an important public health hazard, Susceptibility of viruses to antimicrobial agents can depend and indeed the annual number of tuberculosis cases on whether or not the viruses possess a lipid envelope. continues to rise in many countries. The greatest risk of Non-­lipid viruses are frequently more resistant to acquiring infection is from the undiagnosed patient. disinfectants and it is also likely that such viruses cannot Equipment used for respiratory investigations can become be readily categorised with respect to their sensitivities to 348 18 Chemical Disinfectants, Antiseptics and Preservatives Table 18.2 Antibacterial activity of commonly used disinfectants and antiseptics. Activity against Class of compound Mycobacteria Bacterial spores General levela of antibacterial activity Alcohols Ethanol/isopropyl + − Intermediate Aldehydes Glutaraldehyde + + High o-­Phthalaldehyde + + High Formaldehyde + + High Biguanides Chlorhexidine − − Intermediate Halogens Hypochlorite/chloramines + + High Iodine/iodophor + + Intermediate, problems with Ps. aeruginosa Peroxygens Peracetic acid + + High Hydrogen peroxide + + High Phenolics Clear soluble fluids + − Intermediate Chloroxylenol − − Low Bisphenols − − Low, poor against Ps. aeruginosa Quaternary ammonium compounds Benzalkonium − − Intermediate Cetrimide − − Intermediate a Activity expected per manufacturer’s instructions and will depend on environmental conditions and bioburden. Table 18.3 Antifungal activity of disinfectants and antiseptics. Time (minutes) to give >99.99% killa of Antimicrobial agent Aspergillus niger Trichophyton mentagrophytes Candida albicans Phenolic (0.36%) 5 × 105 2.6–4.5 min at 54 °C, 60% relative humidity and 600 mg l–1 EO Formaldehyde Bacillus atrophaeus ATCC 9372 >5 × 105 – 7 Ionising radiation Bacillus pumilus ATCC 27.142 >1 × 10 1.9 kGy a American Type Culture Collection (ATCC). aseptically transferred to an appropriate nutrient medium, the ability of a filter to produce a sterile filtrate from a which is then incubated and periodically examined for ­culture of a suitable organism. For this purpose, Serratia signs of growth. Spores of G. stearothermophilus in sealed marcescens, a Gram-­negative rod-­shaped bacterium (mini- ampoules of culture medium are used for steam sterilisa- mum dimension 0.5 μm), has been used for filters of tion monitoring, and these may be incubated directly at 0.45 μm pore size, and a more rigorous test involving 55 °C; this eliminates the need for an aseptic transfer. Brevundimonas diminuta ATCC 19146 (formerly Aseptic transfers are also avoided by the use of self-­ Pseudomonas diminuta) having a minimum dimension of contained units where the spore strip and nutrient medium 0.3 μm is applied to filters of 0.22 μm pore size and are present in the same device ready for mixing after use. Acholeplasma laidlawii is applied to filters of 0.1 μm pore The bacterial species to be used in a BI must be selected size. The 0.22 μm pore size filters are defined as those capa- carefully, as it must be non-­pathogenic and should possess ble of completely removing B. diminuta from suspension. above-­average resistance to the particular sterilisation pro- This test uses a realistic inoculum level of B. diminuta of cess. Resistance is defined by a spore destruction curve 107 cells cm−2 of filter area. The extent of the passage of obtained via test exposure to the sterilisation process; rec- this organism through membrane filters is enhanced by ommended BI spores and their decimal reduction times increasing the filtration pressure. Consequently, success- (D-­values; Section 21.2.2.1) are shown in Table 21.7. Care ful sterile filtration depends markedly upon the challenge must be taken in the preparation and storage of BIs to conditions. Such tests are used as part of the filter quality ensure a standardised response to sterilisation processes. assurance process, and a user’s initial validation proce- While certainly offering the most direct method of moni- dure. They are not employed as a test of filter perfor- toring sterilisation processes, it should be realised that BIs mance in use. may be less reliable monitors than physical methods and they are not recommended for routine use, except in the case of gaseous sterilisation. Additionally, there is a need to 21.13 ­Sterility Testing maintain the resistance characteristics of the BI which can be lost on repeated laboratory subculture. A sterility test is essentially a test which assesses whether a The long-­standing criticism of BIs is that the incubation sterilised pharmaceutical or medical product is free from period required in order to confirm a satisfactory sterilisa- contaminating microorganisms by incubation of either the tion process imposes an undesirable delay on the release of whole or a part of that product with a nutrient medium. It the product. This problem has been overcome for steam therefore becomes a destructive test and is of questionable sterilisation by using a detection system in which a spore suitability for testing large, expensive or delicate products enzyme, α-­glucosidase (reflective of spore viability), con- or equipment. Furthermore, by its very nature it is a statis- verts a non-­fluorescent substrate into a fluorescent product tical process in which part of a batch is sampled and the in as little as 1 hour. chance of the batch being passed for use then depends on Filtration sterilisation requires a different approach the sample passing the sterility test. Random sampling from biological monitoring, the test effectively measuring should therefore be applied to products that have been 428 21 Sterilisation Procedures and Sterility Assurance processed and filled aseptically. With products sterilised in sterilisation process may be impaired and thus must be their final containers, however, samples should be taken given the best possible conditions for growth. from the potentially coolest or least sterilant-­accessible part of the batch. 21.13.1.1 Direct Inoculation Limitations are inherent in any procedure intended to The direct inoculation method involves introducing test demonstrate a negative, and as the sterility test is intended samples directly into nutrient media. The European to demonstrate that no viable organisms are present, fail- Pharmacopoeia recommends two media: (i) fluid mercap- ure to detect them could simply be a consequence of the toacetate medium (also known as fluid thioglycolate use of unsuitable media or inappropriate culture condi- medium), which contains glucose and sodium mercaptoac- tions. To demonstrate that no organisms are present, it etate (sodium thioglycolate) and is particularly suitable for would be necessary to use a universal culture medium the cultivation of anaerobic organisms (incubation tem- suitable for the growth of every possible contaminant and perature 30–35 °C); and (ii) soyabean casein digest medium to incubate the sample under an infinite variety of condi- (also known as tryptone soya broth), which will support tions. Clearly, no such medium or combination of media the growth of both aerobic bacteria (incubation tempera- is available and in practice only media capable of support- ture 30–35 °C) and fungi (incubation temperature ing non-­fastidious bacteria, yeasts and moulds are 20–25 °C). Limits are placed upon the ratio of the weight or employed. Furthermore, in pharmacopoeial tests, no volume of added sample relative to the volume of culture attempt is made to detect viruses, which, on a size basis, medium so as to avoid reducing the nutrient properties of are the organisms most likely to pass through a sterilising the medium or creating unfavourably high osmotic pres- filter. Nevertheless, the sterility test does have an impor- sures within it. Other media may be used provided that tant application in monitoring the microbiological quality they can be shown to be suitable alternatives or if the iden- of filter-­sterilised, aseptically filled products and does tification of specific organisms is required. For example: offer a final check on terminally sterilised articles. In the UK, test procedures laid down by the European MacConkey agar, which is a selective and differential Pharmacopoeia remain applicable and must be followed; medium for the detection of coliforms and enteric patho- this provides details of the sample sizes to be adopted in gens according to the European Pharmacopoeia (agar particular cases. medium H). The Harmonised European Pharmacopoeia proposes reinforced Columbia agar medium containing gen- 21.13.1 Sterility Testing Methods tamicin sulphate (20 mg l–1) as a selective enrichment Three alternative methods are available when conduct- media for Clostridia. ing sterility tests according to the European Cetrimide agar is used in the isolation and identification Pharmacopoeia: direct inoculation, membrane filtration of Pseudomonas aeruginosa. Cetrimide is a quaternary and addition of concentrated culture medium. When ammonium compound (see Chapter 18) that inhibits undertaking sterility tests, it is necessary to conduct the growth of a wide range of Gram-­positive and some control tests that confirm the adequacy of the testing Gram-­negative microorganisms. facilities by sampling of air and surfaces in addition to Mannitol salt agar is used to isolate pathogenic staphylo- carrying out tests using samples ‘known’ to be sterile cocci, selectively inhibiting the growth of most other (negative controls). In practice, this means samples that bacteria as a consequence of its high salt concentration. have been subjected to a very reliable sterilisation pro- Pathogenic strains of staphylococci (coagulase-­positive cess, for example, radiation, or samples that have been staphylococci) produce yellow colonies with yellow subjected to repeat sterilisation procedures. In order to zones. Non-­pathogenic staphylococci usually produce minimise the risk of introducing contaminants from the small red colonies with no colour change to the surround- surroundings or from the operator during the test itself, ing medium. testing must be carried out under conditions of strict Xylose lysine deoxycholate agar is used for the isolation asepsis, for example, under laminar airflow; isolators are and differentiation of enteric pathogens, e.g., Salmonella, often employed which physically separate the operator which can reduce sodium thiosulphate producing hydro- from the materials under test. gen sulphide which creates a complex with ferric ammo- The culture media employed in sterility tests should pre- nium citrate giving black or black-­centred colonies. viously have been assessed for nutritive (growth-­ Sabouraud dextrose agar is used to cultivate yeasts, supporting) properties and a lack of toxicity using specified moulds and aciduric microorganisms as designated by organisms. It must be remembered that any survivors of a the European Pharmacopoeia. Dextrose is fermented  21.13 ­Sterility Testin 429 providing carbon and energy. The high dextrose concen- Table 21.8 Inactivating agents (neutralising agents). tration and acidic pH make this medium selective for fungi. Modification with cycloheximide, penicillin and Inhibitory agents Inactivating agents streptomycin additions aids selection from heavily con- taminated sources. Phenols, cresols None (dilution) Alcohols None (dilution) Parabens Dilution and Tween 21.13.1.2 Membrane Filtration Membrane filtration is the sterility testing technique rec- Mercury compounds Sulphydryl-­containing compounds ommended by most pharmacopoeias and, consequently, is the method by which most products are examined. The Quaternary ammonium Lecithin + Lubrol W; Lecithin + compounds Tween (Letheen) method involves filtration of fluids through a sterile mem- Benzylpenicillina, β-­Lactamase from Bacillus cereus brane filter (pore size 0.45 μm) and any microorganism ampicillin present will be retained on the surface of the filter mem- Other antibioticsa None (membrane filtration) brane. After washing in situ, the filter is divided aseptically Sulphonamides p-­Aminobenzoic acid and portions are transferred to suitable culture media (such as those above) which are then incubated at the a See text. appropriate temperature for the required period of time. Water-­soluble solids can be dissolved in a suitable diluent and processed in this way, and oil-­soluble products may be possible inactivating agents, for example, chloramphenicol dissolved in a suitable solvent, for example, isopropyl acetyl-­transferase (inactivates chloramphenicol) and myristate, before filtering. enzymes that modify aminoglycoside antibiotics (acetyl- transferases, nucleotidyltransferases or phosphotrans- 21.13.1.3 Addition of Concentrated ferases which catalyse modifications at hydroxyl or amine Culture Medium groups of the 2-­deoxystreptamine nucleus or the sugar The concentrated culture medium sterility test is a highly moieties). sensitive method for detecting low levels of contamination in intravenous infusion fluids. A concentrated culture 21.13.2.2 Dilution medium is added to the fluid in its original container, so that The antimicrobial agent is diluted in the culture medium the resultant mixture is equivalent to single strength culture to a level at which it ceases to have any activity, for exam- medium; this enables the sampling of the entire volume. ple, phenols, cresols and alcohols (see Chapter 19). This method applies to substances with a high dilution coeffi- cient, η, and may be accompanied by specific inactivation 21.13.2 Antimicrobial Agents (Table 21.8). Where an antimicrobial agent comprises the product or 21.13.2.3 Membrane Filtration forms part of the product, for example, as a preservative, its This method has traditionally been used to overcome the activity must be nullified in some way during sterility testing activity of antibiotics for which there are no inactivating so that an inhibitory action in preventing the growth of any agents, although it could be extended to cover other prod- contaminating microorganisms is overcome. This is achieved ucts if necessary, including preservatives if no specific or by specific inactivation, dilution or membrane filtration. effective inactivators are available. Basically, a solution of the product is filtered through a hydrophobic-­edged 21.13.2.1 Specific Inactivation membrane filter that will retain any contaminating micro- An appropriate inactivating (neutralising) agent (Table 21.8) organisms. The membrane is washed in situ to remove any is incorporated into the culture medium. The inactivating traces of antibiotic adhering to the membrane and is then agent must be non-­toxic to microorganisms, as must any transferred to appropriate culture media as outlined in product resulting from an interaction of the inactivator and Section 21.13.1.2. the antimicrobial agent. Although Table 21.8 lists only benzylpenicillin and ampi- cillin as being inactivated by β-­lactamase (from Bacillus 21.13.3 Positive Controls cereus), other β-­lactams may also be hydrolysed by β-­ lactamases. Other antibiotic-­inactivating enzymes are also It is essential to show that microorganisms will actually known (see Chapter 13) and have been considered as grow under the conditions of the test. For this reason, 430 21 Sterilisation Procedures and Sterility Assurance positive controls must be carried out; in these, the ability of Example: small numbers of suitable microorganisms to grow in If a sample of two items is taken from a large batch con- media in the presence of the sample is assessed. The micro- taining 10% contaminated containers, then the probability organism used for positive control tests with a product con- of a single item taken at random being contaminated (p) is taining or comprising an antimicrobial agent must, if at all possible, be sensitive to that agent, so that growth of the p 0.1 where10% 0.1 organism indicates a satisfactory inactivation, dilution or removal of the agent. The European Pharmacopoeia sug- whereas the probability of such an item being uncontami- gests the use of designated strains of Staphylococcus aureus nated (q) is given by ATCC 6538, B. subtilis ATCC 6633, P. aeruginosa ATCC q 1 p 0.9 9027, Escherichia coli ATCC 8739 and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028 as appro- The probability of both items being contaminated is priate aerobic organisms, Clostridium sporogenes ATCC given by 11437 as an anaerobe and Candida albicans ATCC 10231 or Aspergillus brasiliensis ATCC 16404 (formerly Aspergillus p2 0.01 niger) as fungi. A positive control (medium with added test sample) and a negative control (medium without the test and of both items being uncontaminated by sample) are inoculated simultaneously, and the rate and 2 extent of growth arising in each should be similar. However, q2 1 p 0.81 the negative control without the test sample is, in effect, exactly the same as the growth promotion control that is The probability of obtaining one contaminated item and also described in the test procedure, so, for the organisms one non-­contaminated item is concerned, it is not necessary to do both. 1 0.01 0.81 0.18 2 pq All the controls may be conducted either before, or in parallel with, the test itself, providing that the same batches of media are used for both. If the controls are carried out in In a sterility test involving a sample size of n containers, parallel with the tests and one of the controls gives an the probability P of obtaining n consecutive ‘steriles’ is unexpected result, the test for sterility may be declared given by invalid, and, when the problem is resolved, the test may be n qn 1 p repeated. The European Pharmacopoeia states for batches of 21.13.4 Specific Cases greater than 500 items that the sample size required is 20. Values for various levels of p (i.e., the proportion of con- Specific details of the sterility testing of parenteral prod- taminated containers in a batch) with a constant sample ucts, ophthalmic and other non-­injectable preparations size are given in Table 21.9, which shows that the test can- and surgical sutures will be found in the European not detect low levels of contamination. Similarly, if differ- Pharmacopoeia. These procedures cannot conveniently be ent sample sizes are employed (also based on (1 − p)n), it applied to items such as surgical dressings and medical can be shown that as the sample size increases, the proba- devices because they are too big. In such cases, the most bility of the batch being passed as sterile decreases. convenient approach is to immerse the whole object in cul- It is clear that a sterility test can only show that a propor- ture medium in a sterile flexible bag, but care must be tion of the products in a batch is sterile. The correct conclu- taken to ensure that the liquid penetrates to all parts and sion to be drawn from a satisfactory test result is that the surfaces of the material. batch has passed the sterility test, but this is not a guaran- tee that the entire batch is sterile. 21.13.5 Sampling A sterility test attempts to infer the state (sterile or non-­ 21.13.6 Retests sterile) of a batch from the results of an examination of part of a batch, and is therefore a statistical procedure. Under certain circumstances, a sterility test may require Suppose that p represents the proportion of contaminated repeating, but the only justification for a second iteration containers in a batch and q the proportion of uncontami- of testing is unequivocal evidence that the first test was nated containers, then, p + q = 1 or q = 1 − p. invalid; a retest cannot be viewed as a second opportunity  Further Reading 431 Table 21.9 Sampling in sterility testing. Contaminated items in batch (%) 0.1 1 5 10 20 50 p 0.001 0.01 0.05 0.1 0.2 0.5 q 0.999 0.99 0.95 0.9 0.8 0.5 Probability (P), of drawing 20 0.98 0.82 0.36 0.12 0.012 500 items. for the batch to pass when it has failed the first time. monitoring of a particular sterilisation process. The steril- Circumstances that may justify a retest would include, for ity test on its own provides no guarantee as to the sterility example, failure of the air filtration system in the testing of a batch; however, it is an additional check that will facility which might have permitted airborne contami- detect gross failure, and continued compliance with the nants to enter the product or media during testing, non-­ test does give confidence as to the efficacy of a sterilisation sterility of the media used for testing, or evidence that or aseptic process and this can contribute to sterility assur- contamination arose during testing from the operating per- ance. Failure to carry out a sterility test where required, sonnel or a source other than the sample being tested. despite the major criticism of its inability to detect other than gross contamination, may have important legal and 21.13.7 The Role of Sterility Testing moral consequences. The techniques discussed in this chapter comprise an attempt to achieve, as far as possible, the continuous References DH (2014). Health Technical Memorandum. Decontamination DH (2016b). Health Technical Memorandum. HTMs. London: Department of Health. Decontamination of Linen for Health and Social Care (HTM DH (2016a). Health Technical Memorandum. 01-­04). London: Department of Health. Decontamination of Surgical Instruments (HTM 01-­01). ISO 11140-­1:2014 (2014). Sterilization of Health Care Products – London: Department of Health. Chemical Indicators – Part 1: General Requirements. Geneva: International Organization for Standardization. Further Reading Baird, R.M., Hodges, N.A. and Denyer, S.P. (2000). European Pharmacopoeia, 10. (2021) Council of Europe, Handbook of Microbiological Quality Control: Strasbourg. (This pharmacopoeia consists of volumes and Pharmaceuticals and Medical Devices. London: Taylor & supplements. The most recent should be consulted.) Francis. Fraise, A.P., Maillard, J.-­Y. and Sattar, S.A. (2013). Russell, British Pharmacopoeia (2021). The Stationery Office, London. Hugo and Ayliffe’s Principles and Practice of Disinfection, (The most recent edition should be consulted). Preservation and Sterilization, 5e. Oxford: Wiley-­Blackwell. Denyer, S.P. and Baird, R.M. (2007). Guide to Microbiological McEvoy, B. and Rowan, N.J. (2019). Terminal sterilization of Control in Pharmaceuticals and Medical Devices, 2e. medical devices using vaporised hydrogen peroxide: a London: CRC Press. review of current methods and emerging opportunities. EDQM (2020). Chapter 5.8 Pharmacopoeial harmonisation. J. Appl. Microbiol. 127: 1403–1420. In: European Pharmacopoeia, 10e. Strasbourg: Council MHRA (2022). The Green Guide: Rules and Guidance for of Europe. Pharmaceutical Distributors, 5e. London: Pharmaceutical Press. 432 21 Sterilisation Procedures and Sterility Assurance Sandle, T. (2013). Sterility, Sterilisation and Sterility Assurance and distinctive characteristics. In: BIODEVICES 2020 -­ for Pharmaceuticals. Cambridge: Woodhead 13th International Conference on Biomedical Electronics Publishing Ltd. and Devices, Proceedings; Part of 13th International Joint Zhdanov, A.E., Pahomov, I.M., Ulybin, A.I. and Borisov, V.I. Conference on Biomedical Engineering Systems and (2020). Low temperature plasma vacuum sterilization of Technologies, BIOSTEC 2020 (ed. Y. Xuesong, A. Fred and medical devices by using SterAcidAgent®: description H. Gamboa), 86–93.  433 Part 6 Pharmaceutical Production  435 22 Sterile Pharmaceutical Products and Principles of Good Manufacturing Practice Tim Sandle Head of Compliance and Quality Risk Management, Bio Products Laboratory, Elstree, UK CONTENTS 22.1 ­Introduction, 436 22.2 ­Defining Sterility, 437 22.3 ­Sterilisation Methods, 437 22.3.1 Factors Affecting Sterilisation, 438 22.3.1.1 Number and Location of Microorganisms, 438 22.3.1.2 Microbial Quality of Starting Materials, 438 22.3.1.3 Innate Resistance of Microorganisms, 438 22.3.1.4 Physical and Chemical Factors, 439 22.3.1.5 Organic and Inorganic Matter, 439 22.3.1.6 Duration of Exposure, 439 22.3.1.7 Storage, 439 22.4 ­Demonstrating Sterility, 439 22.5 ­Types of Sterile Product, 440 22.5.1 Injections, 440 22.5.1.1 Formulation Considerations, 440 22.5.1.2 Intravenous Infusions, 441 22.5.1.3 Small-volume Injections, 441 22.5.1.4 Freeze-­dried Products, 441 22.5.1.5 Water for Injection, 442 22.5.2 Non-­injectable Sterile Fluids, 442 22.5.2.1 Non-­injectable Water, 442 22.5.2.2 Urological (Bladder) Irrigation Solutions, 442 22.5.2.3 Peritoneal Dialysis Solutions, 442 22.5.2.4 Inhaler Solutions, 442 22.5.3 Ophthalmic Preparations, 442 22.5.3.1 Formulation Considerations, 442 22.5.3.2 Eye Drops, 442 22.5.3.3 Eye Lotions, 443 22.5.3.4 Eye Ointments, 443 22.5.3.5 Contact Lens Solutions, 443 22.5.4 Dressings, 443 22.5.5 Implants, 443 22.5.6 Absorbable Haemostats, 443 22.5.7 Surgical Ligatures and Sutures, 443 22.5.8 Instruments and Equipment, 444 22.5.9 General Considerations, 444 22.6 ­Good Manufacturing Practices for Sterile Products, 444 22.6.1 Regulatory Framework, 444 22.6.2 In-­process Controls and Quality Control (QC), 446 22.7 ­Sterility Assurance and the Manufacture of Sterile Products, 446 Hugo and Russell’s Pharmaceutical Microbiology, Ninth Edition. Edited by Brendan F. Gilmore and Stephen P. Denyer. © 2023 John Wiley & Sons Ltd. Published 2023 by John Wiley & Sons Ltd. Companion website: https://www.wiley.com/go/HugoandRussells9e 436 22 Sterile Pharmaceutical Products and Principles of Good Manufacturing Practice 22.8 T­ erminal Sterilisation and Aseptic Processing, 446 22.9 ­Cleanrooms and Facility Design, 447 22.9.1 Design of Premises, 448 22.9.2 Internal Surfaces, Fittings and Floors, 448 22.9.3 Services, 449 22.9.4 Air Supply, 449 22.10 ­Operating Principles for Aseptic Processing, 451 22.10.1 Sterile Filtration, 451 22.10.2 Managing Aseptic Assembly, Connections and Interventions, 452 22.10.3 Transfer of Materials into and out of Aseptic Processing Areas, 452 22.11 ­Minimising Human Intervention, 453 22.11.1 Blow–Fill–Seal Technology, 453 22.11.2 Restricted Access Barrier Systems, 453 22.11.3 Isolators, 453 22.11.4 Single-­use Sterile Disposable Technology, 454 22.12 ­Personnel, 455 22.13 ­Media Simulation Trials, 455 22.14 ­Quality Risk Management, 457 22.15 ­Environmental Monitoring, 457 22.16 ­Release of Sterile Products, 459 22.16.1 Assessments of Sterility, 459 22.16.2 Assessments of Pyrogenicity, 459 22.16.3 Visible Particulates, 459 22.17 Summary, 460 Acknowledgements, 460 Reference, 460 Further Reading, 460 22.1 ­Introduction pharmaceutical medicines. This is not necessarily due to any intrinsic formulation complexities in the products Injections, infusions and pharmaceutical forms for appli- themselves but because those medicines, due to their route cation to eyes and mucous membranes must meet the of administration, are required to be sterile at the point requirement to be sterile; this is also the case for a range of they are administered to the patient. It is not possible to medical devices. Sterility is important, since these products determine to what extent compromised sterility would generally breach or compromise host defences exposing affect an individual patient. This is because people are indi- the patient to infection from contaminating microorgan- vidually unique in relation to form and physiology; it is isms (see Chapter 17). A large proportion of sterile phar- also because the context of administration and treatment maceutical preparations are given by injection (see will vary widely between individuals. Nonetheless, a con- Section 22.5). Products administered by injection are often taminated product, especially one administered intrave- called parenteral products, deriving from the Greek and nously (via a vein) or intrathecally (via the brain or the meaning any route other than through the gut. Thus, cer- spinal cord), is likely to cause harm or even death. tain medicines such as peptides, proteins and many chem- In addition to the medicinal product, the various compo- otherapeutic agents which would be inactivated in the nents required for the production, safe transport, storage gastrointestinal tract are given parenterally. and use of sterile products are equally as important. These Achieving sterility is not straightforward, whether the items also need to be sterile when required for aseptic pro- product is produced by terminal sterilisation (see cessing post-­sterile filtration, whether they are large Chapter 21) or by assembly from sterile ingredients in a stainless-­steel mixing vessels subjected to steam sterilisa- clean environment (aseptic processing). The development tion using an autoclave, product packaging, or ready-­ and production of sterile medicinal products, whether in assembled sterile disposable kits, sterilised using radiation large-­scale pharmaceutical processing or small-­scale bio- or gas. For other parts of pharmaceutical manufacturing, technology, as medicines made on a named-­patient basis ‘sanitised’ equipment (of a low bioburden, typically