T E C H N O L O G YCLEANROOM

December 2013

THE INTERNATIONAL JOURNAL OF CONTAMINATION CONTROL

GETTING TO GRIPS WITHCHLORINE DISINFECTANTS

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● Single-use solutions for scaling up biopharmaceutical manufacturing

● Pet project: modular cleanroom for veterinary cancer treatments

● Innovative pack makes dispensing ofophthalmic drops safer and easier

CLT1213frontcover1_001clt1007cover 29/11/2013 13:57 Page 1

30 CLEANROOM TECHNOLOGY December 2013 www.cleanroomtechnology.com

The science of chlorine-baseddisinfectantWhen selecting the ideal cleanroom disinfectantthere are many criteria to consider and a goodunderstanding of the product characteristics isessential. Karen Rossington takes a look at thehistoric development and latest advancements inchlorine disinfection

Asuccessful cleanroom disinfectantneeds to meet many criteria, not onlyin terms of its efficacy but also in

terms of packaging, ease of use and operatoracceptability. Much has been written on howto specify and select a cleanroomdisinfectant and this article thereforeprovides a brief overview of requirementsthat will help when comparing availablechlorine disinfectant chemistries.

Taking as a given good broad spectrumefficacy, including highly resistant bacterial

spores, the requirements for the idealcleanroom disinfectant are quite lengthy: asterile option for grade A and Benvironments;1 non-inflammable so can itbe used over large areas with no health andsafety concerns; also fast drying with shortcontact times to reduce the time taken forbiodecontamination. However, in our idealworld this cannot be traded for problemswith equipment or our operators, or thewider environment in terms of disposal.

Another requirement is that cleaning

processes leave no disinfectant residue or, asa minimum, free rinsing residues. This isbecause no residue means there is no needfor residue removal and this, in turn, canshorten the decontamination process.

The product will need to have in excess ofa 12 month unopened shelf life and in excessof three months in-use shelf life to bepractical to store and use.

This ideal disinfectant would then need tobe manufactured to the requirements ofcGMP, be notified to the BPR2 and providedin cleanroom compatible packaging in avariety of formats so that it is suitable for usein all areas of the cleanroom.

It goes without saying that this all needs tobe achieved in a cost-effective formulation.Many people believe that this ideal sporicidedoes not exist and a compromise mustalways be made.

On first reflection, a chlorine-baseddisinfectant would not necessarily spring tomind as the disinfectant that meets all ofthese ideal needs. Sodium hypochlorite isthe most widely used chlorine-based surfacedisinfectant but has some known drawbacks,notably: inactivation in organic matter; it iscorrosive to some metals; and it will leave aparticulate residue. However, all chlorinedisinfectants are not the same. Advances inproduction methodologies have allowed thecreation of a disinfectant that meets all of therequirements above.

Chlorine’s long historyOne of the first known uses of chlorine fordisinfection was in the form of hypochloriteknown as chloride of lime. Snow used it in18503 after an outbreak of cholera to attemptto disinfect the Broad Street Pump watersupply in London. Berthollet, in 1785,prepared a bleaching agent by dissolving‘Scheele’s gas’ in water and in 1789 improvedit by mixing it with a solution of causticpotash (KOH). This was carried out in aFrench chemical plant in Javel and is stillknow as Javelle water to this day. A shortwhile later Labarraque replaced theexpensive potassium hydroxide with causticsoda; this development resulted in what wasprobably the first use of sodium hypochloriteas bleach.

Chlorine first began to be used as adisinfectant in the late 1800s to early 1900s.Liquid bleach – sodium hypochlorite – cameinto widespread use in the 1930s and today itis the most widely used of all the chlorinatedbleaches. Hypochlorite solutions were usedfor the treatment of open wounds duringWorld War I and led to the use of on-sitegeneration of hypochlorite in hospitals.These went out of favour until the 1990s,which saw a great surge in the interest of on-site generation of chlorine.

These on-site generators provide asolution containing only 0.8% chlorine,

Cleanroom disinfectantsmust have good materialcompatibility

disinfectants

www.cleanroomtechnology.com December 2013 CLEANROOM TECHNOLOGY 31

which is non-hazardous; however,hazardous hydrogen gas is produced as a by-product. On-site generation is also quiteinefficient compared with bulk productionand has high associated electricity costs.

Dry calcium hypochlorite appeared on theUS market in 1928. This bleaching agentcontains up to 70% active chlorine and isalso known as High Test Hypochlorite. It isavailable in a variety of forms includingpowder, granules, briquettes and tablets.

Calcium hypochlorite systems tend to beused for small water treatment plants andwell systems. Calcium hypochlorite has avery strong oxidising potential andconsequently is very dangerous to store anduse. Calcium hypochlorite dissolved in watergenerates 2 mols of hypochlorous acid forevery 1 mol of calcium hypochlorite.

Chlorine as a disinfectantNot all chlorine species are equally effectiveas disinfectants. Many studies have exploredthe mechanism of chlorine disinfection andalthough it is not possible to explainprecisely how each particular chlorinespecies works, current theory4 believes thatinactivation occurs by means of one or moreof the following mechanisms: inactivation ofthe key enzymes; disruption of nucleic acidsrendering them non-functional; andoxidative damage to cell walls or other vitalcell components. For each of themechanisms described above theeffectiveness of each disinfecting agent is afunction of both its rate of diffusion throughthe cell wall and it reactivity with the cellwall, proteins and nucleic acid.

Hypochlorous acid (HOCL) is the mosteffective disinfectant in the chlorine familyavailable in dilute solution. It is suggestedthat HOCL is 80 to 120 times moreefficacious than sodium hypochlorite.5

Because HOCL has no charge and has arelatively low molecular weight it is betterable than the other chlorine baseddisinfectants to penetrate the cell walls. Italso reacts more rapidly than other chlorine-based disinfectants to oxidation reactionswith organic matter, i.e. the criticalcomponents of microbial cells. Converselythe hypochlorite ion is a relatively poor

disinfectant because of its inability to diffusethrough the cell wall. Since it is negativelycharged it is electrostatically repelled fromthe cell walls, which are also negativelycharged. It is much larger in size than anHOCL molecule so it also diffuses moreslowly due to its larger size.

Chlorine chemistryChlorine is added to water in one of threeforms: elemental chlorine (chlorine gas),sodium hypochlorite solution or calciumhypochlorite powder (high-testhypochlorite). Chlorine gas reacts rapidlywith water to form two compounds:hypochlorous acid (HOCl) and hydrochloricacid (HCl).

Cl2 + H2O X HOCl + HClChlorine water hypochlorus hydrochloric

acid acid

Hypochlorous acid (HOCl) is a weak acidthat further dissociates into the hypochlorite

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Test Description Log Time Test Description Log Time (min)reduction reduction

EN1276 E.hirae >log 7 1min EN13704 C.sporogenes > log 6 1 min

EN1276 S.aureus >log 7 1min EN13704 C.difficile > log 6 1 min

EN1276 P.aeruginosa >log 7 1 min EN13704 B.subtilis > log 6 1 min

EN1276 E.coli >log 7 1 min EN13704 B.pumilis > 3.88 1 min

EN14476 Poliovirus 4.33 30 secs EN13704 B.cereus > 3.24 1 min

EN14476 Adenovirus 4.67 30 secs EN13704 P.glycolyticus > 3.12 1 min

EN1650 A.niger >log 6 1 min EN13697 B.subtilis (stainless steel) > log 6 1 min

Table 1 Efficacy data for Contec ProChlor 2000ppm hypochlorous acid

ion (OCl-) and hydrogen ion according to thefollowing equation:

HOClXH+ + OCl- (hypochlorite ion)

These three species exist in an equilibriumwhich is both pH and temperaturedependent; the sum of these is referred to asthe total available chlorine. At 25°C and pH 7.5, half of the total chlorine is present asHOCl and the other half as OCl-. Thedissociated hypochlorite ion (OCl-)predominates at higher pH values, above 7.5, while the undissociated hypochlorousacid (HOCl) predominates at lower pHvalues. At pH 5, nearly all the chlorine ispresent as HOCl, while a pH value of 10drives nearly all the chlorine to be present asOCl- (see Fig. 1). At low pH and high chlorineconcentrations the hydrolysis is notcomplete and a significant fraction remainsin the form of molecular chlorine Cl2.

In a sodium hypochlorite solution whichnormally sits at a pH of 11–13, all available

Hypochlorous acidis 80 – 100 moreefficacious thanhypochlorite

disinfectants

disinfectants

CONTACT

Karen Rossington is a marketing consultant forContec Inc● info@contecinc.com● www.contecinc.com

References1. EudraLex. The Rules Governing Medicinal Products in theEuropean Union Vol. 4 EU Guidelines to Good ManufacturingPractices for Medicinal Products for Human and VeterinaryUse. Annexe 12. EU Biocides Regulation (528/2012) 3. White’s Handbook of Chlorination and AlternativeDisinfectants Black and Veatech Corporation, Wiley Publishing4. GM Fair et al. Journal of the New England Water WorksAssociation 1947 61 285–3915. G M Fair, et al. Journal American Water Works Association1948 40 1051–10616. BS EN1650:2008 +A1:2013 Chemical disinfectants andantiseptics — Quantitative suspension test for the evaluation offungicidal or yeasticidal activity of chemical disinfectants andantiseptics used in food, industrial, domestic and institutionalareas — Test method and requirements (phase 2, step 1)

chlorine is in a form of hypochlorite ions(OCl-) which, as previously discussed, is farless efficacious than hypochlorous acid.

Until recently it has not been possible tocreate hypochlorous acid in a stable solutionwith a usable shelf life. Hypochlorous acidgenerated from dissolving dry calciumhypochlorite has a shelf life of approximatelyfour hours, therefore has not been suitablefor use in pharmaceutical or healthcareenvironments.

Advances in chlorine chemistry have ledone company to stabilise a solution based oncalcium hypochlorite, at a pH of 3.5–5.5 so allavailable chlorine is in the form ofhypochlorous acid with a shelf life of 18months. This product is available to thepharmaceutical industry as ContecProChlor.

If the science was correct this productshould be more efficacious and faster actingthan the equivalent sodium hypochloriteproduct. To validate this, Contec carried outthe usual panel of TEC 309 EN disinfectanttests. Table 1 shows a summary of theseresults for a 2000ppm active chlorinesolution. Complete kill against spores wasachieved in both clean and dirty conditionsagainst a modified EN 13697 surface tests inone minute. This is a significant increasewhen compared with sodium hypochloritein both the log reduction achieved and the

kill rate. Various micro-organisms have been

tested including a particular resistant houseisolate which was “typed” as P. glycolyticus.Test work was also repeated against thenewest version of EN 16506 for fungi andmoulds, which has a requirement for >75%of the A. niger (now A. brasiliensis) spores tobe in the form of “spiny spores” which aremore resistant.

Due to its low pH and available chlorineconcentration, the product also carries nohazard and thus requires no specialised PPEor disposal consideration. It is non-corrosiveand current testing shows goodcompatibility with cleanroom materials.

Although not residue-free, because a smallamount of residual calcium salt will remain,it is free-rinsing and present at very lowlevels. A residue on evaporation result (EP)gave 1200ppm per ml compared with

4500ppm for a quat or 21,368ppm forsodium hypochlorite.

So the evidence and data suggest thatstabilised hypochlorous acid ticks most, ifnot all, the ideal disinfectant functionalrequirements. When manufactured by acompany with 25 years of experience ofmanufacturing products for thepharmaceutical industry, the packaging,manufacturing and quality aspects of theideal disinfectant are also met. CT

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32 CLEANROOM TECHNOLOGY December 2013 www.cleanroomtechnology.com

Fig.1: The species exist in an equilibriumwhich is pH and temperature dependent

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