INTERNATIONAL Iso STANDARD 14698-1 Frstedtion 2003.00.01, Cleanrooms and associated controlled environments — Biocontamination control — Part 1: General principles and methods Salles proores et environnements maltris6s apparentés — Maltrise de 1a biocontamination — artis 1: Prinsipes géaéraux 0! méthodes Refoconce number 90 14698-42006) 2180200310 14688-1:2003(E) POF asciaimor ‘Tris POF Sle may cota embase typetaces In aocrtance wih Adee’ leasing ali, ts fie may Be ped oF wowed but shat not oo eiled uneee the Ypotacee whic aro embeded ate leereos to anc ral or te computer perm the eating Ih ‘Suricacing ths fle, paras steopt aren tho fespencniy of rat neingng Adobe: eaneng paley Tha ISO Cena Secetanat ‘accepts ne isblty nite sre, cba a tadorark of Adete Systoms Inoorperaiod, Detais ofthe sofware proculs unod t crate ts POF fe can be feund tthe Genet Ini relative ic the i; the POF creation ppramclers were operized for geting vey care es Deen lent ensure tht fa We suave fr use by ISO member Dodi. Ih the ely svant ia! prob vlabtg tsa und plage inform the Corel Secretariat at tha adiexe given below © Isom ‘Allrightsrasaned Unless otherwise specited, ne part of his publication may be regroduced or ulized in any frm or by ary meer, ‘Severe er mechanical. itcudin photocopying and micofim. wiheut permission in wring fom ether ISO atthe adress Deon oF 190s member booy nthe cour ofthe resumen 'S0 copyightefics (Cats ponte 55 « CH1277 Geneva 20 Tet ea1z2 740911 Fax $4122 140 0947 Emal eopyai@ienog Web wenn eo o%p Pubisnedin Satzestans i 12180 2009 — al nets retoreeContents Page Foreword... Introduction. Scope. Normative referencos ‘Terms and definitions Principles of biocontamination control Establishing the Formal System . Expression, intorprotation and reporting of results... scscciscslll Verification of the Formal System. Training. Documentation... Annex A (informative) Guidance on determining airborne biocontamination . Annex B (informative) Guidance on validating air samplers... ‘Annex (informatie) Guidance on determining blocontamination of surfaces ‘Annex D (informative) Guidance on determining biocontamination of textiles. Annex E (informative) Guidance on validating laundering processes. ‘Annex F (informative) Guidance on determining biocontamination of liquids Annex G (informative) Guidance on training . Bibliogrephy (2180 2009 — args reserves oForeword 150 (the Intemational Organization for Standardization) is a workiwice federation of national standards bodies: (SO member bodios). Tho work of proparing International Standards is normally carried out through ISO. technical commitees. Each member body interested in a subject for which a technical committee has been ‘established has the right to be represented on that committee. international organizations, governmental and ‘ron-governmental. in liaison with ISO, also take part in the work. 1SO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standard.zation International Standards are drafted in accordance with the rules given in the iSOAEC Directives, Part 2 ‘The main task of technical commitzes is to prepare International Standards. Draft International Standarcs ‘adopted by tho technical committoes are eireuiatod to tho mambor bodies for voting. Publication as an Inte‘national Standard requires approval by atleast 75 % of the member bacies casting a vote. Attention is drawn {o the possibilty that some of the elements of this document may be the subject of patent ‘ighis. [SO shail not be held responsible for identifying any or all such patant rights. 1SO 14658-1 was prepared by Technical Committee ISOITC 209, Cleanrooms and associated controlled environments 1SO 14608 consists of the folowing parts, under the general tile Cleanrooms and associated controled environments — Biocontarination control — Part 1: General principles and methods — Part 2: Evalvation and interpretation of bvocontamination data ” (0180 2003 — Al ners reserveIntroduction ‘The principles descrited here are intended to promote appropriate hygienic practices. This part of SO 14698 is, ‘one of @ number of standards considering factors important forthe ereation of clean, controlled environments. Hygiene has become increasingly important in many areas of modern socioly. In such area, hygione or biocontamination control methods are, or will be, used to create safe and stable products, International trade In hygieno-sensitive products has greatly increased, At the same time, tre use of antimicrobial agents has been reduced or forbidden, creating a need for increased biccontamination contro ‘This part of IS 14896 js the first general International Standard for biocontamination control, However, many factors besides cleanliness must be considered in the design, specification, operation and control of cloanrooms and associated controlled onvironments. In some circumstances, relevant regulatory agencies could impose supplementary policies or restrictions. In such situations, appropriate adaptations of the standard testing procedures might be required 18180 2009 — Aight reaerved vLede EVE Grea Eo hte wipe afer > ies aa Segui Waverly Cocabe al laetiaaheseietINTERNATIONAL STANDARD 180 14696-1:2003(E}) Cleanrooms and associated controlled environments — Biocontamination control — Part 1: General principles and methods 1 Scope This part of |SO 14698 establshos the principlos and basic methodology ot a formal systom of biocontamination control (Formal System) for assessing and controling biocontamination when cleanroom technology is apples for that purpose, This part of ISO 14888 specifies the methods required for monitoring risk zones in @ consistent way and for applying cortrol measures appropriate to the dearee of risk involved, In zones where risk is low, I can be used as a source of information, Acplication-epecifie requirements are not given. Noither are fre and safety issues addressed for these, seo regulatory requirements and other national or local documentation 2 Normative references The following referenced documents are indispensable ‘for the application of this document. For dated Feferences, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. SO 14644-4:2001, Cleanrooms and associated controled environments — Part 4: Design, construction and Startup 180 14608.2:2003, Cleanrooms and associated controlled onvirenmonis — Biooontamination contro! — Part 2: Evaluation and interpretation of biocontamination data 3. Terms and definitions For the purposes of this document, the following terms and definiions apply. 3.4 General aad action level lovol sot by the user in the context of controfed environments, which, when exceeded, requires immediate intervention, including investigation of cause, and corrective action B42 alert level lovel sat by the usor in the contoxt of controlled environmants, giving early warning of a drft from normal conditions, which, when exceeded, should result in increased attention to the process 16180 2003 — Al aghis reserved 1ISO 14698-1:2003(E) 343 bioaerosot ‘lispersed biological agents In @ gaseous environment 4 biocontamination contamination of materials, devices. individuals, surfaces, liquids. gases or ar with viable particles 345 ‘cleanroom room in which the concentration of airborne particles is controled, and which is constructed and used ine manner to minimize the intoduction, generation, and retention of particles inside the room, and in which other relevant parameters e.g. temperature, humidity, and pressure, are controled as necessary [ISO 14644-1:1999, 2.1.1) 348 contact device ‘specially designed appiance holding an appropricte, sterile, culture medium with an accessible surface used for surface sampling 3A7 contact plate contact device where the container is a rigid dish 348 control point point n 2 controlled environment at which control is applied and a hazard can be provente reduced to acceptable levels ‘olminated or 348 controlled environment defined zone in which scurces of contamination are contrlied by specified means 34.40 corrective action ‘action tobe taken when the results of monitoring indicate tha ft or action levels are excoeded 3441 Formal System ‘system of blocontamination contol with estabished and documented procedures 3442 hazard potential source of harm [ISONEC Guide 511999, 3.5)21 3443 impact sampler device designed to sample partces in the air, or other gas, through a colision with a solid surface 34.44 impingement sampler device designed to sample particles in the air, or other ges. through a colision with a liquid surface and the subsequent entering into the liquid 2 (0180 2003 — rats eserveeISO 14698-1:2003(E} 34.15 ‘qualification process of demonstrating whether an entity — activity oF process, product, orgarization, or any combinat thereof — is capable of fulfilng specified requirements 34.46 tisk ‘combination of the probabilty of occurrence of harm and the severity of that harm [ISOMEC Guide 51:1999, 32)21 3447 risk zone defined and delimited space where indwvidusls, products or materiale (or any combination of these) are particularly vulnerable to contamination 34.48 sotto piate ‘suitable container (e.g. a Pett dish) of appropriate size, containing an appropriate, sterile, cuture medium, \which is left open for a defined period to collet viable particles depositing from the air 3449 swab siefle collection device. nom-toxic and non-inhbitory to the growth of the microorganisms being sampled, ‘consisting of a specific matrix of sutable size, mounted on an applicator 3.1.20 target level dofined love! set by the user as a goal for routine operations, for te user's own purpose 34.21 validation confirmation, trough the provision of objective evidence, thal the requirements for a specifc intended use or application have been fultilod {iSO 9000:2000, 3.8.5} 34.22 verification Confirmation, through the provision of objective evidence, that specified requirements have been fulfited {IS 9000-2000, 3.8.4] NOTE —__Wonttonng anc aucttng methods, procedures and tests, inclucing random samping and analysis, can be used in the voificaion of tte Formal Syetom. tas viable particle panicle that consists of, or supports, one or more live microorganisms 34.26 viable unit wu ‘one or more vaabie paises which are enumerated as 2 single unit NOTE When viable units are enumerated as colonies on agar media, Is common usage 10 name them colony foming unite (CFU), One CFU might canest of one or more VU, 1150 2009 — A aghts rasan 33.2 Occupancy states 324 as-built ‘condition where the installation is complete with all services connected and functioning, but wth no production ‘equipment, materials or personnel present [SO 14644-1:1999, 24,1]! 322 atrest ‘condition where the installation is complete with equiomert installed and operating in a manner agreed upon by the customer and supplier, but with no personne! prosent, [ISO 14644-1 1999, 2.4.2)! 323 ‘operational ‘condition where the installation is functoning in the specified manner, with the specified number of personne! prasent and working in the manner agreed upon [150 14884-11999, 2.4.3)" 4. Principles of biocontamination control 4.1 & formal systam of biscontamination control (Formal System) shall be established, implemented and maintained within cleanrooms and associated environments. The Formal System will assess and control factors thet can affect the microbiological quality ofthe process and product. ‘There are 2 number of accepted mathods for achieving this goal by risk assessment, The hazard analysis entical control point (HACCP) system 17.8.8) = commonly used, Faulk tree analysis (FTA), or the falure mode and effect analysis FMEA)", or any other validated equivalent system can be used Ip many such methods, any type of hazard can be considered. Within this part of ISO 14699, only ‘mierobiclogical nazards ara addrassea, 42 To assess and control the microbological hazards, any salected system shalt address the following principles: 2) ‘Identification of potential hazard) to the process or product, assessment of the Ikelihood of occurrence: of these hazerd(s), and identification of measures for their prevention or control, b) designation of risk zones and, in each zone, determination of the points, procedures, operational steps ‘and environmental concitione that can be controlled to aliminato the hazard(e) or minimize the likelihood of thar occurence, ©) establishment of limits to ensure control; 4d) establishment of a monitoring and observation schedule; 2) esteblichment of corrective actions to be taken when monitoring results indicate that © particular point, procedure, operational step or environmental condition is not under eonirol 1) establishment of procedures, which may include supplementary tests and procedures, to verify that the ‘chosen Formal System is working effectively, 9) establishment of training procedures; 1h) estabishment and maintenance of appropriate documentation, 4 © 150 2002 — Al gts reservaaISO 14898-1:2003(E} 5 Establishing the Formal System 5.1 General requirements It is the responsibilty of the user to develop, initiate, implement and document a Formsl System for biccontamination control that allows detection of adverse conditions in a timely fashion. kt is imperative that ‘such a programme be talored to the fold of application, to the specific facility and to specified conditens, and that this system be an inlegral part of a quality management system. The gualily management system shall include an appropriate training programme for he selected Formal System. In adation, itis essential that a monitoring programme (see 5.3) be designed and implemented in. a manner that minimizes the possibilty of the sampling activites themselves contrbuting fc the contamination of the produst or risk zane or bath, Risk zones shal be classified according to relevant guidelines, regulations (where these exist) and the chosen Formal System, Risk zones may slso be classified according to the level of aenal and surface bioconlamination, for example, low, medium, high or very high risk. NOTE The frst tho parts of e Formal Sysiem, as given in 4.2 a} end b), are not discussed jn deta in bis part of SO 14688, but information on how ta deni, assess and contol hazards is gen in aher sources. See, for example, 12] 5.2. Alert, action and target levels The user of a cleanroom or controlled environment shall sot microbiological alort and action levels. Those levels shall be appropriate to the field of application, to the classification of the risk zones and to what is achievable using current technology, Microbiological target levels may be used as an alternative. to ‘microbiological alert and action levels in some spect fields of application. During initial start-up and at intervals established according to the Formal System, deta on biocontamination levels should be reviewed to establish or confirm a baseline for the determination of alert and action levels. ‘Alert and action levels may be related to the target levels in any spectfic applications where these are set ‘Alert and action levels should be reviewed and adjusted as appropriate. 5.3. Monitoring of biocontamination 53.4 General Detection and monitoring of blocontamination in risk zones shall be catried out by sampling and enumerating ‘able units with appropriate methods in accordance with a sampling plan. Examples of sources of biocontamination that can constitute a hazard are air, surfaces, textes and liquids (see Annexes A, C, D and F) Microbiological sampling may be useful for providing baseline data as new installations are constructed and ‘commissioned, incuding, as relevant, in the as-bull state. Monitoring in risk zones shall be performed when the installation is in the as-buit and atest states. Moritoring shall also be performed routinely in the ‘operational state according to the selected Formal System. 53.2 Sampling 53.21 General ‘The appropriate sampling method and related procedures shall be selected and performed 10 reflect the complexity and variety of situations. Sampling shall be corried out using a device and method selected in accordance with the writen procedure and in accordance with the instructions provided by the davice manufacturer (6180 2003 — al rights reserved 55.3.2.2 Sampling device ‘A camping davice shail be selacted accorsing 10 the area being montored, The selection for a particular ‘pplication shall take into consideration the folowing factors: 2) type of viable particles for which to sample; b) sensitivity of the viable particles to the sampling procedure; ©) expected concentration ofthe viable particles; d) indigenous microbial flora; ©) accessibility of the risk zones: 4) ability to detec ow levels of biocontamination ) ambient conditons in the sk zone being sampled ‘h) time and duration of sampling: i} samptng method, matoral and proper of he sampling medium |) fect of the sampling devies on the process or envronmantto be montored: ) collection accuracy and etelency, 1) Incubation and visto panicle cetecton and evalustion method: 1) ype of information to bo obtained (ag. qualitative or quanttativa aspects); 1) otfiiancy of extrastion/ise fds, where appropriat. 6.3.23 Sampling plan ‘A sampling plan shall be developed through the selected Formal System and shall be documented. A ‘Gocumented samping plan is essential for accurately assessing and interpreting biocantamination dala ‘Sampling shall be carried cut when the area is in the operational condition and during periods of greatest ‘tress in the system, for example, before the end of 2 chit or when the greatest amount of acbivty is taking place. Sampling in the at-rest condition may also provide useful information about the facity design and Performance. “The sampling plan shall comprise the folowing 2) initial sampling plan to provide a reference point or baseline within the framework of the chosen Formal ‘Systom; )_ routine sampling plan resulting from the implementation of the chosen Formal System. 2.4 Design of the sampling plan ‘The sampling plan shall take into account the cleantiness level of the risk zone and the degree of biocontamination control required for the activity being conducted, to protec: individuals, the environment, the process and the product. The following are examples of elements fo be considered: ) choice of the sampling location, taking account ofthe location and function of the risk zone: 6 (6180 2003 — A rgps ecervesb) number of samples (limitad or small sample volumes may not provide representative resuts, although, in some cases, large numbers of samples may compensate forthe size of the sample volumes) ©} frequency of sampling: 4) methods of sampling, including whether the tests will be quaitative or quantitative: 6} volume to be taken or the area that should be covered to constitite a sample; ) diluents, rinse fuids, neutralzers, etc. 9) factors pertinent to a particuler station that could affect culturing results; 1h) impact of operations, personnel and equipment in risk zones which contribute fo biocontamination, such 1) compressed gases, 2) room air, 3) manufacturing equipment, 4) monitering’measuring devices, 5) storage containers, 8) number of persons present in zone, 2) unprotected surtaces of personnel, 8) personal atte, 9) protective clothing, 10) wailsteeiings, 11) feore, 12) doors, 19) benches, 14) chairs, or 45) alr admitted from other sources. 5.3.25 Froquoncy of sampling ‘Tho froquancios of sampling shal be developed using the solacted Formal System and shall be confirmed or modified 2s necessary in the folowing cases: @) when alert or action levels are exceeded consecutively ) efter prolonged shut-down of activities; 9) on detection of infectious agents in risk zones: ‘after any significant maintenance work has been undertaken on the ventilation system, (© 580 2003— Al ighsresaned T1s0 14698- -2003(E) )) after changes to the process tnat affect tne cleanroom environment; 1) after recording of unusual results; {9) after changes 10 the cleaning or disinfection procedures; 1h) after unp'anned incidents that could contribute to biecentamination, 53.2.6 Sampling sites ‘Sampling sites shall be determined through the selected Formal System, and included in the sampling plan. ‘More than one sample may be taken at each site and different numbers of samples may be taken at different locations, ‘Samping shall be cartied out at the microbiological control points defined in a writton procedure. 32.7 Identification of samples: The labelling of each sample shall carry the following information or a coding that provides traceability ofthe information: 8) collection site; b) date and time of collection; ©) person collecting the sample; 4) currant activity at tho time of sampling 2) culture medium type; 1) any deviations from the sampling plan 53.3. Validation ‘The selected monitoring system shall be used as part of the quaification and validation process for cleanrooms and assooated controlled environments in accordance with |SO 14898.2 and ISO 14644-4:2001, Annex C. NOTE Information onthe vatdation of some microbiological metheds ' gen in ISO 14698-2. 5.4 Processing of samples The collection, transport and processing of samples shall not affect the viabilty and number of the collected organisms. Factors to be considered arc a) transport/storage conditions and duration, b) uso of neutralizing agonte, and ©) use of osmotic solutes. ‘Samples shall be collectod in a manner and in containors such as net o add to, or inhibit, biooontamination. 8 1 150.2003 —Al tants reserveISO 14698. -2003(E) 5.5 Culturing of samples 55.4 General Culture media and incubation conditions (e.g. temperature, duration, oxygen tension, relative humidity) shall be selected according to the types of microcrganisms expected, This selection will also depend upon the ‘sampling environment and the procedure and equipment used, 55.2 Culture media Culture media shal, if not otherwise indicated, be non-selective. Appropriate additives shall be included to ‘overcome, or minimize, the effects when residual antimicrobial activity at ihe sampling point is expected. When culture media are used within cleanrooms or associsted environments, the external surface of their ‘containers shall be maintained in a state of cleanliness appropriate to thei use NOTE The adoption ot double- er trpe-wrapping may be nécetsary to maintain the stats of earliness. ‘Appropriate quality control procedures for the culture mecia shail be ensured! "91" 5.3 incubation When selecting a suitable incubation temperature and time for the inoculated culture media, conditions that favour the growth of the types cf organisms expacted to enter the clean environment shall be considered, whenever possible. Total incubation periods of two to five days for bacteria and five to seven days for fungi are generally acceptable, especially when the number of YU Is low. Whan anaerobic, thermophilic, micto-aerophilic, or Nutritionally deficient or fastidious bacteria, and fungi, are of concem, specific atmospheric condtions and incubation times could be necessary. Plates should be observed al appropriate intervals over the incubation Poned. 5.8 Evaluation of sampling data 5.6.4 Genoral ‘The evaluation of biocontamination data shall provide sufficient information for effective corrective actions. Further information cn the evaluation of biocontamination data is given in ISO 14698-2. NOTE Monitoring of microbial contamination may be performed by the measurement of indract indicators, e adencsine tishosptete (ATP) measurements. However, I should be noted that ere may be Po rec: relaton between resanca of sich indicators and biocontamination Ite thavefors essential wnen the Formal System ie Daing veri oF the montoring eyetom is boing validated, hat thor be a crect cetrmation of biocentarnation 5.6.2 Enumeration I's generally accepted that, as with other microbiological counts. the estimation of biocontamination can be Influancee by instruments and procedures used to perform these counts. Therefore the enumeration of viable particles from the samples shall be performed only by appropriate validated methods. NOTE 1 Infermaton on enumeration of viable pericies is given in other sources!4 NOTE 2. This part of ISO 14608 doos not imply or accopt any citect constant or causal lnk bolwaon concentrations of vable and ron-~viable parties. Conta levels for those parameters can be set separately as require. (© 180.2003 — Al ighis reson 9ISO 14898-1:2003(E) 58.3 Characterization Microbial monitoring cannot identify and quantify all the microbial species found in controled environments. Evaluation of results shal include choice of an appropriate level of characterization. NOTE The level of characterization wil depend upon the citcaliy of the area involved end whether Investigation warrants futher Identfcation Broad calegories based on cell morphoiay, slaining properties ard other characierstcs ‘may de suficent When required, icentification, at leas! 1 the genus level, can be carted out using established laboratory ‘metheds. Information gathored through characterization can help inthe evaluation of claaning and disinfecting procedures and in cetermining a source of contamination or an apprepriste corrective action. Idenifieaton of iolaies from eric! ‘areas will usuely ieke precedence over denificlion from non-ctitcal ersas. 6 Expression, interpretation and reporting of results Quantitative resuits are expressed as viable units (VU) or as colony-forming units (CFU), depending on the method used, using appropriate SI units (7. information on data evaluation is given in ISO 14698.2, To assist in interpretation, results shall ba reviewed aver extended parods to datermine trends. Based on tha review of these investigations and specific testing results, decisions shall be made on the significance of Unusual results, and the acceptability of the operations or products processed under those conditions. ‘The test report shall include or make reference to the following 2) type of sample b) method{s) used and, here appropriate, the number and tile ofthe standard: ©) collecting device used; @) sampling site: ©) type of activity underway at the fime of sampling, including occupancy state; ) number of persons within the sampling area, where appropriate; 9) sampling date and time of sampling; 1h) ¢ampling curation, whore appropriate; 1) tme of examination of samples; ) conditions and duration of incubation; k) variations from the described test method, as well as any factor hat may have influenced the results; 1) test results fiom the examination of the collected samples after inital and final reading 'm) when quantitative tests have been performed, the results, expressed using appropriate SI units; 18) desciiption ofthe isolate(s), if characterized, c) name of the organization responsible forthe test report and the dae of completion of the test P) name and signature ofthe individual(s) sponsible for performing the test 10 (01802003 — Argh reservedISO 14698-1:2003(E) 7 Verification of the Formal System The results of monitoring biocontamination shall be examined periodically in order to confiem that the system ‘chosen is functioning in accordance with the established procaduras and the spectfied requirements have been fufiled [see 4.2). NOTE This examination could requre use of monitoring and audting methods, procedures end taste, cluding random samping and analysis. It could elso require the systematic verification of all working sleps ard equipment to ‘ersure the proper functioning of the Formal System. It verification indicates deviations from the established limits or @ change in the microbiological status of the controlled environment, corrective acton shail be intiated. if appropriate, the Formal System shall be modi. 8 Training ‘training programme shell be implemented (soe Annex G), 9 Documentation Dooumentation shell inchide descriotion of the Formal System, — tisk assessment report, — sampling plan, — action, alert and target levels, as applicable — test and sampling procedures, — test report, — verification report, and — training records. (0180 2003 — irghisrosoved "ISO 14698-1:2003(E) Annex A (informative) Guidance on determining airborne biocontamination A.A Introduction This annex provides guidance on the determination of airborne biocontamination in situations where microbial controls coneiderad desirable ar nacassary. This measurement invclvas collection of representative camplos: forthe detection of those viable particles that need to be controlled and monitored. This assessment of airborne biocontamination is carried out in acco‘dance with the basic principles ofthis part of ISO 14698, which requie the establishment of a Formal System to assass and control blocontamination where eloanroom technology is appiog. “Techniques for the validation of a camping device are given in Annex B. A2 Principio Detection and monitoring of microbial cantaminaton of the air in a risk zone is carried out by collecting viable Particles with appropriate sempiing devices, according to a sampling plan, when the risk zone isin the as-built ‘and at-rest siates, as appropriate, anc routinely under normal operation inthe risk zone, A.3 Sampling devices A341 General ‘There ara a_great variety of methods available for the collection and enumeration of airbome viable partcles"4 The selection of a pertioular method and device will depend uncon the purpose for which the Sample is required, The collection eiflciency of samplers will vary, an appropriate method or methods. anc equipment should be cerefuly selected ‘Sampiing devices fall into two categorios: 2) passive semoling devices, such as setle plates; ) active samping devices, such as impact, impingement and fitration samplers, “The manufacturer of these devices should provide instructions for thair use as well as information on their limitations, The collection efficiency of active sampling devices is discussed in Annex B. A.3.2 Selection of a sampling device ‘The sampling rate, duration of sampling and type of sampling device can strongly influence the viability of the microorganisms that are collected. Impingement devices may not be sullable for sampling airborne viable particles because of their low sampling volume and low rate of sampling, and their tendency to alstupt clumps. of vieble particles. 12 18180 2003 — Al rors records‘Bacause of the number and variety of microbial air eampling systems commercially available, the selection for a particular application should consider, as a minimum, the following factors: a) type and size of viabie particles to be sampled: 'b) sensitivity of the viable particles to the sampling procedure ©) _expacted concentration of viable particle )__abilty to detect high or low levels of biocontamination; 2) appropriate culture media (see 5.5.2)" 1) time end duration of sampling, ‘9) ambient conditions in the environment being sampled: h) disturbance of unidirectional irflow by the sampling apparalus: sampler properties such as 1) appropriate suction fiow rate for iow levels of viable airborne particles, 2} appropriate impacvairtiow velocity, 3) collection accuracy and efficacy, 4) case of handling (weight, size) and operation (ease of use, auxiliary equipment, dependence on vacuum punips, water, electricity, 5) ease of cleaning and disinfection or sterilization, and 6) possible intrinsic addition of viable particles to the biocontamination to be measured. ‘The exhaust ei from the sampling apparatus should not contaminate the environment being samled or be reaspirated by the sampling device. A.3.3 Passive microt ‘sampling devices (sedimentation sampling devices) Passive microbial air sampling devices such as settle plates do not measure the total number of viable paficles in the air; thay measure the rate at which vable particles settle on surfaces. Settla plates may therefore bo used for the qualiatve and quantitate ovaluation of airborne contamination of products, This ‘can be done by determining the sete plate count per time: then, by relating both the area and time of exposure of the product to that of the settle plate, the possible contamination of the product can be calculated 01 211, A.3.4 Active microbial sampling devices A341 General ‘The use of active alr sampling devices in risk Zonas is essential for the assessment of the microbial qualty of arr There ere several types of actve devices commercially available, each having its own limitations. Based on the princes of sampling, the two main types of apparatus considered auitabe for riak zones with normal (ow leve!) bisconiaminaticn are impact samplers and filrtion samplers, (2180 2003 — Al ngs reserved 8180 14898-1:2003(E) A342 Impact and impingement samplors Because there are a variely of impaci and impingement samplers available for the detection of viable panicle the device selected for use should have the following charactersties: 2) impact velocity ofthe air hiting the culture medium thet is @ compromise between 41) being high enough to allow the entrapment of viable particles down to approximately 1 ym, and 2) boing low enough to ensure viability of viable particles by avoiding mechanical damage or the break- up of clumps of bacteria or micromycetes, b) sampling volume that is a compromise between being large enough fo detect very low levels of biocontamination and being email enough to avoid physical cr chemical degradation of the collection medium, In areas of high biocontamination, the impaction method and sample volume should be selected in way appropriate to achieving separate colonies, fo allow the resus o be interpreted. “The device should mest the folowing minimum requirements: — sufciont tow rate to cotect 1m? in 2 reasonable time, without signifeant drying of the sampling medium; — appropriate ar impact spees tothe culture medium, ‘A343 Filtration samplers Filtration sampling devices are widely used for air sampling. By appropriate choice of pump. fiker medium and ‘ter size, almost any desired sample quantity can be collected in 2 given sampling period. For the design and use of a fitrtion sampling device, the following factors should be considered: a) ensure that the fitration conditions do not affect the viabllty of the microorganisms collected, 0.g. by dohyaration; b) oliminata static electicty that will interfere with the rate of impact of viable particles onto the fier membrane: ©) apply the sarne constraints or suction flow rale and impact air velocity as in 3.4.2; 4) ensure that the fiter membrane holder can be connected to @ vacuum source, fitted with @ device for measuring the suction rate, wthout contamination ofthe fiter materia ©) ensure thatthe fiter membranes can be placed aseprically in the fiter holder, can be removed asepticaty ater fitering the desired quantity of air and can be placed on solid culture medium o in liquid euure medum. A.4 Expression of results The number of viable particles should be expressed in viable units per cubic metre 14 (0180 2003 — Al nets reserves180 14698-1:2003(E) Annex B (informative) Guidance on validating air samplers B.1 Introduction ‘This annex describes a technique for determining the collection efficiency of samplers used for counting sitborne microbes. Mansfacturers or third-party testing organizations will usually partorm this evaluation. ‘The collection efficiency of microbial air samplers can be considered in two ways: physical efficiency and tiological etficency, Physical efficiency is the ability of the sample to collect various sizes of particles. Physical efficiency is the same whether the partide is @ microorganism, carties a microorganism or is. an inanimate particie, Biological effcioncy is the officency of the sample in collecting microbs-carrying paricies. Biological efficiency will be lower than physical effciency for a number of reasons, such as the survival of the microorganisms during collection and the abilty of the colection medium to support their growth. The test method desorbed in this annex is mainly concemed with physical efficiency. B.2 Experimental method B.2.1 Test chambor ‘A test chamber approximately 11 m wide by 1 m high by 2m ong is suitsble, but any small, enclosed space would be acceplable. A test aerosol should be generated ether within this area, or supplied to the area. The test aree should be supplied wth HEPA-fitered air and maintained at negative pressure by extracting ar at a suitable raie, The exiracted air should be HEPA filtered. “The airflow within the test chamber should be non-unidirectional with an air-change rate equivalent to that found in 2 typical cleanroom, ie. between 20 and 60 per hour. To void local concentrations of unmixed ar, care should be taken with the method of supplying snd extracting air. A useful approach is to supply and extract sufficient ar to ensure negative pressure conditions and to recroulate the remainder by means of paddle or fan within the test area, A patticl counter and matnod of sampling air within the chamber should be provided to encure the aerosol is well mixed and to check on its concentration. Temperature and relative humidity should be maintained at (22 + 2) *C and (60 + 10) % RH, respectively. The apparatus witin the test area shall be able to be manipuated ftom ouside of the chamber, 2.9. By use of gounets orhal outs, 5.2.2 Test strains of microorganisms 5.22.1 Strains for testing physical efficiency ‘The test sirin should be Baciis subtits vat niger NGTG 10073 (=DSM 2277), which survives well the collection conditions. The test stran should be prepared in a culture medium meeting the nutritional requirements ofthe fest strain and used as a viashed spore suspension. NOTE Polystyrene spheres and olber pes of nonable parle canbe used to deterrne the physical efiiency fair samplers!) The results obiained are similar to nose produced by miciebiiegical parices. Honever, In some Samplers is Imoossilo to datect al the Mon-viable partelas, wheres f microbes are usec, tay wil grow ito colonies that can be easily seen ans dontiiog © 180 2003 — At nights recone 15ISO 14698-1:2003(E) 822.2 Strains for testing biological ofticioncy Many of the microbes in the air of occupied areas come from the skin of personnel; coagulase-negative Staphyiococe predominate. However, t is also possible to fd in some rooms a substantial number of ‘microorganisms that come from the process. If biological testing is to be carried out, bacteria typical of those found in tho room air chould bo tested. An organiem euch as Staphylococcus opidermidis (NCTC 11047, ATCC 14980) can be used. However. it should be noted that changes in the spray solution, the spray method and the collection concitions might cause the biological efficiency to vary anc make this method less reliable ‘than the physical efficiency. 8.2.3 Generation of microbe-carrying particles Asrosols ofa contolled partole size ere produced by an apparatus such asthe spining-tap or spinning ise ‘aerosol generator], The dsc or tops spun at high speed and fed by a liquid Suspension of microbes that are spun out as a fine homogeneous spray, By varying the speod, te ize ofthe wot croplos can be variod, The droplets dry quickly and, depending upon the amount of insoluble material in the liquid, various sizes of mat ory pariclae can bo produced. Tha wet paricla tamoter can be dotormined by use of an aquaton that ‘elates to the censty and surface tension ofthe quis and the rotational speed and ciamoter of fe spinning disc. Aternatwely, I can be measured microscopcaly ‘After formation, the wet particles will be reduced by evaporation to 2 size that depends upon their solid Content. It is useful to increase their size and weight by incorporating poassium iodide into the spraying solutions. The diameier of the dry particle can be calculated using the following equations or determined microscopically by sampling the air inthe test chamber by means of a fier membrane. The radius (r) of any sphere is related to ite volume (1) by the following equation @1) 62 where 1yg_ isthe volume of spore (approximately 0.5 um) jg, isthe volume of particle after evaporation; jp. can be caloulted by __ Volume of wet particle (m®) x concentration of solid in particle (gim?: es ‘density of the solid materialin solution (gim?) Having determined the radius of the dry particle the diameter is easily ascertained. The aerodynamic behaviour of a particle will vary according to its density. Therefore, it is necessary to calculate the equivalent partcle diameter of the dry particle, ie. the size the dry partice would be if it were of unt density, as follows, 1 Equivalent particle diametor= 4 (p)2 es 16 150 2003— Al ahtsraseros'S0 14898-1:2003(€) where 416th diameter of he ory panice; is the density of incorporated solid material B.2.4 Testing The tests take place inside the chamber or in an area adjacont to it that has turbulent air conditions similar to 1 cleanroom. The sampler to be tested and a membrane-fiter holder. containing a 0.45 um filer, should be placed close to one another but far enough away from the aeroso| Generator to ensure that the particles are dry when sampled (approximately one metre). The perticle counter shoud be used to check that the particle concentration is the same at the sampler and membrane-fiter hoider postions. The membrane sampler, operating at = fiow rate of approxmately § min, should not face upward but should face to the side or downward. preventing depestion by gravity of particles onto the membrane. Both samplers should be jsnitched on together. The sampling time wil cepend on the concentration of microbe-carrying particles n the air but a few minutes should suffice. After the test, place the membrane on a plate containing casein-peptone Soya meal-peptone agar or a validated, equivalent medium. After incubation of both sets of samples for two days at 37 °C, court the colonies Prior to use in the test, suspension of the washed spores, at a concentration not greater than 10®/m to 107ml, in an 80 % solution of ethanol will ensure single spores are obiained in mast of the particles, The necessary ‘amount of aerosol generated will depend on the size of he chamber and the amount of air supplied and extracted. However, the azrosol concentration should noi prolong sampling time and there should be no Coincidence of bacterial colonies on the sampling medium DDitferant concentrations of solids should be dispersed in tho solutions to provide a range of partcie sizes when sprayed. The concentrations of solids required can be calculated using the equations given in B23. Five solutions should be prepared ta provide particle sizes over a range of equivalent partic clameters of approximately 0,8 um to 15 um. For each particle size, atleast 10 experiments should be carried out. B.3 Interpretation of results Use the yield from the test semler and the membrane fiter sampler from the sane volume of air to calculate the efficiancy using the folowing equation: test sampler count ——_—_ wtvornploenaunti 100 {otal count (from membrane sample) Efficiency of sampier (%) = es The results may be plotted as particle size against efficiency, with all points plotted es means with standard deviations of effidencies, (2160 2008 — Al nahteresenes 7180 14698-1:2003(E) Annex C (informative) Guidance on deter ing biocontamination of surfaces ‘This annex provides guidance on the determination of biocontamination of surfaces in situations, particularly risk zones, where biocontamnination control is considered desirable or necessary. This measurement involves the collection of representative samples for the detection of viable particles that are oresert and that may need to be controlled or monitored. These methods right not give the total number of viable microorganisms present but, under controlled concitions, can give relevant and comparable results. These methods are ~ppied routinely in the operational condiion and, f appropriate, in as-buil and al-est conditions. “This assessment of airbome biocontamination is carried out in accordance with the basic principles ofthis part of ISO 14698, which requre the establishment of a Formal System to assess and contro! biocontsmination ‘where cleanroom technoloay is applied. C2 Principles ‘A-count of microorganisms on a surface al a point in time is obtained by a contact device or a swab. A contact device can apply a sold nutrient medium of known area to the surface, which is then incubated, The resultant Colonies aive @ rnirror-image "map" of the original viable units. A swab can be used {o wipe a surface and the umber of microorganisms removed by the swab can be counted. ‘A count of the rate at which microorganisms are fang on tha surface is obtained by exposing, for a known period, a nutiient surface of known area, which is then incubated. The resultant colonies give a rate of deposition per area per period. €.3 Sampling devices €.3.4 Contact sampling devices Contact plates or other devices can be used that allow a nutrient medium, held in a suitable flexible or rigid Container, to make contact with the surface lo be sampled. The accessible contact surface should be > 20cm ‘The nutrient medium should be applied to the surface for a fow seconds with a uniform end steady pressure to the whole ares, without allowing any ciccular or linear movement. The device is then retuned to its container and the sampled surface is cleaned to remove any nutrient residues, ©.3.2 Swabs Collection of viable units may also be achieved by appropriate application of a swabbing technique. The use of sterile moistened swabs, sponges or wipes is particularly convenient for sampling large, non-absorbent, lireguler or recessed surfaces not accessible to contact devices “The swab should be pre-moistaned with a sterile rinse medium. The swab should be stroked in close parallel ‘swoops over the defined sampling araa, while being slowly rotated. Sampling of tho samo area should be repeated, sticking the same swab perpendicular to the intial sweep. The swab should then be piaced in @ 18 10180 2003 — lt rghit eserves1S0 14898-1:2003(E) specitied amount of rinse liquid and agitated. The rinse qué should de assayed for viablo unis, After sampling, the sample site surface should be cleaned to remove any residue of the rinse medium. C.3.3 Sottle plates ‘Sottlo plates are suitable for the qualitative and quantiative evaluation of possible surfaca contamination by arbome viable parties depositing from the air Where appropriate, the number of microorganism-carrying particies depositing from the air onto surfaces in @ given time can be determined by settle plates containing a suitable culture modium; the plates are then incubated. This technique does not measure the total rumber of microorganisms present in the air, t ‘measures the number that have seitied onto a surface during the sampling period, The sensitvty of this method may be enhanced by using large-diameter Pett dishes (.e. 14 cm dameter) and extending exposure time, while taking care to avoid dehydration of the culture mecium(2#| C.4 Expression of results The number of viable particies on surfaces should be expressed in viable units per 1 dm2, or inthe case of settle plates, per 1 dm? per hour (1 dm? = 100 am) 1 1S0 2003 — A rights reserved 19180 14898-1:2003(E) Annex D (informative) Guidance on determining biocontaminat D.1 Introduction D.t.4_ This annex provides guidance on the determination of biocantomination of textiles in situations whore microbial contamination control is considered desirable or necessary, This assessment of the biocontamination of textiles is carried out in accordance with the basic principles of this part of ISO 14698, which require the establishment of a Formal System to assess and control bicoontaminetion where cleanroom technology is applied This assessment involves the collection of representative samples for detection end monitoring of viable patices that are present on, or are shed by, textiles, Textiles used in 'sk zones should be of an adequate cleantinass level as appropriate for the work activity, the purpose for which they are usod or both, Textlos chould be monitored for biocontamination in order to ‘minimize the risk of adversely influencing the activities, products, devices, etc. in risk zones, D.1.2 _In relation to the selection of textiles for, and the assessment of. related biocontamination in risk Zones the folowing factors should be considered. a) type and form of textle(s), e.g, protective clothing, wipes, etc; b) choice of fabric; ©) partite generation and dispersion characterstics of the fabric, d)_ inadequate barrier effect due to inadequate fabric fitrtion properties, ©) cleaning, decontamination or sterilization ofthe textiles 1) particle removal efficiency from the textile: 9} gatment design: fh) textile permeability, surface condition and abrasion resistance. 1D.A.3_ Ifthe biocontamination of a textile is found to be excessive, appropriate methods will be required to find possible causes, Typical causes include p00 particle retention due to fabric properties such as foe type, weave or design b) incorect usage, eg. clothing not changed frequently enough, insufficient decontamination, or ineffective cleaning or both, inappropriate textile washing cycles fr the microbiological constraints of he sk zone, and ©) recontamination after laundering, 20 (© 180 2003 — Arrant reienesISO 14698-1:2003(E) ‘This annex Is not intended to give guidance on the determination of the pormeabilty of fabrics to viable particles. Nor does it cover specific aspects of textes that may be required for ceriain fields of application Such as sterilized ans departculated textiles, nor textle quality as juoged by visual inspection or by touch. D.2 Principle Detection and mantoring of microbial contamination of a tex in a risk zone is carried out by collecting viable particles with appropriate sampling devices, according to 8 sampling plan D.3 Contact sampling devices For detetmination of viable particles on textiles, suitable contact devices may be used (see Annex C). including those appropriate for tasting small textile toms. If possible, the fabric should bo hold againet a hard, fiat, smooth surface before the contact plate is applied When using sampling devices based on dehydrated media on a support, rehydration may be carried out by using the quantity of quid indicated by the manufacturer; alternatwvely, this can be dane by using solutions that inactivate or neutralize detergents or disinfectants or that neutralize both detergents and disinfectants, NOTE _Whera tentiles are requited to oe sterile before use, ee pert of the eleriizaten veldation the misrebia ‘contamination of the tecies can be determined by usin mechanical agtation (e.g. using @ slomacher) fo remove the merooiganisins from samples of the ‘extle placed into an exttacton soluton. Ths extraction souton s then membrane ‘eres D.4 Expression of results ‘Tho number of viable particles should be expressed in viable units per 1 dm? of the textile. sampled 100 om}, (01802003 —Arertsrosoved a|S 14898-1 :2003(E) Annex E (informative) Guidance on validating laundering processes E.1_ Introduction This annex provides guidance on, and describes a technique for, the validation of laundering processes in situations whare eontrel of biocontamination is considered desirable and necessary £.2 Test method £.2.4 Principle ‘The validation involves using pieces made of a textile of the same type as those that undergo the laundering procass. These places are contaminated by known microorganisms in a meacured quantity, The pioces aro then put through the laundering procass thal is to be validated. The abiliy of the process to reduce by a factor (of 10° the number of bacteria and 10* the number of yeasts and fungal spores is checked, ‘The following controls are performed: '8) Control A: enumeration ct viable units in initial microorganism suspension. Control A is dasigned to demonstrate that intial numbers of microorganisms are high enough to allow measurement of the desired reduction of the microorganism population ) Controt B: enumeration of viable units on control pieces that have undergone exactly the same procedure {as tho test piece with tho excoption of the laundering process. Control B is dosignod to demonstrate that the viabiily of microorganisms does not change aver the validation period. ©) Control C: enumeration of viable units on control pieces that have undergone exacly the same procedure 2 the lest piece, including the laundering process, Dut have been contaminated wit) the microorganism ‘suspension only after the laundering process. Control C is designed to demonstrate that the technique for counting the number of surviving microorganisms is appropriate to the process conditions (time, ‘mechanical effect, temperature, presence of residues of washing product(e) on the taxtiles, otc). For the tast proper, a suspansion of known microorganisms is prepared in a protein solution. A known volume (of the suspension is applied to the test pieces. The test pieces are subjected to the laundering process as part Of a simulated normal texte load, Enumeralion of microorganisms on test pieces is done afer the laundering process. The reduction of the microorganism population is measured and compared to tho values mentioned above. tis essential that garments used in the simulated normal toad be made stele before reuse or are destroyed E.2.2 Microorganisms E221 Bacteria ‘Ata minimum, the following bacterial strains should be used: 2) Enteropoceus hiras ATCC 10841 b) Escherichia coll ATCC 10596 22 (0180 2008 — Arig esenesISO 14698-1:2003(E) £2.22 Fungi It fungicidal activity is claimed, at a minimum the folowing fungal strains should be used: a) Saccharomyces cerevisiae ATCC 9084, b) Asporgilus niger ATCC 16404, 2.2.9 Bacterial spores If sporocidal activity is claimed, at a minimum the spores of the folloning strain should be used: Bacilus ‘subtlis var. niger ATCC 6633. £.2.3 Microbial suspensions E23 Suspension medium Sterile peptone saline water should be used as the suspension medium for the suepensian of boctors. For fungi, add 0,05 % (vA) of polysorbate 80 or other validated chemical. Sterile cistlled water should be used for bacterial spores. £232 Recovery medium ‘The suspension medium, distilled water or any solution thal can be fitered in test conditions can be used. {fa disinfectant neutralizing agent must be used, iis can be added to the recovery medium, £.2.3.3 Protein solutions The following aqueous solutions are orepared. — Solution A: 3% (wi1) bovine albumin (Cohn's fraction V), adusted to pH sterlized by membrane fitrstion 820.2 I necessary, Solution B: 15 % (w/) yeast extract adusied to pH ~7+ 0.2 steriized by a validated procedur. — Solution C: solutions A and B aro mixod in tho ratio 100:20, se that tho concentration of gach protein is 25% (wil) £.2.4 Control and test pieces The pieces, made ‘ram a de-sized textile, must be representative of the textles that undergo the laundering process to'be validated. They should be used only once. The pieces should have an overall size of 410.cm x 5 om, including a contaminated area of 5 cm x § cm and free enc(s) used to attach them to a texte in the load, Pieces are wrapped in a material permeable to steam and sterlized by a validated procedure. E.2.5 Preparation of the inoculum A suspension of = 108 bacterial cells, or 2 107 fungal cells or bacterial spores per miliire is prepared. © 80 2003 — Al ignsrasened 23180 14698-1:2003(E) E.26 Procedure £261 Controls “These are 3s follows. — Control A: enumerate VU in duplicate in an agar medium after appropriate dilution of the inoculum ‘suspension. The mean of the two counts in the dilution containing 30 VUim! to 300 VUIml is called Check that the number in the orginal suspension was > 108im for bacterial lis or > 107/ml for fungal ‘alls or bactorial epores, — Control 8: using appropriate diutions, inoculate two control pieces with 0,§ ml ofa suspension containing 30'VUrm! to:300 VUrml and two other control pieces with 0,5 mi of a suspension containing 300 VLml © $3000 VUlmi. Those four conitol pieces are handled and testod togethor with tho test pieces throughout the test wih the exception of the laundering process. When returned to the laboratory, they are incorporeted in 2 nutritive agar mecium and incubated. VU are counted. The mean count corresponding to the most heavily contaminated control pieces is called and the other is called N’, — Control G: 0,5 mi of protein solution G (see E:2.3.3) I$ applied to one control plece. The latter uncergoes ‘he whole laundering process. The control piece is then immersed in 100 mi of 8 recovery medium, agitated ‘or 15's 10 30 & and deposited in a Petri dish. Then 1 ml of a suspension containing 30 VU/m! 10 300 VUim is applied to the control piace. The jattor is covered with 10 ml of agar medium, incubated ard ‘hen counted. The count is caled 7, The 100 ml of recovery medium used previously is fitered through a ‘itration membrane capabie of retaining microorganisms. After rinsing three times, the mambcane is covered with 50:mi of new recovery medium. The 1 ml of the suspension containing 20 VUiml to 300 VUim| Is added to the 60 mi of recovery medium and then ftered. The membrane and the flirtion apperatus are rinsed with another 50 ml of cacovery medium and then fiterod, efter which the membrane. 's transferred onto an agar medium and incubated. The count is called 72. Caleulate n= (nj +%,)/2 ‘= N'p=n, the experimental conditions are validated for the test proper. WW’ < 0.5 N andlor 1% < 0,05.N andlor n < 0,8 N, the experimental conditions are not validated forthe test proper. Redo the contibis, for example, adding approptiale compounds to neutralize chemical residues in ‘conttol places submitted tothe laundering process. E262 Test proper Mix and leave in contact for 5 min at ambient temperature 3 ml of the microorganism suspension (E 2.5) and 2 mlof the protoin solution C of (E 2.3.3). Apply 0,5 ml of the resuting suspension to the test piece. For each ‘microorganism tested, contaminate three test pieces. ‘Afier the laundering process, the contro! pieces are brought back to the laboratory as quickly as possible Each piece is transfered into 100 mi of recovery medium and agitatad for 15's to 30's. Then the following ‘steps ere partormed. '9) Transfor 0,1 ml in 9,9 ml recovery medium and agitato. Transfer those 10 ml onto a fitration mombrane three times with £0'mi new recovery medium. Place the membrane on an agar nutiive medium and incubate. ) Transfer 1/mi onto a fitration membrane and then rinse three times with 0 mi new recovery medium. Place the membrane on an agar nutritive medium, and incubate ©) Transfer the remaining 98,9 ml onto a fitration membrane and rinse three times with 60 ml new recovery medium. Piace the membrane on an agar nutritive medium and incubate 4) Transfer each test place aseptically to a Pei dish, cover with agar medium and incubate 24 16150 2003 — A nghts asorvedISO 14898-1:2003(F) 14 [¢ tho number of VU determined on the membranes — the mean of counts resulting from €.2.5.2 a), 8) and), 1 isthe mean number of VU determined on he test pieces in E262 ¢). ‘= n4, + nis therefore the number of residual microorganisms following the laundering process. .2,7 Interpretation of results: Calculate the ratio of the number \v of microorganisms applied to the contiol pieces to . Check if the \eundoring process ensures reduction by a factor of at least 105 of the numbor of bactaria and reduction by at ‘east 10" of the number of yeasts and fungal spores. 180 2003 — At rights reserved 25ISO 14898-1:2003(E) Annex F (informative) Guidance on determining biocontamination of liquids F.1 Introduction This annox provides guidanco on the determination of biocontamination in liquids (aqueous or non-aqueous) in situations where control of biocontamination is considered desirable and necessary. This measurement Involves the collection of representative samples for the detection of viable partcles that are present and that ‘could need fo be controlled or monitored, This assesamant of the biocontamination of a quid is carried out in accordance with the bssic principles of this part of ISO 14688, which require the establishment of a Formal System to assess and contiol Diocontamination where cleanroom tachnology is applied. In additon, the following factors should be considered: 8) microbial ecology and related parameters in risk zones: ») expected concentration ofthe viable particles in the spectc liquids); ©) condition ofthe liquid!s); @) accuracy and effisiency of collection. F.2 Principle Cotection of samples for the detection and monitoring of microbial contamination of liquids ia risk zones is Carried out with appropriate sampling devices and according to a sampling plan, when the risk zone is at-rest and routinely under normal operation. Qualitaive and quantitative detection of viable particles may be achieved by diract or indiract measurement techniques. F.3 Procedure F.3.4 General There ere a variety of methods avallatle for the determination of clocontaminaton of liquids. The selection of 2 particular mathod will dapand on the nature of the iquid and the volume of sample required. For example, our plates, spread plates, membrane fitration and other methods can be used?) Pressure of liquids shall be suitably reduced for sampling. Attention should be paid to the liquid condition and the expected concentration of viable units in the Squid F.3.2 Sample preparation Depending on the liquid and biocontaminalion level, the sample can be assayed directly or after appropriate treatment 26 (© 1802008 — At igh resonISO 14698-1:2003(E) F.3.3. Sample investigation Methods for the detection of biecontamination should be selected that are appropriate to the nature of tha ‘iquid(s) to be sampled. F.4. Expression of results ‘The number of wabie particles should be expressed in viable units per 1 mi (1 cm®), (9180:2003— Al ngnsresenes or180 14898-1:2003(E) Annex G (informative) Guidance on training G.1 Introduction This annex provides guidance on taining personnel in the context of the control of biocontamination of cleanrooms and associated controled enviroments. ‘An essential contribution to all elements of qualty management systems and the key to quality management is 2 continuous, organized and appropriate training programme for all persons who work with the system chosen according to this part of ISO 14396. All persons involved, including sub-contractor personnel, should be trained in an appropiiaie manner to enable consistent, reliable and reproducible results and delivery of services. Paiticular consideration should te given to the training of persons involved in microbiological ‘monitoring and laboratory analysis. Training requires the development of usable procedures and training materials, coupled with documentation and record keeping of the performance elements applied, as well as a system for verification of training Training may be conducted within the organization, or outside by en independent organization This annex is not intended to supply criteria for assessment of competence, level of performance, performance appraisals or a complete personnel training programme, but to point out the most important {ctivitios and elements to include in a training and verfiction cycle in the field of biocontamination control, G.2 Elements for standardized training programmes G.21 General Training documents should be prepared wherever the procedure warrants it, and in that case should be a detailed presentation of the single steps of procedures. Each step should be divided inta its consituent parts to desenibe fully the content and scope of the training required for these stops. 6.22 Training documents A training document should consid the folowing aspects a) listing of documents and references to be used during training; 8) description and definition of training objectives and methods to be used ©) descriptions of the details of each procedural step. 6s appropriate, for a full understanding of the requirements for performing that specific, 4) results of measurements, if appropriate; @) scheduled training courses, whether held in-house or elsewhere, 1) description of the evaluation of the effectiveness of training, 28 © 180.2003 —Al sgnisreseneaIS 14898-1:2003(E} {A unified training manual should be developed for the selected system, rather than production of indWidual ‘uides for each procedure or assay. A uniform document format should be adopted and should focus on the procedure concerned, avoiding excessive comprehensive background information 6.2.3 Training manual ‘The department or organization should collate all the training Gocuments related to a particular area or facity into a singlo manual to serve as the training manuel. This manual shou'e transtate and standardize the details ofa procedure into seperate, clearly understandable steps to be used to achieve acceptable performance. A training manual should typically be used for the following purposes: a) training new employees, b) implementation of new methods, instruments and sampling devices: ©) good microbiological and hygiene practices and laboratory safely, 4) risk analysis: fe} changes in the sampling and monitoring plan: 4) retraining when less than satisfactory performance is reported: 3} periodic verification G.2.4 Microbiological and biocontamination control procedures Formal training for all porsons assigned to controlled environments. and individuals responsible for the administration and general operation of the environmertal control programme, including sampling personnel and ‘aboratory technicians, should include 4) basic principles of microbiology, >) fundamentals cf applied microbiology, hygiene and epidemiology, 12) good employee aseptic techniques and precautions, 4) principles of environmental control processes, 2) microbiological sampling techniques, 1) basic principles of microbiological hazard analysis, 9) understanding microbiotogical target, alert and action levels, h)prinples of trend analysis, specialized training on all required laboratory methods, and additionally on any automated system to be Used to aesist microbial idantification, and 1) instruction on writing clear roporte (2180 2003 — Al rghts reserved 29ISO 14898-1:2003(E) G.3 Training verification G.3.1 General Verification should be made thet training has taken place and is documented. Verification that personnel have been trained in the specified system should be procedure-based. Therefore, veriication of training should focus on the epocfics of procedure performance. To maka it aatior to very an omployee. performance on the procedures associated with the employee's job, @ systematic approach to implementation of training verfication is recommended. G.3.2 Assessment tools Aer the training verification procedure is developed, itis necessary to dasign an assessment tool to verity the affectiveness of training, The employee's pactormance should be assessed against an established performance standard as defined by the laboratoryidepertment management/supervision or training institution. A variety of “measurng tools” may be used to verify performance, for example: a) assessment against the objectives of the training programme: ©) itn tests; ©) assessment of response to 1) case studios trom the fields of concern, 2) problems, and 3) situations related to procedure; J) response to oral questioning related fo procedure; 2) testing of known and unknown samples. Pass/failciteria should be known to all paricipants before assessment is carried out G.3.3 Documentation 3.3.1 General Documentation of the results of training may be supported on a variety of systems. Consistency in record keeping and formatting of employees! records is important to the documentation of the traning verfication. Documentation should comply with regulatory and accreditation requirements and standards, with ‘orgenizational (employer) or departmental polices or both, and with laboratory dopartment or saction sorvicas: ‘and guidelines. 6.3.3.2 Retention of records Records should be held in a clear, writen form indicating a) the ently of trainee, b)_ who was responsible for training, ©) where records are kept and who has access, ) when records can be discarded, and ©) when and how records ars reviewed, 30 (©180.2003 — Alirgnisresavesio} 3 4 a co) a 10) mi 112) 13) 114] 115) 116) 171 (18) 19] (20) 180 14698-1:2003(E) Bibliography ISO 14544-1, Cleanrooms and essociated controlled environments — Part 1: Classifeation of air cleanliness ISOMIEC Guide 51, Safely aspects — Guidelines for thei inclusion in standards 1S0 9000, Quality managenent systems — Fundamentals and vocabulary 180 14971, Medical devices — Aoplicalion of risk management to medical devices COVELL, V.T., MERSHOFER, MW. eds). Risk assessment methods. Approaches for assessing health and environmental risks. New York, Planum Prass, 1993 PIERSON, M.D., CORLETT, DA (208). HACCP — Principles and applications, New York Van Nostrand Reinhold, 1982 Hozard Analysis Crticel Control Point (HACCP) system and guidelines forts application. 1995 Cedex Alimentarius: Commission. Ainorm 97/13. Annex to Appendix ll. Joint FAOWHO Food Standards Programme. Rome, Food and Agricultural Organization of the United Nations, 1995, IAANKE, M, Use of the HACGP concept for the sk analysis of pharmaceutical manufacturing process. European Jourmal of Parenteral Sciences, 2(4), pp. 113-117, 1997 'S0 15161, Guidelines on tho application of ISO 9001:2000 for the food and drink industry IEC 61025:1900, Fault tree analysis (FTA) IEC 60812:1985, Analysis techniques for systom roliabilty — Procedure for failure modo and effects ‘analysis (FMEA) Wu, W. Operating @ cleanroom: Contamination control. From Chapler 15, Cleanroom technology — The fundamentals of design, testing and operation. Chichester, U.K. Jonn Wiey and Sons, 2001, DIN 58849-9, Quality management in medical merobiology — Part 9 Requiremems for use of contrat strains for tasting cufture media Conny, JEL (ed): Quality assurance and quality control of microbiological culture modia. Darmstadt, GIT Veriag, 1962 IsoaRo, P.. CALOF, J., CoNTAMN, C. La contamination microbiologique des atmosphéres closes. COngines: methodes o'etudes. Journa de Chirurgre (Paris), 119, pp. 503-512 1807218, Microbiology of food and animal feeding stufls— General res for microbiological ‘examinations 'S0 31 (allparts), Quantities and units HENNNGSON, EW. AKLEERG, M.S. Evaluation of microbiological air samplers: a review. Joummal of Aerosol Science, 28, pp. 1458-1492, 1884 Marti, B, LiGHTHART, B. Effects of betaine on enumeration of alrbome bacteria Applied and Environmental Vierobiology, 68, pp. 1286-1289, 1990 \WaYTE, W. In support of settle plates. PDA Journal of Pharmaceutical Science and Technology, 60, pp. 201-204, 1696 15180 2003 — A egms eserves auISO 14898-1:2003(E) 1 (22) (23) a) (25) 32 PirzuaRe, Mt, PASQUARELLA C., PITZURRA, O., SAVIN, A, La misura dela contaminazione microbica ‘dell aria: ufcn’. elo IMA Nota 2. Ann. la, 8, pp. 447-452, 1996, Macien,.M., FiRsT, MW. Reuter contfugal air sampler: Measurement of effective airlow rate and collection efficiency. Applied and Environmental Microbiology, 48, pp. 1980-1262 CLARK, RP, GOFF, MR. The potassium iodide method for determining protection factors in open- fronted microbiological safety cabinots. Joumal of Appliod Mecrabiolegy, 51, pp. 439-460, 1981 WHYTE, W., NIVEN, L. Airborne bacteria sampling: the effect of dehydration and sampling time. Journal Of Parenteral Science and Technology, 40, op. 182-187, 1986, Tavion, RLHIM., ALLEN, JIM., GELDREICH, E.E. A comparison of pour plate and spread plate methods. Journal ofthe American Water Works Association, 78, pp. 35-37, 1933 (01802003 — AI ngs resoneeSeerISO 14898-1:2003(E) les 13,040.35, Price based on 22 paves (©180:2000 — Al ngteresorved