Tiêu chuẩn iso 11138 1 2017

4.1.2.2 Man facturing comp nents shal inclu e al mate ials incorp rated in, or coming into dir ect contact with, the tes or ganism suspension, the inoculated car ie an / r it primary p c

Sterilization ofhealth car e products —

Part 1:

Stérilisation de s produits de santé — Indicateurs biolo iques —

Partie 1: Exigence s g n rale s

T ir d edition

2 17-0

Reference n mb r

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© ISO 2017, P blshed in Sw itz rlan

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written permis ion Permis ion c n be req esed from either ISO at the ad r es below or ISO’s member bod y in the c u try of

F reword i v

Introduction v

1 Sc ope 1

2 Nor mati ve r eferenc es 1

3 Terms an definitions 1

4 General manufacturing r eq ir ements 4

4.1 Man facturing contr ols 4

4.1.1 Q ualty manag ement ys ems 4

4.1.2 Trac a i ty 4

4.1.3 Finished pr od ct r eq ir ement 4

4.1.4 Pe son el 4

4.2 Tes or ganism 4

4.2.1 Strain 4

4.2.2 Orig inating inoculum for suspension .5

4.2.3 Tes or ganism cou t 5

4.3 Information to be pr ovided b the manufactur er (la el ng ) 5

4.4 Storag e an transp rt 6

5 Specific manufactur ing r eq irements 7

5.1 Suspensions 7

5.2 Car ie , primary and se on ary p ckag ing 7

5.3 Inoculated car ie 7

5.4 Biolog ical in icators 8

5.5 S lf-contained biolog ical indicators 8

6 Deter minatio of po ulation and r esistanc e 8

6.1 General r esistanc req irement 8

6.2 Tes or ganism 8

6.3 Po ulation of tes or ganisms 8

6.4 Resis anc characte is ics 9

6.5 Tes con itions

9 7 Cultur e c onditions 10

7.1 Incub tor 1

7.2 Gr ow th medium 1

7.3 Incub tion 1

7.4 Sofwar e v ldation 1

7.5 Incub tion time using dete tion sys em 1

A nne x A (normative) Deter mination of viable count

12 A nne x B (normative) Determination of g r ow th inhibitio b carr ier s and primary packag ing materials e x posed to steriization pr oc es es 14

A nne x C (normative) D value deter minatio b survi vor curve metho 17

A nne x D (normative) D value determinatio b fractio negati ve metho .21 A nne x E (normative) Survi val kil r esponse character istic 37 A nne x F (informative) Relatio ship betwe n c omp nents of biolog ical indicator s 39 Biblog raphy 40

ISO (he Int ernational Org nization for Stan ardization) is a worldwidefede ation of national s an ards

b dies (ISO membe b dies) The work of pr p ring Int ernational Stan ards is normaly car ied out

through ISO t ech ical committ ees Each membe b dy int er st ed in a subje t for w hich a t ech ical

committ ee has be n es a lshed has the right t o be r pr sent ed on that committ ee Int ernational

org nizations, g overnmental an non-g ove nmental, in laison with ISO, also take part in the work

ISO cola orat es closely with the Int ernational Ele trot ech ical C mmis ion (IEC) on al matt ers of

ele trot ech ical s an ardization

The proc d r s used t o develo this document an those int en ed for it furthe maint enanc ar

desc ibed in the ISO/IEC Dir ctives, Part 1 In p rticular the dife ent a pro al c it eria ne ded for the

dife ent ty es of ISO document should be not ed This document was draft ed in ac ordanc with the

edit orial rules of the ISO/IEC Dir ctives, Part 2 ( e www iso org dir ctives)

A tt ention is drawn t o the p s ibi ity that some of the element of this document ma be the subje t of

p t ent right ISO shal not be held r sponsible for identifying any or al such p t ent right Detais of

any p t ent right identified d ring the develo ment of the document wi be in the Introd ction an / r

on the ISO ls of p t ent de larations r c ived ( e www iso org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

F or an ex lanation on the v lu tary natur of s an ards, the meaning of ISO spe if ic t erms an

ex r s ions r lat ed t o conformity as es ment, as wel as information a out ISO’ s adhe enc t o the

World Trade Org nization (WTO) principles in the Te hnical Bar ie s t o Trade (TBT) se the folowing

URL: www iso org iso / for word html

This document was pr p r d b Te h ical Committ ee ISO/TC 1 8, Ste ii zation o health c ar produc ts

This third edition canc ls an r plac s the se on edition (ISO 1 1 8-1:2 0 ), w hich has be n

t ech icaly r vised

A l s of al p rt of ISO 1 1 8 can be foun on the ISO websit e

This document spe if iesg ene al r q ir ment for prod ction, la el ng, t es methods an pe formanc

r q ir ment for the man factur of biolo ical in icat ors inclu ing inoculat ed car ie s an suspensions

int en ed for use in v l dation and monit oring of st eri ization proc s es Othe p rt of ISO 1 1 8

pro ide ad itional spe if ic r q ir ment for biolo ical indicat ors for defined st eri ization proc s es

A gra hic desc iption of a biolo ical in icat or an it comp nent is pr sent ed in Ta le F.1 The

pr sentation inclu es the two ty es of biolo ical in icat ors w hich ar co e ed b ISO 1 1 8 (al p rt )

This show s that inoculat ed car ie s can be pr sent ed dir ctly t o the st eri izing ag ent without prior

p cka ing, or inclu ed in a primary packag e that pe mit ac es b the st eri zing ag ent

The r sis anc charact eris ics depen on the ty e of t es org nism, it n mbe s, the method of

pr paration, the subs rat e up n w hich it is inoculat ed, environmental con itions d ring inoculation

an drying an the efe t of the primary p ckag e A dvic on sele tion, use and int erpr tation of r sult

of biolo ical in icat ors can be fou d in ISO 14161

F or any in ivid al st eri ization proc s , inclu ing those co e ed in r lev nt p rt of ISO 1 1 8, the

r sis anc of the biolo ical in icat or wi l also depen on it mic oenvironment d ring t es ing In

theory, this could lead t o an in nit e v riation in the pr p ration of biolo ical in icat ors Mor o e , a

st eri zation proc s could be manipulat ed in inf init e v riety t o suit each p s ible set of con itions t o

w hich prod ct could be ex osed It has, the efor , be n a routine practic t o man factur biolo ical

in icat ors that, w hen ex osed t o a set of con itions in a def ined st eri zation proc s , pro ide r sis anc

charact eris ics ex r s ed as D v lues an , w he e r lev nt, z v lues Such v lues ar set out in the

r lev nt p rt of ISO 1 1 8

The ISO 1 1 8 se ies r pr sent the cur ent “ tat e-of-the-art” ac ording t o the ex e t r pr senting

man factur rs, use san r gulat ory authoritiesinv lved in develo ing this document

Biolo ical in icat ors for spe ific st eri ization proc s es not co e ed b r fe enc t es conditions in

r lev nt p rt of ISO 1 1 8 should comply with the g ene al r q ir ment in this document, inclu ing

the r sis anc t es ing proc d r s Such biolo ical in icat ors might not be wel enough desc ibed,

or might be used for no el st eri zation proc s es, or might be r pr sent ed b isolat ed bio urden

mic o rg nisms If mic o rg nisms othe than risk group 1 (WHO 2 04 )ar inclu ed in these biolo ical

in icat ors, a pro riat e safety measur s (e.g containment) ar ne es ary

Stan ards exis pro iding r q ir ment for the v ldation an control of st eri ization proc s es

( e Biblo ra hy)

NOTE It is pos ible that some cou tries or re ions have p blished other stan ards covering req irements

for st erilization or biolo ical in icat or (se Biblio rap y)

Sterilization ofhealth car e products — Biolog ical

Part 1:

This document spe if iesg ene al r q ir ment for prod ction, la el ng, t es methods an pe formanc

charact eris ics of biolo ical indicat ors, inclu ing inoculat ed car ie s an suspensions, an their

comp nent , t o be used in the v ldation an routine monit oring of st eri zation proc s es

This document spe if ies b sic an common r q ir ment that ar a pl ca le t o al p rt of ISO 1 1 8

R eq ir ment for biolo ical in icat ors for p rticular spe ified proc s es ar pro ided in the r lev nt

p rt of ISO 1 1 8 If no spe if ic subseq ent p rt is pro ided, this document a ples

NOTE National or re ional re ulations can ap ly

This document does not a ply t o mic o iolo ical t es sy st ems for proc s es that r ly on phy sical r mo al

of mic o rg nisms, e.g filtration proc s es or proc s es that combine phy sical an / r me hanical

r mo al with mic o iolo ical inactiv tion, such as use of washe disinfe t ors or flushing and st eaming

of pipel nes This document, howeve , can contain element r lev nt t o such mic o iolo ical t es

sy st ems

2 Normati ve r eferences

The folowing document ar r fe r d t o in the t ext in such a wa that some or al of their cont ent

cons itut es r q ir ment of this document F or dat ed r fe enc s, only the edition cit ed a ples F or

un at ed r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amen ment ) a ples

ISO 1 1 5, Sterili zatio o health-c ar produc ts — Eth lene ox ide — R eq ir ments fr the develo men

v ldatio a d ro tine c ontrol o a s te ii zatio proc es s fr medic al dev ic es

ISO 1 7 7-1:2 0 , Ste ii zatio o medic al dev ic es — Mic ro iolo ic al meth ds — Part 1: Dete min tio o

a p p lation o mic ro rg ni s ms o produc ts

ISO 149 7, Ste ii zatio o health c ar produc ts — G ene al r q ir ments fr charac te i zatio o a s te ii z in

a ent a d the develo men v ldatio a d ro tine c ontrol o a s terili zatio proc es s fr medic al dev ic es

ISO 1766 -1, Ste ii zatio o health c ar produc ts — Moi s t heat — Part 1: R eq ir ments f r the develo men

v ldatio a d ro tine c ontrol o a s te ii zatio proc es s fr medic al dev ic es

ISO 1 47 , Ste ii zation o health c ar produc ts — B iolo ic al a d chemic al indic ators — Tes t eq ipment

3 Terms and definitions

F or the purposes of this document, the folowing t ermsand definitions a ply

ISO an IEC maintain t erminolo ical data ases for use in s an ardization at the folowing ad r s es:

— IECEle tro edia: a aia le at ht p:/ www ele tro edia org

culture col ection number

uniq e identification of the t es org nism alocat ed b a scientif ical y r co niz d se vic cultur

cole tion

3.5

culture co ditio s

combination of growth media an man e of incub tion used t o promot e g ermination, growth an / r

multiplcation of mic o rg nisms

Not e 1 t o entry: The man er of incub tion can inclu e the t emperature,time an an other con itions specif ied

for incub tion

Not e 1 t o entry: Other c itical proces varia le(s) e hibiting f ir t order inactivation kinetic can achieve an

inactivation of 9 % of a pop lation of the t est mic o rg nism u der stated con itions

[ SOURCE:ISO/TS1 1 9:2 0 , 2.1 , modif ied]

3.7

inactiv atio

los of a i ty of mic o rg nisms t o grow an / r multiply

[ SOURCE:ISO/TS1 1 9:2 0 , 2.2 ]

3.8

in culated car ier

sup orting mat erial on or in w hich a defined n mbe of via le t es org nisms ha e be n dep sit ed

Not e 1t o entry: S e An e F

3 9

nominal po ulatio

man factur r’s s at ed n mbe of via le mic o rg nisms

Not e 1t o entry: T is is g neraly e pres ed in lo

1

fu ction (e.g 1

6

)

primary packag e

element of the packa ing sy st em w hich maintains the int egrity of the prod ct

Note 1 to entry: The packa ing system prot ects the inoculated car ier from damag e an contamination without

preventing penetration of the sterilizing a ent

3.1

proces chaleng e device

PCD

it em designed t o cons itut e a defined r sis anc t o a st eri ization proc s an used t o as es

pe formanc of the proc s

[ SOURCE:ISO/TS1 1 9:2 0 , 2.3 ]

3.12

resistometer

t es eq ipment designed t o c eat e def ined r fe enc combinations of the phy sical an / r chemical

v ria les of a st eri zation proc s

[ SOURCE:ISO 1 47 :2 0 , 3.1 , modified]

3.13

seco dary packag e

containe in w hich biolo ical in icat ors ar p cked for transport an st orag e

3.1

self-co tained biological indicator

biolo ical in icat or pr sent ed in such a wa that the primary p ckag e, int en ed for incub tion, contains

the incub tion medium r q ir d for r co e y of the t es org nism

3.1

survivalkil window

ext ent of ex osur t o a st eri ization proc s u de def ined con itions w he e the e is a transition from

al biolo ical in icat ors showing growth t o al biolo ical in icat ors showing no growth

3.1

surviv r curve

gra hical r pr sentation of the inactiv tion of a p pulation of mic o rg nisms with inc easing

ex osur t o a mic o icidal ag ent u de s at ed con itions

3.17

suspension

via le t es org nisms suspen ed in a fluid

Note 1 t o entry: Suspension can b a biolo ical in icator if re d to use in a se led glas ampoule or may b an

intermediate component used t o prod ce an inoculated car ier or biolo ical in icator

4 General manufacturing requirements

4.1 Manufacturing c ontrols

4.1.1 Qualty manag ement systems

A formal q al ty sy st em (e.g ISO 1 48 , GMPs or othe national or r gional r q ir ment ) t o co e al

o e ations r q ir d b this document shal be es a l shed, document ed an maintained In p rticular,

pr cautions at al s ag es of prod ction t o minimiz contamination that would adve sely afe t the

pe formanc of the biolo ical in icat or shal be taken

4.1.2 Trac eabiity

4.1.2.1 Tr ac a i ity of man facturing comp nent shal be maintained

4.1.2.2 Man facturing comp nents shal inclu e al mate ials incorp rated in, or coming into dir ect

contact with, the tes or ganism suspension, the inoculated car ie an / r it primary p ckag e

4.1.3 Finished pr oduct req ir ements

The f inished prod ct shal comply with the fol owing r q ir ment :

a) la el ing (4.3);

b) man facturing (Clause 5);

c) r sis anc charact eris ics (6.4);

d) st orag e an transp rt (4.4);

e) incub tion (7.3)

NOTE 1 Ad ice on methods for the use of biolo ical in icat or is provided in ISO 1416 1

NOTE 2 National an / r re ional req irements might e ist, for e ample, in the various national or re ional

p armacopo ias

4.1.4 P er onnel

The proc d r s an methods in this document shal be car ied out b suita ly trained and ex e ienc d

la orat ory pe son el ( e e.g ISO 1 48 )

4.2 Test or ganism

4.2.1 Strain

4.2.1.1 Tes or ganisms shal be of a defined strain, a vaia le thr ough a r ecog niz d cultur e cole tion,

and shal be identified b a pr opriate tes methods A statement of trac a i ty shal be provided to the

pur chase up n r eq est

4.2.1.2 The tes or ganism shal be a s rain that is

a) suita le for han lng without spe ial containment faci ities, does not ne d spe if ic containment

proc d r s for han lng and does not ha e spe if ic transp rt or mai ing r q ir ment (e.g Risk

Group 1, WHO 2 04 ), an

b) suff iciently s a le t o maintain it r sis anc charact eris ics for the d ration of the s at ed shelf-l fe

w hen transport ed an st or d in ac ordanc with la el dir ctions

NOTE Traditional y, the test org nisms of biolo ical in icator have b en b ct erial spores, usual y derived

from B ac ilus or G eo ac ilus pecies

4.2.1.3 Tes or ganisms othe than b cte ial sp res ma y be used if they ha ve be n shown to pr ovide

a pr opriate resis anc to thes e i zation pr oc s

4.2.2 Orig inating inoculum for suspensio

4.2.2.1 The initial inoculum for each b tch of tes or ganism suspension shal be

a) trac a le t o ther fe enc cultur an a ai a le through a r co niz d cultur cole tion, an

b) ve if ied as t o it identity and purity

4.2.2.2 The methods used for maintaining tes or ganism cultur es shal be desig ned to pr ote t

them fr om contamination and to minimiz any in uc d chang es in the in er ent pr ope ties of the tes

or ganisms

4.2.2.3 Ve ification tes s ar e spe ific for each s r ain of tes or ganism an shal be documented an

v ldated

4.2.3 Test or ganism c ount

The via le t es org nism cou t of the suspension shal be det ermined in ac ordanc with An ex A

4.3 Information to be provided by the manufacturer (label ing )

4.3.1 T e folowing information shal be pr ovided on the la el of each in ivid al u it of suspension,

inoculated car ie p ckag ing an biolog ical indicator:

— a u iq e code b w hich the man facturing hist ory can be trac d;

— thenameof thet es org nism;

— an in ication of the st eri ization proc s for w hich the suspension, inoculat ed car ie s or biolo ical

in icat orsar suita le;

— theex iry dat e, ex r s ed ac ording t o ISO 8 0 , e.g YYYY-MM-DD;

— theman factur r’ s name, trademark, ad r s or othe means of identification

Int ernational y r co niz d symb ls ma be used w he e a pro riat e ( e 4.1.3, ISO 1 2 3-1, an

ISO 1 140-1)

4.3.2 In ad ition to the information pr ovided in 4.3.1, the information g iven in Ta le 1 shal be pr ovided

on or within the se on ary p ckag ing of each b tch of prod ct

Table 1 — Informatio to be pro ided b the manufacturer

Inoculat ed

car ie

Biolo ical

in icat or

The proces for which the prod ct is suita le for use,

the resistance an the proced re an car ier used to

determine the resistance

a

Instructions for use, especialy data a out he medium,

incub tion an other con itions t o b used for

recov-ery of test org nisms af er e posure t o the steril

iza-tion proces

The n mb r of test org nisms per ml (suspension), or

per u it inoculated car ier or biolo ical in icator)

a

The n mb r of prod ct u its in the secon ary pack — R eq ired R eq ired

a

Tes methodolog used to determine resis anc an pop lation should be su pled b the man fa turer u on req es

4.3.3 If a spe ific instrument isr eq ir ed to dete t outg r ow th of the biolog ical in icator, the instrument

man factur er shal spe ify use of that ins rument an pr ovide a c rtificate of calbration an information

on ther eq ir ed recalbr ation pr oc d r es

4.3.4 If a plca le, information shal be pr ovided on r eq ired maintenanc se vic of the spe ified

ins rument inclu ing detaied ins ructions of how to pe form the maintenanc , se vic and it fr eq ency

4.4 Storag e and transpor t

4.4.1 Storag e and tr ansp rt conditions for the tes or ganism suspension shal be maintained such that

the tes or ganism suspension comples w ith the req ir ement of this document an , wher e r elev nt , a

subseq ent p rt of ISO 1 1 8

4.4.2 If inoculated car ie s ar e p ckag ed for s orag e or transp rt , they shal be p ckag ed in a wa y that

does not adve sely afe t the nominal po ulation or pe formanc of in ivid al inoculated car ie s

4.4.3 Storag e and transp rt con itions for inoculated car ie s shal be maintained such that the

inoculated car ie s comply w ith the req irement ofthis document and where relev nt , a subseq ent

p rt of ISO 1 1 8

4.4.4 In ividualy-p ckag ed biolog ical indicators shal be plac d in a se on ary p ckag e for transp rt

and s orag e Packag ing for transport an s orag e shal ensur e that biolog ical in icators comply w ith this

document an , where r elev nt ,a subseq ent p rt of ISO 11 3

5 Specific manufacturing r equir ements

5.1.1 Cultur e medium an incub tion con itions shal consis ently pr od c tes or ganism suspensions

that me t the pe formanc r eq ir ement of this document an any r elev nt subseq ent part of

ISO 1 13

5.1.2 T e suspen ing medium for the tes or ganism suspension shal not adve sely afe t the s a i ty

of the tes or ganism an shal be comp tible with the proc d r es an mate ials emplo ed in the

man factur e of inoculated car ie s an biolog ical in icators

5.1.3 T e method of harvesting an subseq ent tr eatment of suspensionsto be used in the inoculation

of car ie sshal ensur e that resid es do not ad ve sely influenc the pe formanc of the inoculated car ie

or biolog ical in icator

5.2 Car rier, primary and sec ondar y packag ing

5.2.1 T e mate ials of the car ie an the primary an se ondary p ckag ing shal not contain any

contamination (physical, chemical or micr obial) that would adve sely afe t the pe formanc of the

biolog ical in icator

5.2.2 T e car ie , the primary an se on ary p ckag ing , an the spe ified s orag e conditions shal be

desig ned so that the pe formanc char acte is ics of the biolog ical in icator me t the r eq ir ements of

this document thr oug hout the s ated shelf lfe of the pr od ct T e pur chase shal be pr ovided with a

statement of the max imum an minimum v lues of each dimension of the car ie on req est

5.2.3 During and afe the s e i zation proc s , the car ie and theprimary p ckag ing shal not r etain

or r elease any substanc to such an ex tent tha on trans e to the incub tion medium, u de thecultur e

con itions, the g r ow th of low n mbe sof surviving tes or ganisms wil be in ibited Testing shal comply

with A nnex B

5.2.4 T e car ie , the primary packag ing , an the se on ary p ckag ing shal withs an plan ed

transp rt an han lng at the p int of use, w ithout damag e or br eakag e

5.2.5 Ra w mate ials used for the car ie an the primary packag ing shal withs an ex posur e to the

s e i zation pr oc s for which they ar e inten ed in such a wa y that the pe formanc characte is ics of

the inoculated car ie or biolog ical in icator ar e maintained Complanc shal be tes ed b o se v tion

of the car ie an the primary p ckag ing ex posed to the ex tr eme rang es and rates of chang e of the

chemical an physical v ria les of the s e i zation pr oc s

NOTE R eference st erilization con itions can b fou d in relevant parts of ISO 1 1 8

5.3 Inoculated car ier

5.3.1 T e inoculated car ie shal be made of mate ials that wil withstan ex posur e to the s e i zation

pr oc s without dis ortion, melting , corr osion or othe faiur e that would imp ir the use ofthe inoculated

car ie

5.3.2 Only one s rain of tes or ganism shal be used in a b tch of inoculated car ie s, u les the

man factur er has demons r ated that the use of multiple strains does not sig nificantly afe t tes or ganism

pe formanc in the spe ified s e i zation proc s

5.3.3 Prior to inoculation, the car ie shal be s e i z d in ac or danc w ith ISO 1 6 5-1, ISO 1 1 5,

and ISO 149 7 or othe r elev nt s e i zation methods If s e i zation is not practica le, ac eptable

bio urden lmit of the car ie prior to inoculation ma y be es a lshed in ac or danc with ISO 11 3

-1:2 0 , An ex B

5.3.4 C r ie s shal be inoculated so asto maintain a consis ent micr obial p pulation ( e 6.3)

5.4 Biolog ical indicator

5.4.1 In ividualy packag ed biolog ical indicators shal be prep r ed b placing in ivid al inoculated

car ie s in a primary p ck

5.4.2 T e primary p ckag ing shal be v ldated for it intended use ( e A nnex B)

5.5 S lf-c ontained biolog ical indicator s

The pe formanc of self-contained biolo ical in icat ors shal be v ldat ed, inclu ing the a i ity of the

cultur medium t o promot e growth of the t es org nisms aft er being subje t ed t o the st eri ization

proc s

6 Determination of population and r esistanc e

6.1 General resistanc e r equirements

6.1.1 T e resistanc of each lot/b tch of biolog ical in icators shal be tes ed to demonstrate

conformanc w ith thepe formanc r equir ement spe ified in this document and any r elev nt ubseq ent

p rt of ISO 1 1 8

6.1.2 Resis anc characte istics of biolog ical indicators inten ed for s e i zation pr oc s es not

spe ified in any subseq ent p rt of ISO 1 1 8 shal be defined using the spe ific c itical v ria les of that

s e i zation pr oc s ( e ISO 149 7)

6.1.3 It is r ecog niz d that the v ldation and monitoring of some s e i zation proc s es ma y use

biolog ical in icators that do not me t the minimum p pulation and/ r r esis anc c ite ia spe ified in

ISO 1 13 These biolog ical in icators are ac eptable pr ovided that

a) al othe r q ir ment of ISO 1 1 8 ( inclu ing the method of t es for po ulation an r sis anc )

ar met,

b) theprod ct information inclu es a clear s at ement of the po ulation an r sis anc , an

c) the prod ct la el car ies a clear warning that he p pulation an / r r sis anc (as a pro riat e) is

below the v lue spe if ied in the r lev nt p rt of ISO 1 1 8

6.1.4 Resis anc tes ing shal inclu e dete mination of the via le cou t an dete mination of the

resis anc characte istics ( e 6.3 and 6.4)

6.2 Test or ganism

The t es org nism shal be spe if ied ( e 4.2)

6.3 Population of test or ganisms

6.3.1 T e via le cou t hal be dete mined ( e A nnex A)

6.3.2 Po ulation ve ification shal be achieved when r esult fal within 5 % to 3 0 % of the

man facturer ’s s ated nominal p pulation C nfir mation tes r esult of the p pulation dete mined b end

user s or man facturer s d ring s ated shelf lfe ma y me t the 50 % to 30 % rang e, but could fal below

the minimum p pulation spe ification as defined in this document In these cases, the or ig inal p pulation

is considered to be ver ified if the confirmation tes result are within the 5 % to 30 % rang e

6.4 Resistanc e characteristic

6.4.1 Resistanc characte is ics shal be dete mined b a combination of the folowing methods:

a) det ermination of the D v luethrough the cons ruction of a surviv r curve ( e An ex C); or

b) det ermination of the D v luethrough a fraction neg tive method ( e An ex D); and

c) ve if ication of the surviv l/ki l r sponse charact eris ics ( e An ex E)

6.4.2 T e v lues o tained b these methods shal be within the rang es spe ified in the r elev nt p rt

of ISO 1 1 8 At leas two of these v lues shal be inclu ed in the la el ng of the biolog ical in icators

6.4.3 T e D v lue shal be within ± 0 % of the manufactur er’s stated v lue when dete mined b the

man factur er d ring the stated shelf lfe using the method spe ified b the man factur er

Idealy, the surviv r curve is l near o e the ful rang e of inactiv tion In practic , deviations from

this ideal oc ur, but lnearity shal be maintained within ac epta le lmit Cons ruction of a surviv r

curve b en me ation es a l shes the r sis anc for surviving po ulations gr at er than a pro imat ely

5 × 1

1

, w he eas the fraction neg tive method es a l shes a s atis ical y b sed calculation of surviving

t es org nisms below that level Go d cor elation of the D v lues o tained b the two methods can

the efor be used t o es a lsh that the e ar no se ious deviations from a lnear surviv r curve

R elev nt p rt of ISO 1 1 8 might r q ir ad itional det erminations [e.g z v lue for biolo ical

in icat ors for mois heat t eri zation (ISO 1 1 8-3) or dry heat st eri zation (ISO 1 1 8-4 )]

The r sis anc charact eris ics spe if ied in this document an any subseq ent p rt of ISO 1 1 8 a ply

t o the spe ific t es con itions s at ed in those p rt

Confirmation t es r sult of the D v lue det ermined b en use s or man factur rs d ring s at ed shelf

l fe ma me t he ±2 % r q ir ment, but could fal below the minimum D v lue spe ification asdef ined

in this document In these cases, the original D v lue is conside ed t o be ve if ied if the conf irmation t es

r sult ar within the ± 0% rang e

6.4.4 T e surviv r curve, when ploted as a semi-logarithmic curve of the lo

1

of the via le tes

or ganism cou t agains time, shal be lnear w ith a coeficient of dete mination, r

Table 2 — Minimum t est samples ac ording to metho

Tes meth d ac ording t o

ISO 1 1 8-1

Minimum n mbe of

t es samples

Minimum n mbe of

ex osur con itions

Minimum t otal n mbe

b

1 0b

Minimum t otal n mb r depen ing on choice of combination of methods: 1 4 or 2 4

NO E Common tes c n itions for spe if ic s eri sation methods have developed over time an are presented in relevant

par s of ISO 1 1 8

a

The viable c u t of the u pro es ed ino ulated c r ier or biologic l in ic tor

b

The e t a set of tes in c n itions at the e posure su seq ent o t ( e Table D.1) is not used in the c lculations, b t is

a c n ition for a c ptin the tes result as vald

7 Culture conditions

7.1.1 T e incub tor shal be set to pr ovide, an monitored to confirm, the spe ified cultur e con itions

7.1.2 In ad ition to r outine monitoring of temperatur e, the temperatur e distribution w ithin the

incub tor shal be v ldated

7.2 Gr ow th medium

7.2.1 T e g r owth medium shal be spe ified an demons rated to supp rt the g rowth of an inoculum

of les than 1 0 tes or ganisms

7.2.2 L bel ng shal inclu e information on cultur e conditions afe ex posur e to the s e i zation

pr oc s ( e 4.3)

7.2.3 T e g r owth medium shal be v ldated to ensur e that it can neutralz any s e i zing ag ent

resid als that might influenc the via i ty of the tes or ganism ( e 5.2.3)

7.3.1 T e incub tion time an temperatur e shal be v ldated

7.3.2 Instructions for incub tion ( e Ta le 1) shal be provided A r efer enc incub tion time is

commonly r ecog niz d to be seven da ys for establshed s e i zation pr oc s es, such as mois heat

and ethylene o ide, using wel characte iz d tes or ganisms, such as G eo ac illus stearothe mo hilus

and B acillus atro h eus, r espe tively Wher e suficient data ar e not a vaia le to sup ort a r eferenc

incub tion time of seven da ys for a no el s e i zation method, at leas 14 da ys shal be used as the

refer enc incub tion time on which to b se the v ldation

NOTE 1 It is pos ible t o have a validated incub tion time that is shorter than the reference incub tion time

NOTE 2 National or re ional req irements for incub tion time validation can also e ist

7.4 S f ware val dation

F or incub t ors w hich incorp rat e sof war or w hich ar medical sof war in themselves, the sof war

shal be v ldat ed ac ording t o the s at e of the art taking int o ac ou t the principles of develo ment

l fe ycle, risk manag ement, v ldation an ve if ication

7.5 Incubation time using detection system

If a spe if ic det ection syst em is t o be used with a spe if ic biolo ical in icat or, then the combination of

the two shal be spe ified b the man factur r an used w hen det ermining the incub tion time

A nnex A

(normative)

A 1.1 E umeration te h iq es ar e used to dete mine the n mbe of via le tes or ganisms in

suspension, on inoculated car ie s, or fr om p ckag ed biolog ical in icators, b cou ting of dis inct colony

forming u it (CFUs) This method is used when the ex pe ted n mbe of r eco erable tes or ganisms is

a o e 5 × 1

1

CFUs

A 1.2 T e r elev nt pr od cts shal be examined for r eco er able tes or ganisms in ac or danc with A 2

thr oug h A.4 This method a ples to b th proc s ed an u pr oc s ed tes samples and can be used for

the dete mination of initial via le cou t (u pr oc s ed samples) as wel as for D v lue dete mination

using the surviv r curve method (pr oc s ed samples)

A 1.3 A lte native methods of en mer ation with demonstrated eq iv lenc to dir ect plating te h iq es

ma y be used

A minimum of four t es samplesfrom each lot/ at ch or ex osur shal be used

A 3 Sample pr eparation and cultur e methods

A 3.1 T e tes samples shal be plac d in an a pr opriate v lume of suspen ing medium The tes

or ganisms shal be eluted fr om the tes samples b a v ldated pr oc d r e (e.g mac ration with g las

beads, g rin ing and/ r blen ing in a homog eniz r an / r blen e , v rtexing , ultr asonication or othe

a propriate proc d r e) S e ISO 1 7 7-1

A 3.2 T e conc ntration of micr oor ganisms in the suspensions shal be adjus ed b diution, if

ne es ary, in a pr opriate s e ie diution fluid Numbe s of CFUs should be w ithin a spe ified rang e for

the method used wheneve p s ible

F or cultur s pour d int o molt en a ar or spr ad on soldif ied a ar in r gular siz Petri dishes, n mbe s

of C Us betwe n 3 an 3 0 ar conside ed t o be the mos ac urat e

A 3.3 A n a pr opriate method for the en meration of via le or ganisms shal be used

Ap ro riat e methods inclu e membrane f iltration t echniq es, dir ct spr ading on semi-sold a ar

growth medium or mixing with molt en t empe ed a ar growth medium ( e ISO 1 7 7-1)

A 3.4 T e biolog ical in icator man factur er shal identify or make a vaia le a suita le medium for

reco e y of tes or ganisms an / r complete data and ins ructions for the pr ep ration of such a medium

A 4 Incubation and enumeration

A 4.1 T e plated samples or the membrane fite s shal be incub ted at temperatur es an times

spe ified b the man factur er

In g ene al, the incub tion times an t empe atur s ar 5 °C t o 6 °C for not les than 48 h for

the mo hi ic mic o rg nisms an 3 °C t o 3 °C for not les than 48h for meso hi ic mic o rg nisms

NOTE Desic ation of the growth medium can adver ely afect growth at elevated incub tion temperatures

A 4.2 A fte the a pr opriate incub tion time, the numbe s of colony forming u its on the plates or

membrane fite s shal be cou ted and the mean n mbe of reco erable tes or ganisms pe a pr opriate

u it hal be calculated

A nne x B

(normative)

This method is used t o det ermine the suita i ty of thecar ie an primary packa ing mat erials for the

int en ed st eri ization proc s b identifying p s ible in ibit ory efe t of these mat erials on growth

of the t es org nisms aft er st eri ization The phy sical pro e ties of these mat erials shal ha e alr ady

be n t est ed for suita i ity ( e 5.2 an 5.3) Tes methods ar given in r lev nt p rt of ISO 1 1 8

Spe if ications for r sist omet ers ar given in ISO 1 47

B.2.1 A suspension of tes or ganisms of the same s rain shal be pr ep r ed in the same man e as

the or ganisms to be used for inoculation of car ie s T e suspension shal be of k own p pulation, as

dete mined b via le cou t , to pe mit dispensing of tes samples w ith a p pulation of les than 1 0

via le or ganisms

B.2.2 T e incub tor shal be set to pr ovide, an monitor ed to confirm, the temperatur e spe ified in the

cultur e con itions

B.2.3 T e g r ow th medium shal be as spe ified in the cultur e con itions

B.2.4 Tes samples shal be u inoculated car ie s or primary p ckag ing mate ials pr ep r ed in

ac ordanc w ith B.3

B.3.1 Pr ep r e fife n containe s of g rowth medium an eq i ibrate to the incub tion temperatur e

spe ified in the cultur e con itions Use the same v lume of g r owth medium as typicaly used for the

suspension, inoculated car ie or biolog ical in icator

B.3.2 Take a r epr esentative sample of eighte n pr eviously s e i z d u inoculated car ie s an divide

the car ie s into nine g r oups of two T ey shal be packag ed in the mate ial used in the man factur e of

the biolog ical in icators

NOTE If sterilization is not practica le, se 5.3.3

B.3.3 Take six g r oups of the car ie s fr om the sample taken in B.3.2, each containing two car ie s, an

then ex pose them to the s e i zation pr oc s

B.3.4 S t the o erational conditions of the r esis omete to the v lues spe ified in the r elev nt p rt of

ISO 1 13 , as a pr opriate

B.3.5 At the en of the pr oc s , u wrap the car ie s an asepticaly trans e them to the g r ow th

medium without subje ting them to inte mediate treatment Plac the content of one g roup of two

car ie s into each of the six containe s of g rowth medium previously eq i brated to the incub tion

temper atur e ( e B.3.1) Re or d the timetaken to complete the trans e

B.3.6 Incubate the g r ow th medium containing the car ie samples at the spe ified temperatur e for

2 h ± 1 min to alow any in ibitory substanc s to desor b from the car ie s Remo e the g r ow th medium

fr om the incub tor an inoculate each containe with a v lume of the tes or ganism suspension calculated

to contain les than 1 0 tes or ganisms Return the inoculated medium to the incub tor Incub te for the

v ldated time pe iod spe ified for r eco e y of biolog ical in icatorsu de normal con itions of use

B.3.7 C ntrol is pe formed b tr ans e ring the thr ee r emaining g r oups of two car ie s, not ex posed

to the pr oc s , to the thr ee r emaining containe s of incub tion medium Incub te these containe s for

2 h ± 1 min, then inoculate each containe with les than 1 0 tes or ganisms, an incub te them for the

spe ified incub tion time in the same man e as desc ibed in B.3.6

B.3.8 Micr oor ganism identification ma y be pe formed if the use of non-s e i e car ie s is suspe ted of

influencing the tes results

B.3.9 F r g rowth medium contr ols, incub te thr ee containe s of g r owth medium, without car ie s,

for 2 h ± 1 min Then inoculate each containe w ith les than 1 0 tes or ganisms an incub te for the

spe ified incub tion time in the same man e as desc ibed in B.3.6

B.3.10 T e remaining thr ee containe s of g r ow th medium ar e used as negative media contr ols Incub te

these non-inoculated media containe s for the v ldated time pe iod spe ified for reco e y of biolog ical

in icators u de normal con itions of use

B.4 Interpr etation of results

B.4.1 If “ no g r owth ” oc urs in one or mor e of the g rowth media contr ols, the tes proc d r e shal not

be r egar ded as v ld

NOTE “No growth” in the growth media controls can b in icative of failure to control the pop lation of the

test org nism inoculum or of inap ropriat e recovery con itions (i.e growth medium, incub tion time, incub tion

temperature etc )

B.4.2 If “ no g rowth ” oc urs in one or mor e of the car ie contr ols, the car ie shal not be r egarded as

suita le for the man factur e of inoculated car ie s or biolog ical indicators

NOTE “No growth” in the car ier control where growth oc ur ed in the growth medium can in icate that

the car ier mat erial is in ibit ory to the growth of the test org nism

B.4.3 If “ no g r owth ” oc urs in one or mor e of the six tests of car ie s ex posed to the s e i zation

pr oc s , the car ie mate ial shal not be r egar ded as suita le for the man factur e of inoculated car ie s

or biolog ical in icators

NOTE “No growth” can b caused by either high levels of a sorption of sterilant or by de radative chang s in

the car ier mat erial d ring the sterilization proces

B.4.4 If “growth ” oc urs in one or mor of the neg tive media controls, the t es proc d r shal not be

r g rded as v ld

NOTE “Growth ” in the ne ative media control could in icate that the media samples have b en contaminated

B.5 Determination of g row th inhibition by primary packag ing materials

B.5.1 Samples of theprimary p ckag ing mate ial shal be tes ed in a simiar wa y as the car ie mate ial

(i.e folow s eps B.1 through B.4 using theprimary packag ing mate ials as the tes samples)

B.5.2 T e tes shal be car ied out using samples of primary p ckag ing mate ial eq iv lent to tw ic the

ar ea normaly in contact with the inoculated car ie , or for self-contained biolog ical in icators, eq iv lent

to the ar ea normaly in contact w ith the r eco e y medium T e tes samples shal be imme sed in the

g r owth medium

A nnex C

(normative)

C.1 Principle

This method det ermines the n mbe of surviving t es org nisms b dir ct cou ting of colony forming

unit (C Us) This method is also r fe r d t o as the “dir ct en me ation method” S e also An ex A

NOTE T e method has a practical lower limit of ap ro imat ely 5 × 1

1

C Us

C.2 Materials

C.2.1 Tes samples r epresenting sp re suspensions, inoculated car ie s or p ckag ed biolog ical

in icators shal beinclu ed in the mate ials A minimum of 2 tes samples ar e r eq ired for this method

NOTE Test methods are given in relevant parts of ISO 1 1 8 Specif ications for resist ometer are given in

ISO 1 472

C.2.2 T e incub tor shal be set to pr ovide, an monitored to confirm, the temperatur e spe ified in the

cultur e con itions

C.2.3 T e g r owth medium as spe ified in the cultur e con itions shal be included in the mate ials

C.3.1 Tes samples shal be subje ted to defined ex osur e con itions The rang e of ex posur es shal be

stated S e Ta le 2

C.3.2 A minimum of five ex posur es shal be used an shal inclu e

a) one ex osur in w hich the sample is not subje t ed t o thest eriant (e.g 0 time ex osur ),

NOT T e st erilant can b a sent or replaced by an inert g s or medium

b) at leas one ex osur in w hich the via le p pulation is r d c d t o 0,0 % of the original inoculum

(4 lo

1

r d ction), an

c) a minim um of thr e ex osur s co e ing the int erv ls betwe n ex osur a)and ex osur b)a o e

C.3.3 Not les than four tes samples shal be used for each ex osur e in each dete mination The same

numbe of replcates shal beused for each ex osur e

C.3.4 If the s e i zing ag ent lea ves a r esid e in or on the tes samples, this shal be neutr alz d as

rapidly as p s ible so as not to inte fere with the tes r esult If a neutral zation pr oc d r e is req ired, it

shal be v ldated

C.3.5 Within 2 h of each ex posur e, the tes samples shal be tr eated to r emo e the tes or ganisms from

C.3.6 T e suspensions shal be adjus ed in a pr opriate s e ie diution fluid For cultur es p ur ed into

molten agar or spread on soldified agar in r eg ular siz Petri dishes, n mbe s of colony forming u it

betwe n 30 and 30 ar e considered to be statis icaly v ld

C.3.7 Using al the data o tained, plot the lo

1

of the surviving p pulation agains ex posur e time in

min tes, dose level or othe proc s v ria le an dete mine the best-fit r ecti near curve b r eg r es ion

analysis using the method of leas sq ares Surviv r data p ints within 0,5 logarithms of the initial

p pulation shal not be inclu ed in the reg r es ion analysis Calculatethe negative r ecipr ocal of the slo e

of the lne o tained, which is eq al to the D v lue in min tes, dose level or othe pr oc s v ria le at the

s ated ex posur e con itions

a) The slo e of the bes -f it r cti near curve is calculat ed using F ormula (C.1):

(C.1)

w he e

m is theslo e of the bes -f it r cti inear curve;

n is then mbe of data p int ;

An ex mple of r gr s ion analy sis is given in Ta le C.1

Table C.1 — Examples of re res io analy sis

y

1

1y

1

(t2

)= 0 t (lo

1y

1

1y

1)2

y

2

1y

2

(t2

1y

2

1y

2)2

y

3

1y

3

(t2

1y

3

1y

3)2

y

4

1y

4

(t2

1y

4

1y

4)2

y

5

1y

5

(t2

1y

5

1y

5)2

15

=

=

∑log

1

15

i

ii

i

ii

=

()

=

=

∑2

15

ii

1

15

b

An critic l pro es parameter, e.g time ()