TECHNICAL BULLETIN

TEST OF STERILITY IN ETHYLENE OXIDE VALIDATION

Background

While Tests of Sterility are integral part of he validation of radiation Sterilization Processes (Methods 1 & 2
and VDmax), ISO11135-1:2007 clearly stipulates that the validation of Ethylene Oxide (EO) sterilization
processes utilise biological indicators (BIs) to confirm the validated process delivers Sterility Assurance Level
-6
(SAL) of a minimum of 10 . The BI used for EO sterilization processes is required to have a minimum
6
population of 10 and a D-value of a minimum of 2.5 minutes. The indicator organism, , is a
B a c i l l u s a t r o p h e u s

gram positive, spore forming rod which has been demonstrated to be resistant to EO and this resistance is
standardised. As the natural bioburden of medical devices presented for sterilisation is generally low in
numbers and of organisms present are generally not resistant to EO, there is limited benefit in incorporating
Tests of Sterility in the validation process. It should be noted at this point that there is no specific requirement
within ISO11135-1:2007 to carry out Tests of Sterility as part of the validation of an EO sterilization process
and as such their use should be carefully considered before incorporating into any such validation process.

This technical bulletin is intended to outline when Tests of Sterility may be of value as part of the validation of
an EO sterilization process and when is not appropriate to use them.

When Can a Test of Sterility be of Value in the Validation of an EO Process ?

Before incorporating a Test of Sterility into a validation process, it is important to clearly understand what we
mean by a Test of Sterility as opposed to a Test for Sterility. The fundamental difference between the two
terms relates to what the results of the tests are used for, with the former being used in a validation process
and the latter as part of the release criteria for a fully validated process. Definitions from ISO14937:2008 are
outlined below.
3 . 3 4 T e s t o f S t e r i l i t y 3 . 3 3 T e s t f o r S t e r i l i t y

T e c h n i c a l O p e r a t i o n p e r f o r m e d a s p a r t o f T e c h n i c a l o p e r a t i o n , d e f i n e d i n P h a r m a c o p o e i a ,

d e v e l o p m e n t , v a l i d a t i o n o r r e q u a l i f i c a t i o n t o p e r f o r m e d o n p r o d u c t f o l l o w i n g e x p o s u r e t o a

d e t e r m i n e t h e p r e s e n c e o r a b s e n c e o f v i a b l e s t e r i l i s a t i o n p r o c e s s o r f o l l o w i n g a n a s e p t i c

m i c r o o r g a n i s m s o n p r o d u c t o r p o r t i o n s t h e r e o f . m a n u f a c t u r i n g p r o c e s s .

When validating an EO sterilization process, Tests of Sterility may be of some value in establishing
representative data to meet the requirements of section 8.5 b) of ISO11135-1:2007, which states:
B i o l o g i c a l i n d i c a t o r s u s e d a s p a r t o f t h e e s t a b l i s h m e n t o f t h e s t e r i l i z a t i o n p r o c e s s s h a l l :

a ) c o m p l y w i t h C l a u s e s 5 a n d 9 . 5 o f I S O 1 1 1 3 8 - 2 : 2 0 0 6 ;

b ) b b b b u u c b

e s h o w n t o e a t l e a s t a s r e s i s t a n t t o e t h y l e n e o x i d e a s i s t h e i o r d e n o f p r o d t t o e

s t e r i l i z e d ;

c ) b e p l a c e d w i t h i n t h e p r o d u c t a t l o c a t i o n ( s ) w h e r e s t e r i l i z i n g c o n d i t i o n s a r e m o s t d i f f i c u l t t o

a c h i e v e o r b e p l a c e d w i t h i n a P C D .

I f a P C D i s u s e d f o r p r o c e s s d e f i n i t i o n , v a l i d a t i o n o r r o u t i n e m o n i t o r i n g a n d c o n t r o l , t h e

a p p r o p r i a t e n e s s o f t h e P C D s h a l l b e d e t e r m i n e d . T h e P C D s h a l l b e e q u i v a l e n t o r m o r e c h a l l e n g i n g t o

t h e p r o c e s s t h a n t h e m o s t d i f f i c u l t - t o - s t e r i l i z e p a r t o f t h e p r o d u c t .

N O T E F
o r i n f o r m a t i o n o n t h e s e l e c t i o n , u s e a n d i n t e r p r e t a t i o n o f b i o l o g i c a l i n d i c a t o r s , s e e I S O 1 4 1 6 1 .

In order to make any decision relating to the relationship between the relative resistances of the BI to the
natural bioburden, there is a need to be able to show differential growth between the two. As such the most
appropriate process to obtain such data is on a fractional cycle where a relationship between survival of the
two can be assessed.

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 TECHNICAL BULLETIN
TEST OF STERILITY IN ETHYLENE OXIDE VALIDATION

Tests of Sterility yielding less positive growth than BIs placed on the same fractional cycle gives evidence that
the BIs have a higher resistance to EO than the natural bioburden.

If the results show either all growth or all no growth, then a relationship cannot be made. For this reason, the
only cycles within a validation that Tests of Sterility should be carried out are the Sub-Lethal/Fractional cycles.
There is no benefit of carrying out Tests of Sterility on either Half Cycles or Full Cycles and should NEVER be
carried out on Routine Cycles.

Outlined below are four scenarios which may be encountered along with whether it may or may not be of
value to incorporate Tests of Sterility into the validation process.

Scenario 1 Simple product with clear gas pathway. No real benefit as empirical evidence exists
Natural bioburden < 1,000 and well controlled, relating to the relative resistances of the BI
+
Mainly Gram cocci and no spore formers versus the natural bioburden

Scenario 2 Simple Product May be of limited value in providing

Natural Bioburden > 1,000 and not well controlled evidence that the natural bioburden is less
Wide range of organisms, including spore formers. resistant than the BI.

Scenario 3 Complex product whose design may May be of value to confirm that the EO is
Have narrow bore lumen, penetrating and delivering lethality to all
Have tight interference fit components areas of the product.
Due to its design be difficult to place a BI in the
most difficult location to sterilize.
Incorporate a coating that may act to protect
viable microorganisms.

Scenario 4 Device containing cotton of Chinese origin and without a Specific test for Pyronema will be required to
claim of Pyronema* free. demonstrate that there is no presence of this
organism.

* , a fungus found on cotton products of Chinese origin and has been demonstrated to
P y r o n e m a d o m e s t i c u m

be highly resistant to EO.

The vast majority of medical devices will fall into Scenario 1, so as such Tests of Sterility should be the
exception rather than the rule.

Conclusion

When establishing the validation of an EO sterilization process, Tests of Sterility have only limited
value in the majority of cases.
Personnel responsible for validation of EO processes need to use sound judgement based on their
knowledge of the product and its microbiological quality before making any decision to use Tests of
Sterility in the validation of their product.
Medical Device Manufacturers need to ensure that they have data pertaining to the product
bioburden demonstrating low and consistent bioburden with adequate characterization of the
natural bioburden.
For products containing cotton of Chinese origin, specific controls need to be put in place to screen
for .
P y r o n e m a d o m e s t i c u m

Tests of Sterility in EO Sterilization validation processes should be the exception rather then the rule.
Results from Tests of Sterility / Tests for Sterility should never be used as part of the release criteria
for product sterilized using EO.

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