UNIVERSITI PERUBATAN ANTARABANGSA

INTERNATIONAL MEDICAL UNIVERSITY

MALAYSIA

General Microbiology
(BCH2232)

Project

Name / Student ID: Alvinder Kaur (PC090728605)

Fabian Mok (PC090726528)
Ngai Kok Leong (PC090727319)
Lee Jia Yin (PC090727720)
Hii Khang Jiet (PC080725452)

Experiment 2: Validation of Sterilization Process

(Gas and Chemical Sterilization)

Date of Project: 20th August 2010

Date of Submission: 3rd October 2010.

 Sterilization is a term referring to any process that eliminates or kills all forms of life,
including transmissible agents such as fungi, bacteria, viruses, spore forms that is present on a
surface, contained in a fluid, in medication, or in a compound such as biological culture media..
Sterility is the complete absence of viable microorganism from a product. The product
achieved is called sterile product and the method employed to attain sterility is termed
sterilization. Sterilization is an essential stage in the processing of any product destined for
parental administration, or for contact with broken skin, mucosal surface or internal organ, where
the treat of infection exists. Sterilization of microbiological material, soiled dressing and other
contaminated items is necessary to minimize the health hazard associated with these articles. For
sterilization process to be effective, Validation on the sterilization methods must be conducted.
Validation of sterilization methods has proven to be vital and most useful in insuring safety of
the product outcome. Validation of sterilization method is a very reliable way to secure the
effectiveness of pharmaceutical products. In the case of sterilization processes, routine
bioburden monitoring, preventive maintenance, calibration, cycle review and approval and
annual reviews are activities that make up validation of sterilization methods.

Chemical sterilization and Gas sterilization are the few method of sterilization of
pharmaceutical product that is recognizes by British Pharmacopoeia (2010).Chemical
sterilization also known cold sterilization is an alternative to high-pressure steam or dry-heat
sterilization. Chemical sterilization is used to the heat sensitive materials such as biological
materials, fiber optics, electronics, and many plastics which would be damage if high-pressure
steam of dry-heat steam sterilization is apply. Low temperature Chemical sterilizers function by
exposing the articles to be sterilized to high concentrations (typically 5 - 10% v/v) of very
reactive chemical and oxidizing agents such as hydrogen peroxide and ozone. Liquid sterility and
high disinfectants typically include oxidizing agents such as Hydrogen Peroxide, Peracetic Acid
and Aldehydes such as Glutaraldehyde and more recently o-phthalaldehyde. Glutaraldehyde
and formaldehyde are the most common disinfectants for chemical sterilization used in practical
sterility for some instruments, such as laparoscopes which cannot be heated. The formaldehyde
is widely use in pathology and surgery department. The mixture of steam and formaldehyde at
temperature of 80, 65, 60, 55 or 50°C will penetrate and kill any microorganisms.
 Both glutaraldehydes and formaldehyde require special handling and leave a residue
on treated instruments; therefore, rinsing with sterile water is essential if the item must be kept
sterile.The advantages of glutaraldehydes and formaldehyde solutions are not readily
inactivated by organic materials and used for item that will not tolerate heat sterilization such as
laparoscopes and some heat sensitive materials. The disadvantages for both solutions can cause
skin irritation. The vapors from formaldehyde (classified a potential carcinogen), and to lesser
degree glutaraldehydes are irritating to skin, eyes and respiratory tract.

In Gas sterilization, chemically reactive gases like ethylene oxide and formaldehyde
possess broad spectrum biocidal activity in many industries .These include the sterilization of
re-usable surgical instruments, medical, diagnostic and electrical equipment as well as the
surface sterilization of powders. On a regular basis, the usage of ethylene oxide is far more
common than the usage of formaldehyde. Ethylene oxide treatment is also considered a good
alternative to radiation sterilization in the production of medical devices. However, these two
techniques differ in the degree of sterility assurance as heat methods so they are usually
reserved for heat sensitive items. The mechanism of antimicrobial action of ethylene oxide and
formaldehyde is assumed to be through the alkylation of sulphudryl, amino, hydroxyl and
carbonyl groups on proteins and amino groups of nucleic acids. Concentration ranges (given as
weight of gas per unit chamber volume) are usually in order of 800-1200mg-1 for ethylene oxide
with operating temperatures of 45oC-63oC while 5-100mg-1 for formaldehyde with operating
temperatures of 70oC- 75oC . Organisms are more resistant to ethylene oxide in a dried state,
as are those protected from the gas by inclusion in crystalline or dried organic deposits, therefore
a minimum level of 30%-70% humidity in the immediate product environment is wanted. The
disadvantages of Ethylene oxide and formaldehyde gases is, it’s potentially mutagenic and
carcinogenic level and acute toxicity including skin irritation, conjunctiva and nasal mucosa. A
major disadvantage of formaldehyde is low penetrating power and this limits the packaging
materials that can be employed to principally paper and cotton fabric.
 The basic definition of a validation is to formulate a documented proved that a method
being used is able to give a reliable results and the consistency is being achieved.
Consequently, is it crucial to conceive and elucidate the methods to achieve a certain results
which are being concerned. For a new method that is being validated, it consists of two levels.
The primary validation is done on a summary of new alternative methods which suppose to be
validated. This is to validate the suitability and applicability of the new method. This is done
before the validation of the actual method.

A validation test is done in to an alternative route to prove that it is acceptable. This

validation is then matching towards the pharmacopoeia methods. Both methods should give
an equivalence results and this conclude that the alternative route is acceptable. Validation on
the qualitative test for the presence or absence of microorganism can be done to prove the
efficacy of the sterilization methods .Result from validation can be used to judge the quality,
reliability and consistency of the sterilization method.

There are few fundamental parameter of validation that requires being access to prove
the reliability of the sterilization method. Accuracy describes the closeness of mean test result
obtain from the sterilization method. Accuracy of the sterilization method can be determine by
running the alternative method analysis and then is compare with the pharmacopoeia method.
Both the method should give a degree of equivalence. Mean value of the accuracy should be
within 15% of its actual value. Precision of the sterilization method can be judge by repeating
the method under same condition, the value obtained should fall into a agreed range. This is
governed by the variance, standard deviation and coefficient of variation. A sterilization method
is done to kill microorganism to produce sterile product. Sterilization cannot be governed by
complete absence of microorganism; therefore Limit of Detection (LOD) parameter is
important to produce the lowest number of micro-organisms that the methods are able to detect
in a given sample. The robustness of the sterilization technique should remain unaffected when
being subjected towards an operational and environmental variable. This indicates the reliability
of the sterilization methods. Other parameter under observation is the linearity where the results
obtained from the alternative methods should be proportional towards the concentration of
micro-organisms from the sample being used.
 The Process of Validation in Chemical Sterilization and Gaseous Sterilization for
Pharmaceuticals products. Chemical and Gaseous sterilization is mainly used with the aim of
discharging the preliminary treatment of bioburden which further accommodating an adequate
safety margin. This is done by exposing the components of concern towards a chemical sterility
which present in gaseous phase for gaseous sterilization and in liquid phase for chemical
sterilization. Validation of the Chemical and Gaseous Sterilization are conducted to confirm
Chemical and Gas Sterilization process is evaluated and under control. By using a sequence
of reliable Biological, Chemical and Mechanical Indicators at regular intervals, the
sterilization procedures are able to be monitored.

Biological indicator is common being used for medical devices, isolators, chambers, etc.
A standardized preparation of chosen micro-organism is used to assay the efficiency of a
sterilization process that is conducted. The micro-organism being used employs a population of
spores that is placed on an inert carrier. The carrier used should be shield by any chance of
contamination or degradation at the same time allowing the contact between the sterilizing agent
and the micro-organism. Sterilization of Glutaraldehyde and Formaldehyde as Chemical
Sterilizer is being validated with the spores of Bacillus licheniformis. This microorganism is
chosen based on its defined and stable resistance towards Glutaraldehyde and formaldehyde.
Hence, they are selected to test the sterilization procedure of Chemical sterilization. The micro-
organism used as the indicator for the gaseous sterilization is added directly towards the gas
being sterilized. Besides that, it could also be administered into the gas which mimics the one
which is in concern to be sterilized. However, it is crucial that the gas product produce no
inhibiting effect towards the micro-organism used. Sterilization of Ethylene Oxide a Gas
sterilizer is being validated with the spores of Bacillus subtilis. This micro-organism is chosen
based on its defined and stable resistance towards ethylene oxide. Hence, they are selected to test
the sterilization procedures.

This biological indicator is packed in a way that the sterilizing agent is readily penetrated into it.
The resistance of microorganism is determine by the Minimum Inhibitory
Concentration(MIC) ,Minimum Bactericidal Concentration(MBC) and the extinction time of the
microorganism.
 Other method of Validation of Chemical and Gas sterilization include the use Chemical
indicator. This includes heat sensitive tape, labels or glass vials containing pellets. Chemical
monitors that indicate if a glutaraldehyde ,formaldehyde in solution in chemical sterilization and
ethylene oxide solution in Gas sterilization meets the manufacturer's recommendation for a
minimum effective concentration (MEC) needed to effectively kill organisms. Chemical
indicator are used for equipment control as a way to find out whether or not the solution is doing
its job or is at the minimum concentration (MEC) needed to effectively kill organisms. Solution
should be tested prior to immersing into medical devices into the solution to ensure that the
solution is at its Minimum concentration (MEC).

Other method of validation of Chemical and Gas sterilization include Mechanical

indicator. This validation is crucial to show something has gone inappropriate. It is carryout
outside the sterilizer such as observing gauges, computer printouts that evaluate the sterilizer’s
function by providing an apparent record of the time, temperature and pressure for that specific
sterilization procedure. Mechanical monitoring may be the first indicator to show that something
has gone awry.

Sterilization processes must be revalidated at least annually in the absence of any change-
driven revalidation. The revalidation can be a subset of the original validation work. Some
acceptable approaches include the use of single runs rather than 3 consecutive runs are sufficient
in the absence of recurring problems, the selection of a worst case load pattern for revalidation ,
revalidation of each type of cycle ,the revalidation of the worst case loading pattern .

The entire sterile item should be stored in an area and manner whereby the packs or
containers will be protected from dust, dirt, moisture, microorganism, animal and insects.
This storage area is best located next to or connected to where sterilization occurs, in a separate
enclosed area with limited excess that is used to store sterile and clean patient care supplies.

A sterilization process should always be considered to compromise between achieving

good antimicrobial activity and maintaining product stability. It therefore must be validated
against a suitable test organism and its efficacy is continuously monitored during use.
Sterilization control and sterilization assurances are comprise an attempt to achieve as far as
possible the continuous monitoring of a particular sterilization process. The sterility test on its
own provide no guarantee, however it is an additional check and continued compliance with the
test does give confidence as to the efficacy of a sterilization or aseptic process.

Reference :
http://findarticles.com/p/articles/mi_m0BPC/is_1_29/ai_n8708450/
http://www.pharmacopeia.cn/v29240/usp29nf24s0_c1035.html#usp29nf24s0_c1035
http://www.pharmacopeia.cn/v29240/usp29nf24s0_c1211.html#usp29nf24s0_c1211
www.engenderhealth.org/ip/instrum/inm12.html
www.microtestlabs.com/.../steam-eo-chemical-sterilization.html