This document provides an overview of the theoretical approach to autoclave validation. It discusses key concepts like the D-value, which is the time required to reduce a microbial population by 90% at a given temperature. The Z-value describes how the D-value changes with temperature. The F0-value represents equivalent sterilization time accounting for varying temperatures. Probability of a non-sterile unit (PNSU), also called sterility assurance level (SAL), can be calculated from initial and final microbial populations and the F0-value. Understanding these parameters is important for validating sterilization processes and ensuring a high probability of sterility.

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2. Theoretical approach of autoclave
validation
Module-3
Prepared by: Surafel Kebede (Bpharm,MBA)
safokebede@gmail.com
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3. Table of contents
1. Objectives
2. Introduction
3. Mechanism of microbial death
3.1. D-value (decimal reduction time)
3.2. Z-value (thermal resistance constant)
3.3. Thermal death time (TDT), (F value)
3.4. Lethal Rate
3.5. Mathematical F0 value
3.6. Probability of Non sterile unit (PNSU)
3.7. Determination of minimum required Fo
4. Biological indicators (BI)
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4. 1. Objectives
After this training trainees are able to understand:
 Mechanism of microbial death.
 The concepts of D,Z and F values and their relationship.
 How D-value, Z-value and F-value be determined.
 The difference between clock time and thermal death time/F
value.
 How minimum Fo value and lethal rate can be determined.
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5. 2.Introduction
 Sterile pharmaceutical products should be:
Free from microorganisms
Free from pyrogens/endotoxin
Free from particulates, and chemicals
 The ultimate goal is absolute absence of microbial contamination
(since sterility specification is an absolute value)
 But sterility can not be assured by end product testing (sterility
testing).
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6. Limitation of Sterility Test
It is destructive test.
Depends on sample size.
Low concentration of microbial contamination may not be
detected.
False positive result (contamination of culture media by
personnel).
•With the sterility test method to ensure absolute sterility, all
samples would have to be tested.
•And hence validation is important to minimize the reliance on
end-product testing.
•An alternative approach to predicting sterilization is the
definition of sterility as a probability of survival. This
probability is related to knowledge of the mechanism of
microbial death and the condition causing it.
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7.  Based on this approach Sterilization can be defined as a process
used to render a product free of viable organisms with specified
probability.
The most prevalent description of sterility used today is the
reduction of anticipated levels of contamination in a load to the
point at which the probability of survival is less than
1/1,000,000 (1 in 1 million).
Once the levels of microbial contamination and resistance to the
sterilization process are known, probability of survival can be
calculated.
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8. Significant research data support the theory that microbial death
may be described as a first order chemical reaction. This leads to
the conclusion that death is essentially a single molecule
reaction.
First order reaction: is a chemical reaction in which the rate
depends on the concentration of one reactant. The rate of
reaction is governed by the concentration of the reactant (spore).
It is a chemical reaction in which the reaction rate is
proportional at all times only to the amount of reactant still to be
degraded.
3. Mechanism of microbial death
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9. From chemical kinetics, if the disappearance of a
species/spores, A follows first order kinetics, then the rate
equation is expressed as;
d[A]/dt=-k[A].........Rate of concentration decrease of spores.
Where; A-reactant(spore/bacteria), B-product/dead spore, k-
rate constant
Mechanism...
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10.  If we use N for number of microbial survivors in a suspension
subjected to heating,
DN/dt=-KN
DN/N=-dKt
After integrating the equation we can get,
lnN/N0=kt where, N- final microbial concentration, N0- initial
microbial concentration, k-rate constant and t-time.
If we plot the number of spore survivors against time, we can
obtain exponentially decreasing graph as shown below.
Mechanism ...
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Survivor curve

11.  The concept of first order chemical reaction seems obvious for dry
heat sterilization, but not for moist heat sterilization, in which steam
or superheated water would appear to take part in the reaction. A
actually, this bimolecular reaction is a first-order reaction, because an
excess of steam or superheated water is always present and its
concentration can be considered constant.
 Regardless of the type of lethality induced by a sterilization process
whether it be heat, chemical or radiation microorganisms upon
exposure to adequate level of such treatment, will die according to a
logarithmic relationship between the concentration or population of
spores and the time exposure or radiation dose to the treatment.
Mechanism ...
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12.  If a homogeneous suspension of microorganisms is heated at constant
temperature, the microorganism destruction commonly follows
logarithmic order of death.
Mechanism ...
12
logarithmic /exponential function with negative slope (eg)

13.  It is possible to use semi logarithm plot.
 Semi logarithm plot: having one scale logarithm and other arithmetic
plot.
 By converting number of spores to logarithmic value and plotting it
against exposure time we can get straight line graph.
Mechanism ...
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14.  Heat sterilization is a function of probability that is dependent on;
i. The number of challenge microorganisms.
ii. The heat resistance of these microorganisms.
iii. The amount of heat exposure.
 When microbes/spores are subjected to heating or radiation , they
will show some resistance to the sterilant. Such resistance can be
expressed as D-value (decimal reduction time).
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Mechanism ...

15. 3.1. D-value (decimal reduction time)
 D-value is used to describe the relative resistance of particular
microorganism to a sterilization process.
 It is heating time in minutes at constant temperature that will result in
reducing microorganisms by a factor of 10.
 It is time in minutes required to inactivate 1 log of a challenge
microorganism.
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Mechanism ...

16.  It is the time in minutes required to reduce 1,000 spores to 100.
 It is the time required for a 90% reduction in the microbial population.
i.e. No=1,000 spores, N= 100 spores, % change=(1000-
100)(100%)/1000 =90%
NB. -D-value remains the same for each log cycle.
-D-value does not depend upon the initial number of
microorganisms present.
- It is expressed in time unit (in minutes).
D-value...
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log N= -k(t)/2.303+ log No
Let No=1000 and N=100, then
Log 100=-kt/2.303+log 1000
Log 100/1000=-kt/2.303
Log 0.1=-kt/2.303
-1=-kt/2.303
t=2.303/k, but t=D
D=2.303/k
D-value...

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 The D value is important in the validation of sterilization
processes for several reasons.
1. It is a specific kinetic expression for each micro-organism in a
specific environment subjected to a specific sterilization agent
or condition.
 In other words, the D value will be affected by;
a. The type of microorganism used as the biological indicator.
b. The formulation components and characteristics (e.g., pH).
c. The surface on which the micro-organism is exposed (glass,
steel, plastic, rubber, in solution, dry powder, etc.).
D-value...

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d. The temperature, gas concentration, or radiation dose of the
particular sterilization process.
3. Knowledge of the D value at different temperatures in heat
sterilization is necessary for the calculation of the Z value.
4. The D value is used in the calculation of the biological F value.
D-value...

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How to determine D-value
There are different methods of D-value determination but it can
be easily determined by using Semilog paper and using spread
sheet.
The spreadsheet method uses the concept of simple regression
analysis.
Eg. Determine D-value of microorganism from the following
data at 120˚C. (ans=3.025 min).
TIME •
•
•
•
•
•
(minutes) NUMBER OF SURVIVORS
0 106
3 1.2X105
6 1.1X104
9 1.2X103
12 1.1X102
D-value...

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How to relate D values at different temperatures
 D values at different temperatures can be related to each other.
For example the D value at temperature T can be related to
121.1˚C.
D-value...

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Eg. If the D value of a certain spore is 5 minutes at 121.1˚C,
what will be the D value of the spore at 111.1˚C (Z=10˚C).
Ans=50 min.
NB. D value increases when temperature decreases.
D-value...

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3.2. Z-value (thermal resistance constant)
 Z-value describes the influence of temperature on decimal
reduction time, D for microbial population.
 It is the increase in temperature necessary to cause a 90%
reduction in D value (1 log reduction).
 Recall that D value is obtained at a constant temperature and if
we plot log D value against temperature on a semilog paper then
the temperature change for one log cycle reduction will give us
Z value.
Thermal resistance plots of log D versus temperature, showing slopes equivalent
to Z = 10°C and Z = 20°C

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Z-value…
 The most commonly used value of z for the destruction of
microbial spores is 10˚C (18˚F). This is based on experimental
observations for Geobacillus stearothermophilus and Clostridium
botulinum, both highly heat resistant organisms. These
organisms are chosen for divergent reasons. C. botulinum was
the subject of the pioneering experiments by food scientists
attempting to destroy this deadly cause of botulism in canned
foods. G. stearothermophilus is a readily available and safe
indicator organism for use in sterilization studies and has similar
resistance.

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Z-value….
Eg. Determine Z value for spore suspension if the following D
values were obtained for different temperature. (ans=16.58˚C)
Temperature (°C) D value(minutes)
102 28.5
106 15.6
110 8
114 5.1
118 3.1

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3.3. Thermal death time (TDT), (F value)
 The usefulness of the temperature dependent model in moist
heat sterilization autoclave is to calculate the lethality of the
cycle over a range of temperature (including heat up and cool
down). To do this a new variable ,closely related to D value is
introduced. This is called F, value (Thermal death time).
 It can also called process lethality.
 F value is defined as the number of minutes required to
destroy a given number of organisms at a given temperature.
 But D value is the number of minutes required to destroy 90%
of organisms at a given temperature.

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Thermal death time…
Microbial death (survivor) curve

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Thermal death…
 Reduce microbial population from 10,000
to 1,000, F=1D
 Reduce microbial population from 10,000
to 100, F=2D
 Reduce microbial population from 10,000
to 10, F=3D
 So F is nothing but it is a multiple of D
value.
 And hence we can say that F=nD, where n
is log reduction.
 Both the thermal resistance curve (log D
vsT) and the thermal death time curve (log
F vs T) are dependent on z, have the same
curve.

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Thermal death…
(logF1-logF2)/ T1-T2= 1/-z
logF1-logF2= (T2-T1)/z
10(T2-T1)/z =F1/F2 , let F1 be FT and F2 be F 121.1˚c
(F121.1 ) 10
(121.1-T 1)/z=FT

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3.4. Lethal Rate
Lethal rate: is defined as the equivalent time for any specific
temperature relative to another temperature (usually 121.1˚C).
 This reference temperature (121.1˚C) is chosen as a base
because it is an economical and effective one for moist heat
sterilization. But it should not be assumed that 121.1˚C is
required to achieve effective sterilization.

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Lethal Rate…
 Example1: the lethal rate for 117.0˚C relative to 121.1˚C
(assuming a z value of 10) is 0.4. This means that for every full
minute (60 seconds) of process time at a temperature of 117˚C,
the process is “credited with” the equivalent of only 0.4 minute
at 121.1˚C.
 Example 2: for a process that ran for 12minuets at exactly
119.1˚C, what will be the equivalent time with respect to 121.1
˚C? (7.56min)

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Lethal Rate…
10
(121.1-T )/z=FT/F121.1˚c ), where F121.1˚c /FT)=L and FT =1 minute
And then; 10
(T -121.1)/z=L, z =10˚c
 The logic behind is that for every 1 minute exposure of a spore at
a certain temperature has given an equivalent exposure time
(“credit”) of less or greater that one minutes at 121.1ºC.
 Temperatures below 100˚C generally add insignificant credit to
the overall sterilization assessment.(L = 0.008 min).
Heat penetration curve

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3.5. Mathematical F0 value
 F value measures equivalent time, not clock time, that a monitored
article is exposed to the desired temperature.
 F0 is a summation over time of the instantaneous lethal rates at a
series of temperatures. In integral form this is;
where ∆t is the chosen time interval and T is the average temperature
over that interval. The smaller the interval chosen, the more accurate
the calculation will be.

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Mathematical F0 …
 An F-value is the number of minutes to kill a specified number of
microorganisms with a specified Z-value at a specific temperature.
An Fo value is the number of minutes to kill a specified number of
microorganisms with a Z-value of 10°C (50°F) at a temperature of
121.1°C (250°F).
 When the assumption of z=10˚c is used F is written as Fo. This is
the most commonly used measure of the lethality of a sterilization
process spanning a range of temperatures.
 It is a common practice to use the F0 equation to determine the
probability of sterility or SAL.
 Eg. What is the F0 value for a process that ran for 12 minutes at
exactly 121.1˚C (ans. F0=12min).

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Mathematical F0 …
Equivalent sterilization time

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Mathematical F0 …
 Another equation for the F value as depicted below given in the
following expression:
 Where L= 10(T−T0)/Z which is the lethality constant integrated
over time limits between time 1 and time 2. Integrating the above
question between two time points will yield the area under the
10(T−T0)/Z versus time curve.

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Mathematical F0…
Plot showing the difference between chamber temperature versus time
(___ ) and lethal rate in the product versus time (----). F is the area under the dotted line curve

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A Manual Calculation of F0 Value
Mathematical F0…

39. 3.6. Probability of Non sterile unit (PNSU)
 It is also known as sterility assurance level (SAL)
 From the above graph we can derive the following equation,
Log No/N=F/D
Where, No is initial population
N is final population
F is total destruction time
D decimal reduction time
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40. SAL…
 Note that in microbial challenge test, a certain number of
spores (usually 106) is inoculated in a single container, however
in actual scenario the microorganisms/spores might be
distributed and found in all container (eg bags).
 Lets say r= number of bags sterilized, No=number of spores per
bag , rNo= total number of spores at the beginning and rN=
total number of spores at the end of sterilization process
respectively.
 Substituting in the above equation;
Log rNo/rN=F/D
 If we want only one bag at the end of heating to contain a
spore, then rN=1, the equation become Log rNo=F/D
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41. SAL…
 After rearranging we can get;
rNo=10F/D or 1/r= No and this is the equation of PNSU(SAL)
10F/D
 Eg. No =104 and if we use 12D, 1/r= No = 104/ 1012 = 10-8
1012D/D
 Therefore incidence of survival is 1 bag in 108 bags
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42. 3.7. Determination of minimum required Fo
 Quite often the process designer will need to know precisely how
much Fo to provide for in a new sterilization cycle to meet a
desired sterility assurance required, together with the bioburden
of the product being sterilized and the resistance of indigenous
microorganism in the bioburden using formula.
Fo= D121.1˚c (log No-log N)
 Where, No-initial bioburden, N- maximum acceptable SAL.
 Eg. The product being sterilized has a bioburden of 100 spores
per container, the D value of the spore is 3.3 minutes and the
desired SAL is no more than 1 unit in 1 million units will be non-
sterile.(ans ,26.4 min.).

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43. 4. Biological indicators (BI)
 A Biological Indicator: is a characterized preparation of specific
microorganisms resistant to a particular sterilization process. It is
used to assist in the qualification of the physical operation of
sterilization
There are three forms of BI’s:
SPORE STRIPS: Paper strips inoculated with spores and
placed inside a glassine envelope.
AMPOULE: glass vial filled with spore suspension and
chemical indicator.
SUSPENSION: solution of spores suspended in Ethanol or
Water used for direct surface inoculation.
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44. Biological …
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SPORE STRIPS AMPOULE SUSPENSION
Reading assignment :
SAT,FAT ,IQ,OQ,PQ of autoclave machine.

45. Thank You
Ver y Much
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