**3.2.1 Decimal reduction dose**

When a suspension of a microorganism is irradiated at incremental doses, the number of surviving cell forming colonies after each incremental dose may be used to construct a dose survival curve, as shown in Figure 5. The radiation resistance of a microorganism is measured by the so-called decimal reduction dose (D10 value), which is defined as the radiation dose (kGy) required to reduce the number of that microorganism by 10-fold (one log cycle) or required to kill 90% of the total number (Whitby & Gelda, 1979). The D10 value

Fig. 5. Typical survival curve for a homogeneous microbial population.

Sterilization by Gamma Irradiation 179

In addition, it has been suggested that some pigments synthesized by microorganisms may play a role in their resistance towards ionizing radiation. For example, carotenoids synthesized by *Exiguobacterium acedicum* were found to be responsible for its radioresistance (Kim et al., 2007). Fungi that synthesize pigments such as *Curvularia geniculata* (melanin) or other *Dematiaceous fungi* that contain melanin and carotenoids have higher D10 values (Saleh et al., 1988; Geis & Szaniszlo, 1984). These pigments appear to be involved in both photoand radio-protection. It was also discovered that a higher amount of Mn+2 in some radioresistant bacteria may partly explain their resistance due to the decrease of protein

It can be defined as the absorbed energy per unit mass ([J.kg-1] = [Gy]). Survival fraction of the microorganisms is reversely proportional with the absorbed dose. Doses for sterilization should be chosen according to the initial bioburden, sterility assurance level (SAL) and the radiosensitivity of microorganisms. A sterility assurance level (SAL) is derived mathematically and it defines the probability of a viable microorganism being present on an individual product unit after sterilization. SAL is normally expressed as 10−n. SAL is generally set at the level of 10−6 microorganisms/ml or g for the injectable pharmaceuticals, ophtalmic ointment and ophtalmic drops and is 10-3 for some products like gloves that are used in the aseptic conditions. Generally for an effectively (F -value) of n = 8 is employed for sterilization of *Bacillus pumilus* for the standard dose of 25 kGy is equivalent to about eight

The process of determining the sterilization dose is intended to establish the minimum dose necessary to achieve the required or desired sterility assurance level (SAL). Sterilization dose depends on: i. level of viable microorganisms on the product before the sterilization process (natural bioburden); ii. relative mix of various microorganisms with different D10 values; iii. degree of sterility, i.e. sterility assurance level (SAL), required for that product. Because of this reason, the optimum sterilization dose is 25 kGy at the above level of

On the other hand, the response of a microbial cell and hence its resistance to ionizing radiation depends of many factors like: i. nature and amount of direct damage produced within its vital target; ii. number, nature and longevity of radiation induced reactive chemical changes; iii. inherent ability of the cell to tolerate or correctly repair the damage

In general, bioburden on any product is made up of a mixture of various microbial species, each having its own unique D10 value, depending on its resistance to radiation; these various species exist in different proportions. A standard distribution of resistances (D10 values) has been agreed upon for the determination of sterilization dose based on Method 1 of ISO 11137 (1995). Thus, 65.487% of the microorganisms on a product has a D10 value of 1.0 kGy, 22.493% of the microorganisms has a D10 value of 1.5 kGy, etc. This is an average distribution based on significant amounts of data. It is not always that this distribution exists; it would depend on the conditions of manufacturing and subsequent processes. Method 1 of ISO 11137 (1995) is based on confirming that this distribution exists. From the reported survival data resulting from numerous investigations carried out on the effects of

and iv. influence of intra and extracellular environment on any of the above.

ionizing radiation on microorganisms, the following observations may be made:

oxidation in presence of higher concentrations of Mn+2 (Daly et al., 2007).

**3.2.2 Sterilization dose** 

times its D10 (2.2-3 kGy).

bioburden (Takehisa et al, 1998).

can be measured graphically from the survival curve, as shown in Figure 5; the slope of the curve (mostly a straight line) is related to the D10 value. With certain microorganisms, a 'shoulder' may appear in the low dose range before the linear slope starts. This 'shoulder' may be explained by multiple targets and/or certain repair processes being operative at low doses.

The decimal reduction dose is affected by irradiation conditions in which the microorganisms exist in dry or freezing, aerobic or anaerobic conditions. The D10 value of some organisms (responsible for selected water-born diseases) irradiated in buffer solution is presented in Table 1.


Table 1. Decimal reduction dose (D10) of some microorganisms

There are many factors affecting the resistance of microorganisms to ionizing radiation, thus influencing the shape of the survival curve. The most important factors are:


can be measured graphically from the survival curve, as shown in Figure 5; the slope of the curve (mostly a straight line) is related to the D10 value. With certain microorganisms, a 'shoulder' may appear in the low dose range before the linear slope starts. This 'shoulder' may be explained by multiple targets and/or certain repair processes being operative at low

The decimal reduction dose is affected by irradiation conditions in which the microorganisms exist in dry or freezing, aerobic or anaerobic conditions. The D10 value of some organisms (responsible for selected water-born diseases) irradiated in buffer solution

*Slamonella typhimurim* 0.30 Gastroenteritis Borrely, 1998

*tuberculosis* 0.30 Tuberculosis IAEA, 1975 *Shigella dysenteriae* 0.60 Dysentery IAEA, 1975 *Vibrio cholerae* 0.48 Cholera IAEA, 1975

There are many factors affecting the resistance of microorganisms to ionizing radiation, thus

b. Compounds associated with the DNA in the cell, such as basic peptides, nucleoproteins, RNA, lipids, lipoproteins and metal ions. In different species of microorganisms, these

c. Oxygen: The presence of oxygen during the irradiation process increases the lethal effect on microorganisms. Under completely anaerobic conditions, the D10 value of some vegetative bacteria increases by a factor of 2.5–4.7, in comparison with aerobic

d. Water content: Microorganisms are most resistant when irradiated in dry conditions. This is mainly due to the low number or absence of free radicals formed from water molecules by radiation, and thus the level of indirect effect on DNA is low or

e. Temperature: Treatment at elevated temperature, generally in the sub-lethal range above 45°C, synergistically enhances the bactericidal effects of ionizing radiation on vegetative cells. Vegetative microorganisms are considerably more resistant to radiation at subfreezing temperatures than at ambient temperatures. This is attributed to a decrease in water activity at subfreezing temperatures. In the frozen state, moreover,

f. Medium: The composition of the medium surrounding the microorganism plays an important role in the microbiological effects. D10 values for certain microorganisms can

g. Post-irradiation conditions: Microorganisms that survive irradiation treatment will probably be more sensitive to environmental conditions (temperature, pH, nutrients,

**(kGy) Desease Reference** 

doses.

is presented in Table 1.

*Mycobacterium* 

conditions;

absent;

**Microorganism D10**

Table 1. Decimal reduction dose (D10) of some microorganisms

a. Size and structural arrangement of DNA in the microbial cell;

the diffusion of radicals is very much restricted;

differ considerably in different media;

inhibitors, etc.) than the untreated cells.

influencing the shape of the survival curve. The most important factors are:

substances may influence the indirect effects of radiation differently;

In addition, it has been suggested that some pigments synthesized by microorganisms may play a role in their resistance towards ionizing radiation. For example, carotenoids synthesized by *Exiguobacterium acedicum* were found to be responsible for its radioresistance (Kim et al., 2007). Fungi that synthesize pigments such as *Curvularia geniculata* (melanin) or other *Dematiaceous fungi* that contain melanin and carotenoids have higher D10 values (Saleh et al., 1988; Geis & Szaniszlo, 1984). These pigments appear to be involved in both photoand radio-protection. It was also discovered that a higher amount of Mn+2 in some radioresistant bacteria may partly explain their resistance due to the decrease of protein oxidation in presence of higher concentrations of Mn+2 (Daly et al., 2007).

#### **3.2.2 Sterilization dose**

It can be defined as the absorbed energy per unit mass ([J.kg-1] = [Gy]). Survival fraction of the microorganisms is reversely proportional with the absorbed dose. Doses for sterilization should be chosen according to the initial bioburden, sterility assurance level (SAL) and the radiosensitivity of microorganisms. A sterility assurance level (SAL) is derived mathematically and it defines the probability of a viable microorganism being present on an individual product unit after sterilization. SAL is normally expressed as 10−n. SAL is generally set at the level of 10−6 microorganisms/ml or g for the injectable pharmaceuticals, ophtalmic ointment and ophtalmic drops and is 10-3 for some products like gloves that are used in the aseptic conditions. Generally for an effectively (F -value) of n = 8 is employed for sterilization of *Bacillus pumilus* for the standard dose of 25 kGy is equivalent to about eight times its D10 (2.2-3 kGy).

The process of determining the sterilization dose is intended to establish the minimum dose necessary to achieve the required or desired sterility assurance level (SAL). Sterilization dose depends on: i. level of viable microorganisms on the product before the sterilization process (natural bioburden); ii. relative mix of various microorganisms with different D10 values; iii. degree of sterility, i.e. sterility assurance level (SAL), required for that product. Because of this reason, the optimum sterilization dose is 25 kGy at the above level of bioburden (Takehisa et al, 1998).

On the other hand, the response of a microbial cell and hence its resistance to ionizing radiation depends of many factors like: i. nature and amount of direct damage produced within its vital target; ii. number, nature and longevity of radiation induced reactive chemical changes; iii. inherent ability of the cell to tolerate or correctly repair the damage and iv. influence of intra and extracellular environment on any of the above.

In general, bioburden on any product is made up of a mixture of various microbial species, each having its own unique D10 value, depending on its resistance to radiation; these various species exist in different proportions. A standard distribution of resistances (D10 values) has been agreed upon for the determination of sterilization dose based on Method 1 of ISO 11137 (1995). Thus, 65.487% of the microorganisms on a product has a D10 value of 1.0 kGy, 22.493% of the microorganisms has a D10 value of 1.5 kGy, etc. This is an average distribution based on significant amounts of data. It is not always that this distribution exists; it would depend on the conditions of manufacturing and subsequent processes. Method 1 of ISO 11137 (1995) is based on confirming that this distribution exists. From the reported survival data resulting from numerous investigations carried out on the effects of ionizing radiation on microorganisms, the following observations may be made:

Sterilization by Gamma Irradiation 181

bacteria 2.9 0°C, Phosphate

bacteria 3.9 Phosphate

*Aspergillus flavus* fungi 0.60 Aerated water,

*Aspergillus niger* fungi 0.42 Aerated water,

*cladosporioides* fungi 0.03-0.25 Aerated water,

Coxsackievirus B-2 viruses 5.3 Water, -90°C Sullivan

Coxsackievirus B-2 viruses 7.0 Meat, 16°C Sullivan

Coxsackievirus B-2 viruses 8.1 Meat, -90°C Sullivan

demonstrating the feasibility of a reaction between the •OH from ice radiolysis and the solute. A comparison was performed with irradiated frozen solutions of metoprolol, which has been studied in liquid aqueous solutions (Crucq et al, 2000). Degradation of metoprolol

On the other hand, the evaluation of the radiosensitivity of bacteria as a function of the addition of radical scavengers is quite difficult since many experiments have been carried out either on isolated DNA, which does not take into account the effects within the cell. For experiments carried out on bacteria, the concentration of the scavenger within the cell was assumed to be equal to that of the extracellular media, which is generally not the case.

It was shown that the protection of bacteria against ionizing radiation in the presence of hydroxyl radical scavengers was highly dependent of the irradiation conditions (Billen, 1984). Scavengers are unable to prevent semi-direct effect due to the hydroxyl radicals from the bound water since the water lattice around DNA does not possess any solvent power (Korystov, 1992). Therefore, scavenging of the radicals from the bound water by an exogenous protector is almost impossible. It was observed that thiols are able to repair DNA

HIV viruses 8.8 Bone, -78°C Campbell

*Curvularia geniculata* fungi 2.42-2.90 Aerated water,

**(kGy) condition reference** 

Grecz et al., 1965

et al., 1965

Grecz et al., 1965

et al., 1965

Saleh et al., 1988

Saleh et al., 1988

Saleh et al., 1988

Saleh et al., 1988

et al, 1973

et al, 1973

et al, 1973

and Li, 1999

Buffer

Buffer, -196°C

20°C

20°C

20°C

20°C

bacteria 4.6 Meat, 0°C Grecz

bacteria 6.8 Meat, -196°C Grecz

**organism classification D10**

*Clostridium botulinum*  spores

*Clostridium botulinum*  spores

*Clostridium botulinum*  spores

*Clostridium botulinum*  spores

*Cladosporium* 

Table 2. Radiosensivities of some micoorganisms

when irradiated in frozen solutions was negligible.

damaged sites before a breakage occurs (ABCRI, 2001).


#### **3.2.3 Effect of temperature and additive on radiosensitivity of living organisms**

Temperature plays a major role in the radiosensitivity of microorganisms. As temperature decreases, water radicals become less mobile. As a general rule, microorganisms are less radiosensitive when irradiated at low temperatures (Thayer & Boyd, 2001). For example, whilst sensitivity of spores from *Bacillus megaterium* was constant between –268 and –148°C, an increase in temperature to 20°C led to a 40% increase in sensitivity. Effect of temperature was observed to be similar for oxic and anoxic spores (Helfinstine et al., 2005**)**.

The indirect effect is partially abolished by freezing the solution. The highest decrease in sensitivity is observed between 0 and –15°C. For example, D10 value of *Escherichia coli* irradiated in meat increased from 0.41 kGy at +5°C to 0.62 kGy at –15°C. For *Staphylococcus aureus*, D10 at –76°C was 0.82 kGy instead of 0.48 kGy at +4°C (Sommers et al., 2002). Subfreezing temperatures offer less protection for spores than for vegetative species since they already have low moisture content. The irradiation of frozen aqueous solutions allowed minimizing the loss of active substance even for a 25 kGy dose. This approach seems to be the most promising method for terminal sterilization of aqueous solutions by ionizing radiations. The major radiolysis product was formed after the attack of the electron. Some of the radiolysis products detected were attributed to the attack of •OH,

1. Generally, bacterial spores are considered more radiation resistant than vegetative

2. Among vegetative bacteria, gram-positive bacteria are more resistant than gram-

6. Anaerobic and toxigenic Clostridium spores are more radiation resistant than the

7. Radiation resistance of viruses is much higher than that of bacteria or even bacterial

8. The majority of fungi have D10 values between 100-500 Gy. *Dematiaceous fungi*, which are found in soils and rotten woods but normally not in pharmaceuticals, are highly radioresistant with D10 values from 6 to 17 kGy. Yeast is more resistant than other fungi. *Candida albicans* for example was found to be quite radioresistant with D10 of 1.1 to 2.3

9. In general, it is observed that viruses are less sensitive towards ionizing radiation than bacteria and fungi. D10 values for most viruses range from 3 to 5 kGy (Grieb et al., 2005), which is far more than bacteria. Radiation sensitivities of single stranded DNA viruses

10. Viruses should not normally be found in pharmaceuticals, except in those originating from biotechnological processes. Biological products are submitted to specific guidelines (IAEA, 2004) and the use of higher irradiation doses may be validated for the elimination of viruses. Inactivation with a sufficient S.A.L. (<10-9) of viruses such as HIV or hepatitis in grafts necessitates high doses from 60 to 100 kGy (Campbell & Li, 1999). Table 2 showed the radiosensivities of some micoorganisms at determined

**3.2.3 Effect of temperature and additive on radiosensitivity of living organisms** 

was observed to be similar for oxic and anoxic spores (Helfinstine et al., 2005**)**.

Temperature plays a major role in the radiosensitivity of microorganisms. As temperature decreases, water radicals become less mobile. As a general rule, microorganisms are less radiosensitive when irradiated at low temperatures (Thayer & Boyd, 2001). For example, whilst sensitivity of spores from *Bacillus megaterium* was constant between –268 and –148°C, an increase in temperature to 20°C led to a 40% increase in sensitivity. Effect of temperature

The indirect effect is partially abolished by freezing the solution. The highest decrease in sensitivity is observed between 0 and –15°C. For example, D10 value of *Escherichia coli* irradiated in meat increased from 0.41 kGy at +5°C to 0.62 kGy at –15°C. For *Staphylococcus aureus*, D10 at –76°C was 0.82 kGy instead of 0.48 kGy at +4°C (Sommers et al., 2002). Subfreezing temperatures offer less protection for spores than for vegetative species since they already have low moisture content. The irradiation of frozen aqueous solutions allowed minimizing the loss of active substance even for a 25 kGy dose. This approach seems to be the most promising method for terminal sterilization of aqueous solutions by ionizing radiations. The major radiolysis product was formed after the attack of the electron. Some of the radiolysis products detected were attributed to the attack of •OH,

4. Radiation sensitivity of moulds is of the same order as that of vegetative bacteria; 5. Yeasts are more resistant to radiation than moulds and vegetative bacteria;

3. *Vegetative cocci* are more resistant than vegetative bacilli;

aerobic non-pathogenic Bacillus spores;

are higher than those of double stranded ones;

bacteria;

spores;

kGy;

conditions.

negative bacteria;


Table 2. Radiosensivities of some micoorganisms

demonstrating the feasibility of a reaction between the •OH from ice radiolysis and the solute. A comparison was performed with irradiated frozen solutions of metoprolol, which has been studied in liquid aqueous solutions (Crucq et al, 2000). Degradation of metoprolol when irradiated in frozen solutions was negligible.

On the other hand, the evaluation of the radiosensitivity of bacteria as a function of the addition of radical scavengers is quite difficult since many experiments have been carried out either on isolated DNA, which does not take into account the effects within the cell. For experiments carried out on bacteria, the concentration of the scavenger within the cell was assumed to be equal to that of the extracellular media, which is generally not the case.

It was shown that the protection of bacteria against ionizing radiation in the presence of hydroxyl radical scavengers was highly dependent of the irradiation conditions (Billen, 1984). Scavengers are unable to prevent semi-direct effect due to the hydroxyl radicals from the bound water since the water lattice around DNA does not possess any solvent power (Korystov, 1992). Therefore, scavenging of the radicals from the bound water by an exogenous protector is almost impossible. It was observed that thiols are able to repair DNA damaged sites before a breakage occurs (ABCRI, 2001).

Sterilization by Gamma Irradiation 183

Collagen is a very variable protein, forming the basis of many connective and support tissues. It is a fibrous structural protein, with a distinctive structure. It has been postulated that polypeptide chain scissions (direct effect) predominate when collagen is irradiated in a dry state due to the direct effect of ionizing radiation, and this, in turn, dramatically increases collagen solubility in vitro and the rate of bone matrix resorption in vivo. It has been found, however, that a crosslinking reaction (indirect effect) appears during the irradiation of collagen in the presence of water (indirect effect), probably due to the action of highly reactive, short lived hydroxyl radicals (• OH) resulting from water radiolysis The Figure 6 shown the simplified scheme illustrating the direct and indirect effects of gamma

Food sterilization by gamma irradiation is the process of exposing food to ionizing radiation to destroy microorganisms, bacteria, viruses, or insects that might be present in the food. Irradiated food does not become radioactive, but in some cases there may be subtle chemical

The treatment of solid food by ionizing radiation can provide an effect similar to heat pasteurization of liquids, such as milk. The use of the term "cold pasteurization" to describe irradiated foods is controversial, since pasteurization and irradiation are fundamentally different processes. Food irradiation is currently permitted by over 50 countries, and the volume of food treated is estimated to exceed 500,000 metric tons annually worldwide.

Fig. 6. Effects of gamma radiation on bone collagen molecules.

irradiation on bone molecules.

(Farkas & Farkas, 2011).

changes.

**5. Gamma sterilization of food** 
