Lab. Anim. (1969) 3, 221-254.

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STERILIZATION OF LABORATORY ANIMAL DIETS USING GAMMA RADIATION by

F. J. LEY U.K.A.E.A.

Research Group, Wantage Research Laboratory, Wantage, Berkshire

J. BLEBY M.R.C. Laboratory Animals Centre, Woodmansterne

Road, Carshalton, Surrey

MARIE E. COATES National Institute for Research in Dairying, Shinfield, Reading, Berkshire, RG29AT and

J. S. PATERSON Allington Farm, Porton Down, Salisbury,

Wiltshire

SUMMARY

Sterilization by gamma radiation has proved to be a suitable method for the treatment of a variety of laboratory animal diets intended for specifiedpathogen-free (SPF) and germ-free colonies. Due to the high penetrating power of this radiation the diets can be packed before treatment in a

manner which prevents recontamination during transport and storage. The main bulk of diets is used for SPF animals and a radiation dose of 2.5 Mrad has proved effective for the control of contaminating organisms. No adverse effects on animals receiving the diets have been noted, observations having been made on growth, reproduction and general health. Irradiated diets formulated according to current laboratory animal practice appear to be nutritionally satisfactory. The rapid development of nuclear power in the U.K. has produced the capacity to make high-power radioactive sources on a large scale. Sufficient quantities of cobalt-60, the most widely used radioactive isotope, have been produced in reactors to allow the introduction of gamma radiation processing on an industrial scale, and 5 large cobalt-60 plants are currently operating in the u.K. for the sterilization of medical equipment and pharmaceutical products.

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Gamma radiation has been used for the sterilization of laboratory animal diets by several U.K. laboratories since 1962, and more recently many others have been testing the suitability of the method. The diet is intended for specified-pat hogen-free (SPF) and germ-free colonies and the radiation treatment competes with the use of either heat or ethylene oxide gas sterilization. The main object of this report is to familiarise those in the laboratory animal field with the details of the radiation process itself and to gather together scientific information directly concerned with this particular application. Apart from data supplied for the report from the authors' own laboratories, other information was obtained by contacting laboratories known to be using irradiated diet. Some 10 of those contacted supplied data and comments which were very valuable. The addresses of those communicating unpublished information (referred to by surname and initials in the text) are included in those listed on pp. 248-9.

It is convenient to divide the subject matter into 3 sections dealing with

technological aspects of the process (including methods of irradiation, packaging and costs), the effect of irradiation on the diet itself (with reference to both nutritional and microbiological aspects), and the effect of feeding irradiated diet to animals in terms of its palatability and influence on growth, reproduction and general health. A variety of diets are mentioned in the text and their formulations, as available, are given in the Appendix. TECHNOLOGICAL

ASPECTS

Type and properties of radiation

Gamma radiation from the radio-isotope cobalt-60 is mainly used. This particular isotope is chosen because of its suitable half-life (5.3 years) and its ease of production in quantity, by neutron bombardment of cobalt-59, in nuclear power reactors. Gamma-rays are in fact indistinguishable from the better known x-rays, both being electromagnetic radiations-the 2 names merely indicate the method of generation, the normal x-ray source being an electrical machine, whereas gamma-rays are emitted by certain radio-active materials. It is important to stress first that gamma-rays from cobalt-60 do not induce radio-activity in the processed material; misapprehension on this point arises through confusion with neutron irradiation which does produce radio-activity. Gamma-rays are, however, ionising, i.e. they displace one or more electrons from some of the atoms of irradiated material leading to the formation of positive and negative ions. By various routes initiated by ionisation, gamma-rays have a lethal effect on living organisms. This sterilizing property is particularly useful when considered in relation to the high penetrating power which allows the treatment of

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pre-packaged materials in considerable bulk. It also gives assurance in regard to the treatment of organisms in even the most inaccessible part of a product. Further, sterilization is accomplished without significant rise in temperature (about 5°C rise at 2.5 Mrad). Electrons from electrical machines such as the linear accelerator or Van de Graaff machine have similar properties to gamma radiation and are used for sterilization on an industrial scale. However, their penetration is more limited and this type of radiation would not be suitable for treatment of diet packaged in the manner to be described. The radiation process

The process is one which can be operated only on a large scale if it is to be economic. Pre-packed materials must be transported to a radiation plant for treatment. The principal features of a plant of modern design are shown in Fig. 1. The packages (usually cardboard cartons) are transported in containers conveyed on a monorail system. They enter and leave the concreteshielded radiation chamber through a labyrinth which prevents the escape of radiation. The radio-active cobalt is contained in a number of stainless-steel

SIDE AND END ELEVATIONS

PLAN

VIEW

Fig. 1.. Diagram of radiation plant suitable for the treatment of prepacked materials.

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tubes mounted in a frame which can be raised and lowered. The safe position for the source is either a dry storage pit (as shown) or a water pond about 20 ft (6 m) deep. The simple operation of lowering the source allows safe entry for personnel into the chamber for maintenance or other purposes. Interlocking mechanisms between the source movement devices and the entrance door to the chamber provide safeguards for plant operators. The conveyor system within the chamber is designed to give the maximum practicable absorption of radiation energy in the packages. The carriers follow a path on each side of the source such that a uniform dose is obtained throughout. Radiation energy is successively absorbed during passage through several layers of packages, which are stacked as close together as possible in order to minimise the escape of unabsorbed radiation. Continuous operation is facilitated by automatic loading and unloading at a convenient point outside the plant. An alternative type of plant which is also suitable for processing materials in cartons operates on the batch principle. A monorail or similar system transports the packages in a closed circuit round the cobalt source for the required dose and then the source is lowered to permit entrance for loading and unloading. This involves increased handling operations and less use of the continuously emitting source, but allows simplification of the conveyor system. Dosimetry

The unit of radiation dose most appropriate for use in radiation technology is the rad. It is a measure of the energy absorbed by the material through which radiation passes and 1 rad is equivalent to 100 erg/g. It is convenient in discussing radiation sterilization to use the multiple unit Mrad (1 million rad). The dose received by material of given density exposed to radiation from a given source is dependent primarily on the time of exposure and the distance of the material from the source. In the plants described the products are conveyed on a fixed route and the dose received is therefore dependent on the exposure time, i.e. conveyor speed. This speed is determined in relation to the required dose by certain dosimetry procedures, and thereafter adjustments are made to allow for the gradual decay of the source, which is of course accurately predictable. In addition, routine dosimetry is carried out as a constant check, particularly when materials of varying density are accepted for processing. A simple, rapid and cheap dosimetry method based on the optical density changes induced in acrylic sheet (red 'Perspex') is currently used in routine operation (Whittaker, 1964). Small pieces of the 'Perspex' are incorporated in various positions within the packages and, after exposure to radiation, change

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in optical density is measured at a certain wavelength using a spectrophotrometer. The 'Perspex' dosimeters are calibrated prior to use by means of a standard source, and hence a quantitative measure of dose is made. Basic dosimetry procedures used in standard source measurement include calorimetry and chemical methods such as measurement of the oxidation of ferrous to ferric ions. Absolute uniformity of dose cannot be achieved (because attenuation is not quite linear). The packages are irradiated from 2 opposite sides, the maximum dose is received by these sides and the lowest dose by the middle. When a dose of 2.5 Mrad is specified for a 56 Ib (25.4 kg) pack of diet, for example, the minimum dose gives is 2.5 Mrad and the maximum dose received is approximately 3.2 Mrad. The dosimetry procedures are normally the responsibility of the plant operator, who keeps appropriate records of source strength, conveyor speeds and dosimeter readings. For the convenience of both the operator and user of irradiated products, qualitative dosimeters are widely used. These consist of small indicator labels which show a distinct colour change as a result of passing through the plant. The label in current use changes from yellow to red and is affixed to the outside of each carton on arrival at the plant or before leaving the manufacturer. The label is made by impregnating polyvinyl chloride (PVC) with an acid-sensitive dye which changes colour due to release of a small amount of hydrochloric acid from the plastic during irradiation (Farrell & Vale, 1963). Use of the label ensures against confusing irradiated and unirradiated products. Packaging of diet

The packaging has been designed to: maintain a sterile product after irradiation, taking into account handling during transport and storage; allow convenient handling at the animal house, particularly in relation to the entry of the diet into SPF environment; facilitate the use of conveying systems at existing radiation plant and to ensure efficient radiation treatment. Packs were developed in relation to the use of radiation facilities at Wantage Research Laboratory where 2 large-scale cobalt-60 package irradiation plants (P.I.P.) are in routine operation; P.I.P. Mk. I is a continuous type and P.I.P. Mk. II is of the batch type. Pelleted diets are packed into polythene bags (1 000 gauge, 0.25 mm) and heat sealed. If required, a further wrap of polythene may be placed around the bags; 250 gauge (0.06 mm) is suitable, and can be closed with tape or rubber band. This will ensure that the outside of the

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inner bag is sterile or at least extremely clean. The bags are then placed in a cardboard carton of appropriate size; the carton must not be distorted by overfilling since this leads to problems with the conveyor at the radiation plant. P.I.P. Mk. I plant can accept cartons measuring 8.5 x 8.5 x 13.25 inch (21.6 x 21.6 x 33.8 em) and these are suitable for 14 Ib (6.35 kg) of diet packed in 12 inch (30.5 em) x 1 000 gauge (0.25 mm) polythene layflat tubing. The carton size for P.I.P. Mk. II is 17 x 17 x 10 inch (43.2 x 43.2 x 25.4 cm) and this will accept 4 x 14 Ib (6.35 kg) bags or 2 x 28 Ib (12.7 kg) bags. The 14 Ib (6.35 kg) pack is popular for its ease of handling, particularly where 'dunk tank' operations are involved. The actual packaging operations are normally carried out by the diet manufacturers on their own premises or by contract arrangement with a packaging firm. Transport of packs to the radiation plant and delivery to the user are also the responsibility of the manufacturer. In fact, the current situation is that packed irradiated diet may be ordered directly from several manufacturers. However, for irradiated diet for germ-free animal work, where the quantity of diet involved is a small proportion of the total demand, individual laboratories normally make their own arrangements. Packs are designed by the user to meet particular requirements, for example in relation to entry port size in isolators. Glass and plastic jars are often used, packed by the user and irradiation arranged directly with the Irradiation Service, the packs being placed inside the cartons which are available from Wantage Research Laboratory.

Cost of irradiation and packaging Irradiation of pre-packaged diet is carried out at Wantage Research Laboratory using both of the large plants referred to previously. The radiation charges are equivalent in both plants and the price varies with the dose required. The bulk of the throughput is being processed at 2.5 Mrad, and for this dose the cost is £0.75 (15 shillings) for the 56 Ib (25.4 kg) box, i.e. £30 per ton; higher doses are charged in direct proportion. Doses lower than 2.5 Mrad would reduce the cost, though not in direct proportion since part of the cost covers handling, and in economic terms there would be little advantage to the user to request doses of less than 1 Mrad. It is interesting that just over 10 years ago the estimate with reference to the use of an electron machine for sterilization of diet, at a dose of approximately 2 Mrad, was about 3 times that quoted above for 2.5 Mrad (Aarons & Hill, 1958). To the irradiation cost must be added the cost of packaging and transport giving a final figure currently in the range £90 to £110 per ton according to

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type of diet. Manufacturers' costs might be expected to be high when demand is small; increased demand might lead to investment in, for example, automatic packing machinery with consequent price reduction. It is not a purpose of the report to compare the costs of the various sterilization methods available. The cost of radiation-sterilized pre-packaged diet is higher than commercially available heat-pasteurised diet, which in turn is higher than untreated pelleted material. Ethylene oxide and heat sterilization processes are normally applied at the site of individual laboratories, and the costs involved will vary with circumstances of operation. These 'on site' methods require capital investment, although they may, of course, be used for purposes other than diet treatment. Labour costs are also involved, and packaging problems of diet handling after sterilization must be considered. Thus, the choice of sterilization method on economic grounds can only be made in the light of each laboratory situation.

EFFECT OF IRRADIATION

ON THE DIET

Microbiology Lethal effect

The lethal effect of ionising radiation on micro-organisms, as measured by the loss by cells of colony-forming ability in nutrient medium, has been the subject of detailed study. Much progress has been made towards identification of the mechanism of inactivation, but there still remains considerable doubt as to the nature of the critical lesions involved, although it seems certain that lethality is primarily the consequence of genetic damage. The role of the cell nucleus as the target for lethal damage is well established, and much evidence points to induced changes in DNA as being responsible for inhibition of cell division. Apart from difficulties in location of the site of primary damage, there is still controversy as to whether the majority of radiation effects on biological systems are due directly to ionisation or to the indirect action of the radiolysis products of water, or both. However, while the work on basic mechanisms continues, much is already known both qualitatively and quantitatively in relation to the radiation inactivation of microbial populations. Just as with heat resistance, there is considerable variability in radiation resistance between microbial species; in general, viruses are more radiation resistant than bacterial spores, which in turn are more resistant than vegetative organisms, yeasts and moulds. Moreover, the inactivation of microbial populations is considerably influenced by conditions of environment during irradiation-for example, gaseous composition, temperature, and nature of the suspending medium.

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Quantitative

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inactivation

As an example, some of the conditions influencing radiation resistance of typhimurium are illustrated in Fig. 2, where inactivation of popu-

Salmonella

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fish meal frozen meat unfrozen meat anoxic buffer aerated buffer

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