NASA/TM-- 208145 Laboratory Animal Science Copyright' 1980 . by the American Association for Laboratory Animal Science

The Manufacture, Shipping and Receiving and Quality Control of Rodent Bedding Materials I

Lisbeth M Kraft

Summary [ The criteria for rodent bedding and nesting materials are discussed. The literature is reviewed regarding sources of bedding materials, manufacturing methods, quality control procedures (microbiological, physical and chemical), storagemethods, shipment, methods of use and disposal, current knowledge concerning bedding effects on animals as related to research and testing and legal aspects. Future needs, especially with respect to the promulgation of standards, also are addressed.

Key Words ] Animal bedding -- Microbiological safety -- Rodentia

In 1974, a journal published for the For the most part, contact bedding laboratory animal field cited 11 commercial will be discussed, although materials placed sources of contact bedding for laboratory ani- under suspended wire cages, as well as no bed- mals in the form of wood shavings or chips and ding at all, also will be mentioned. Most of the six for pelleted products (1). In 1979, the same data will center about mice and rats. publication again listed 11 sources of shavings or wood chips, however, seven of those men- tioned in 1974 were no longer listed 12L Thus seven new vendors had entered the market in Specifications and Requirements: Types of the interim, and, whereas six suppliers offered Contact Bedding pelleted bedding in 1974, there were only four The specifications and requirements in 1979. Of these, three were new vendors. What are we to conclude from this? for rodent contact bedding vary depending on whether one wishes only adequate or optimal, Do bedding vendors tend to be an evanescent lot? Or is the fault to be found in the nature of that is, ideal, conditions for the animals. In our view, adequate conditions are those that will the products? (We assume, of course, that the permit an animal to reproduce and survive in journal personnel were equally industrious in reasonable health. Food and pet animals fall soliciting vendors for listing in both issues). into this category. Optimal conditions, then, While it might be more interesting to are those which follow when all variables, discourse on the vendors themselves, the pur- other than those imposed by experimentation, pose of this presentation is to examine their are known in detail and then are controlled. A products, and even if we cannot reach a conclu- propos of this comment is the fact that the ICLA sion as to the precise reasons for the changes (International Committee on Laboratory between 1974 and 1979, we should be able to Animals) Governing Board (3) strongly urged infer from our discussion if the products them- in 1978 that bedding {type, changing schedule, selves are to blame or tic,perchance, the labora- sterilization status) always be described when tory animal field might be found wanting. the results of experiments using animals are reported. Table 1 indicates a number of desir- 1From the Ames Research Center, National Aeronautics and Space Administration, Moffett Field, CA 94035. able characteristics for contact bedding that

366 Rodent Bedding Vol30,No 2 PartIIofTwo Parts Printedin USA

Table 1 Table 2 particular product, Desirable criteriaforrodent contact bedding Additional desirable criteria for rodent contact polyethylene gran bedding stances and theref, Moisture absorbent Nondesiccating to the animal variability and at Dust free Uncontaminated Remains chemically stable during use Unable to support microbial Non-nutritious Manifests batch to batch uniformity which one is chose growth Nonpalatable Optimizes normal animal behavior "continues to be a Inedible Unlikely to be chewed or Nondeleterioua to cage washers ence" (11). Nonstaining mouthed Noninjurious and nonhazardous to personnel Nontraumatic Nontoxic Ammonia binding Nonma]odorous Even re_ Sterilizable Nestab]e results in a cost responsible fashion. Whether very specific con Deleterious products not Disposable by incineration guidelines. The Go_ formed as a result of Readily available or not we now have all the data necessary to (GLP), for example. sterilization Relatively inexpensive formulate such standards is, as we shall see, Easily stored Fire resistant bedding, indicating somewhat in question. rated from areas hc A number of authors (4-111 have tected against inf mentioned the types of contact bedding and (14); it also states are thought to provide optimal conditions (4 - bedding supplements (as for nesting) that have cages or pens shall 13), while Table 2 shows some personal addi- been utilized in the past or are still in use: pose or conduct c tions. While composing these lists, the author white, ponderosa, sugar and lodgepole pine changed as often a_ was reminded of the "little list" of the Lord shavings; redwood, aspen or other types of pop- mals dry and clear High Executioner in Gilbert and Sullivan's lar, basswood, cedar and maple shavings; white "Mikado" as well as of their patter songs. In- pine sawdust and flakewood; flax stems; shred- The Ani: spiration then brought into being the following ded newspaper, both used and unused; chopped reference to beddin doggerel: and ground corncobs; wood wool, excelsior, Cancer Institute, c I bet you any money that you cannot find a shreds or filaments of aspen; peat moss; paper that its bioassay bedding sheets; pine or hardwood chips; cotton batting; treated hardwood, That will give a mousie comfort from attapulgite (hydrated magnesium aluminum The Gui unmitigated wetting, silicate); clays, such as bentonite; filter paper, Laboratory Anima That will be both hygroscopic but yet dry paper towels; peanut hulls; hay ffor guinea "In general, beddix the critter not, pigs); bagasse (crushed sugar beet or sugar bent and free of s_ That will burn just when you want it to, cane pulp}; cellulose fibers; alfalfa stems; dry animals or persom but not burn very hot, sand; shredded computer printouts; nonabsor- eaten by the anirr That contains no toxins, viruses, no fast bent cotton; and polyethylene granules (pre- cage to keep anima fermenting bug, pared from ethylene polymerized with organic •.. should not com That is stored with ease in cubbyholes or peroxides), it may be safe to say that, of these, tubes. Some bedd underneath your rug, white pine shavings are the most widely used. substances that af_ That smells good both to man and beast Hardwood or pine chips and aspen shreds prob- animals to some ex before it's messed upon ably follow some distance behind. Without an It states further t_ As well as after usage when its wasted extensive survey, however, the precise usage contain substance._ youth is gone, cannot be stated with certainty. on an animal's bic That will do the universal task of cutting A few bedding types mentioned have cedar, for example. variation fallen out of favor for a variety of reasons. in hepatic micros, To give test results with only zero Cedar, and in some instances pine, shavings rats: therefore, th_ standard deviation. i may not be used in pharmacological studies. In the r With aims so high Hay is edible. Peat moss, used newsprint and Long-term Holdin_ And motives so deep, alfalfa stain the animals' coat, although peat bedding criteria I wonder why moss does have the apparent advantage of ment: "The type of ft should also be cheap. relatively high acidity and therefore may con- ability for the dl In any event, fulfilling the criteria trol ammonia. Attapulgite is too hygroscopic should be known.' (some of which seem antithetical as they stand} for some animals, and, together with sand, it is Is it pos_ should provide the basis for standards for the not combustible and thus difficult to dispose of. general nature oft] specific use of a bedding material, with the Pelleted peanut hulls are relatively new on the given above is thai ultimate aim of removing bedding from the list market. Comparative studies with these have contact bedding t} of environmental variables that beset the in- not, to our knowledge, been reported. Regard- therefore, it is not vestigator who attempts to obtain reproducible less of the advantages or disadvantages of a ic? On the other h 367 368 Vo130, No 2 Part II of Two Parts Printed in USA

particular product, it is true that, except for the still afloat in a sea of relative ignorance, and polyethylene granules, all are natural sub- 'therefore, it is prudent not to be too specific? stances and therefore subject to considerable Requirements of the animal: To this variability and at least some contamination. point, we have been almost entirely anthro- Which one is chosen in a particular instance pocentric in dealing with requirements for bed- "continues to be a matter of personal prefer- ding. But what does the animal require? Few ence" (11). studies in which mice or rats have been given Even regulatory agencies are not free choice of bedding for parturition and lacta- very specific concerning requirements or tion or for extended holding periods have been guidelines. The Good Laboratory Practices Act conducted (15-17). Even in these studies, it (GLP), for example, addresses storage areas for must be recognized that the test bedding mate- bedding, indicating that they are to be sepa- rials were first selected by the investigator. Of rated from areas housing test systems and pro- those provided, wood products were preferred tected against infestation or contamination in all cases by the animals, with aspen in the (14); it also states "bedding used in animal form of shreds (excelsior, wood wool) chosen by cages or pens shall not interfere with the pur- all the animals. Of more than passing interest pose or conduct of the study and shall be is the fact that combinations were chosen. Thus changed as often as necessary to keep the ani- all pregnant mice in one study chose aspen mals dry and clean." bedding, 75% added cedar shavings to this, 45% The Animal Welfare Act makes no added pine shavings, 5% added hardwood chips reference to bedding specifically. The National and 5% chose a corncob product as supplement. Cancer Institute, on the other hand, specifies Some chose as many as four different mate- that its bioassay contractors use only heat rials. Rats similarly tested manifested essen- treated hardwood chips. tially the same behavior. The Guide for the Care and Use of In a study using mice over a 10-week Laboratory Animals (12) gives the following: maintenance period, four materials were "In general, bedding or litter should be absor- offered: shredded aspen, cedar shavings, bent and free of substances that could injure ground corncobs and dehydrated alfalfa. On the animals or personnel ... or a type not readily first day, all animals had chosen cedar shav- eaten by the animals. Enough ... placed in ings. Thereafter, the animals chose various cage to keep animals dry between cage changes combinations for the duration of the test, but •.. should not come into contact with watering aspen and cedar combinations predominated tubes. Some bedding materials may contain (17). substances that affect the biologic responses of In another study, pregnant mice animals to some experimental procedures..." were on one of four different bedding materials It states further that "Bedding materials may up to the eighteenth to twentieth day of gesta- contain substances that have significant effect tion (18). Thereupon they were each offered the on an animal's biological responses. Pine and four choices, three of which now differed from cedar, for example, are known to cause changes that with which they had become familiar. The in hepatic microsomal enzymes of mice and choices were: ground corncob, pine shavings, rats: therefore, they should not be used ..." dehydrated and pelleted alfalfa, and flax fiber. , In the report of the Committee on The only clearcut preference in this case con- Long-term Holding of Laboratory Rodents (13), cerned.the alfalfa. Whether the mice had been bedding criteria are restricted to the state- on it previously or not, they almost totally ment: "The type of bedding, source and avail- ignored the product. A study of habitat prefer- ability for the duration of the experiment ence in inbred mice has shown that selection of should be known." birth site for the next generation is affected by Is it possible that the reason for the prior experience of the parents, males being general nature of the requirements and criteria affected more than females by that experience given above is that everything is known about (19). contact bedding that needs to be known, and Other comparative studies, Without therefore, it is not necessary to be more specif- offering free choice to the animals, also have ic? On the other hand, could it be that we are been performed (20-22). Further, comparisons 368 Vo130, No 2 PartII of TwoParts Rodent Bedding Printedin USA

have been made between results in solid bot- trace element research. Suspended wire cages thought at one tim tom and suspended wire cages (23-25). The generally are used for the former, and a num- creased the occurr, results of these are certainly of pragmatic ber of systems have been described for the lat- gland tumors in C3 value, for they took into account several impor- ter (30-32). this was not the c_ tant variables including reproduction, lacta- Requirements during shipping: Ex- tumor incidence sub tion, litter weight and size. Experimental de- cept for recommending that animals be kept the condition of th signs, however, do not always permit us to draw warm or cushioned from shock during ship- general health) rat_ firm conclusions. For example, did the mice and ment, there are no particular requirements or One still finds refel rats in the free-choice trials choose the aspen guidelines. What may be of importance to the nogenic effects of wc because it was aspen or because it was in shreds investigator, however, is to know if there are to their podophyllot_ or for both reasons? Would they have chosen different bedding types in the breeding unit, content (40,41), al pine over aspen if the pine had been shredded? the shipping unit and the research laboratory. have not yet been aq Did they like the cedar shavings because of its Shifting animals from one type to another may firm statement abo_ undesirable pharmacologic characteristics, or have profound effects. the literature also c, would they have accepted it equally if the ced- increased incidence rene and cedrol had been removed? workers in the furni Other questions are also brought to Effects of Bedding tain where the pre mind. Is it a mistake to provide only one kind of Materials on the Animals mahogany, walnut, bedding material as routine? Are we depriving Work on the induction of hepatic hardwoods. The tu_ the animals of an important factor for their microsomal enzyme activity in rodents as a cinomas on the etl well-being by doing so, and what effect might result of bedding them on cedar shavings has with latent periods this have on their usefulness for certain types received considerable attention (33). One is Of additi¢ of testing and research? Can free-choice experi- cautioned against keeping such shavings in the that corncob beddir ments give new insight into rodent behavior vicinity of rodents even though, as we have oxide resulted in ch and therefore increase their usefulness in re- seen, the animals themselves seem to enjoy and increased tumo: search? Suitably designed experiments are them. Further, even when cedar shavings were Ethylene glycol wa,, surely needed in order to answer these as well placed beneath suspended wire cages, the ized bedding. Althc as other problems that may justifiably be threshold for clonic seizures due to pen- gard to carcinogeni posed. tylenetetrazol was significantly decreased (34}. than statistically si Requirements for special rodents: Pine shavings also have been shown to be effec- safety of ethylene Some requirements for special rodents should tive pharmacologically in that they increase ding, nevertheless, be mentioned. In spontaneously hypertensive the activity of sulfabromophthalein sodium rats (SHR), one investigator found that they (BSP)-s-aryl transferase in rats as compared Although seemed healthier on pine shavings as compared with those bedded on polyethylene granules ding, ammonia evol with dehydrated alfalfa, clay or corncobs. He (35). major concern. For ascribed this to the rat's ability to nest on the One report describes increased rat pigs exposed to 170 shavings (26). The Institute of Laboratory pup mortality caused by cedar shavings as com- hours/day, 5 days/wt Animal Resources publication concerning pared with crushed corncob and shredded cant pathological ch these animals makes no mention of special bed- aspen beddings (36). In studying growth and 18 weeks, there wer ding needs for them (27). Bedding should be several hematologic parameters of rats housed in the spleen, kidne carefully chosen for the nude mouse to avoid in suspended wire cages with half of the floor spleen showing the irritation of the mouse's vulnerable skin. The covered with either cotton wool, polypropylene congestion with incr bedding should be dust- and splinter-free. fibers or shavings (type not stated), it was de- swelling and some q Hardwood chips and corncobs are regarded as termined that those bedded on shavings kidneys, and adren_ beddings ofchoice, although pine shavings may showed significantly higher leukocyte counts generative lesions. also be used. Nesting material is regarded as and decreased J3-globulin levels than those kept observed in the cont optional (28). For gnotobiotes, recommenda- on the other materials (37). Growth patterns, of known pathogen-'. tions include that the material be easy to steril- _oncentrations of albumen and of other globu- results in morphol< ize, not be readily eaten by the animals and not lins, hemoglobin, hematocrit, erythrocyte passages, while in yield toxic compounds as a result of steriliza- counts, total serum protein were not signifi- fected rats, the sevel tion (29). cantly different. directly with ammc Requirements for special experiments The question of carcinogenicity of On this basis, it wa and testing: No bedding currently available is cedar shavings, as well as other types of bed- plays an important suitable for most toxicologic studies or for any ding, has been alluded to on occasion. It was murine respiratory 369 370 Vo130, No 2 PartIIof TwoParts Printed in USA

thought at one time that cedar shavings in- Investigators also have shown that creased the occurrence of mouse mammary mice in a dirty environment (one in which gland tumors in C3H sublines (38). However, ammonia levels are high) manifest signifi- this was not the case: the factor in altering cant decreases in some microsomal enzymes, tumor incidence subsequently was shown to be aniline hydroxylase and ethylmorphine N- the condition of the mice (weight gain and demethylase (33). Additional unknowns may general health) rather than their bedding (39). abound. Of some concern is the possibility that One still finds reference to the possible carci- steam sterilization may leave residues on bed- nogenic effects of wooden bedding products due ding. Cyclohexylamine, frequently added to to their podophyllotoxin (in softwood) or lignan water to prevent corrosion of pipes, is such as content (40,41), although these compounds unknown (47). We are "assured" that the dose have not yet been adequately tested to make a of such a chemical will be too low to produce an firm statement about them. On the other hand, effect. But cyclohexylamine is a skin irritant. the literature also contains reports concerning Could it, under the proper conditions, cause increased incidence of nasal cancer in wood- problems in rodents during long-term contact, workers in the furniture industry in Great Bri- for example? Lest we feel too confident that, tain where the predominant woods are oak, since low doses produce no effect, lower doses mahogany, walnut, ash, beech and elm - all will also produce none, attention is called to a hardwoods. The tumors were mostly adenocar- paper which points to a number of dose- cinomas on the ethmoid or turbinate bones response relationships in nature where that is with latent periods of 20-40 years (42). not necessarily true (48). Sometimes less is in- deed more! Of additional interest is the finding that corncob bedding sterilized with ethylene Manufacture of Contact oxide resulted in chronic toxicity in male mice Bedding: Quality Control and increased tumor incidence in females (43). at the Source and by the Ethylene glycol was recovered from the steril- Vendor ized bedding. Although the findings with re- Wood shavings: These are byprod- gard to carcinogenicity were more suggestive ucts of the lumber or woodworking industry than statistically significant, doubts as to the and result from the planing of wood. Except for safety of ethylene oxide sterilization of bed- the fact that the lumber may have been kiln ding, nevertheless, have been raised. dried at some time before the shavings are pro- Although not caused directly by bed- duced, they are not likely to be heat treated ding, ammonia evolution by animal waste is a before shipment to the user. Quality control is major concern. For example, in male guinea in general virtually lacking. A manufacturer pigs exposed to 170 ppm of ammonia up to 6 may be able to claim that his shavings are hours/day, 5 days/week for 12 weeks, no signifi- softer, contain less, debris, are free from pesti- cant pathological changes could be found. After cides or other pollutants depending upon the 18 weeks, there were mild yet definite changes source of the wood, but few if any can assure the in the spleen, kidney, adrenals and liver. The user of microbiological or toxicological purity spleen showing the greatest change: marked by means of assay data. Packaging of the prod- congestion with increased hemosiderin. Cloudy uct is variable. It is not necessarily protected sv_elling and some casts were observed in the from the environment or from vermin. Some kidneys, and adrenals showed some early de- vendors use simple paper bags while others generative lesions. None of these changes were may use only wooden slats and bailing wire to observed in the control animals (44). Exposure contain the shavings. In some cases, deliveries of known pathogen-free rats to ammonia alone are made in bulk. Still others may package results in morphologic changes in the nasal them so that they may be conveniently steril- passages, while in Mycoplasma pulmonis in- ized at the user laboratory. fected rats, the severity of the disease increased Wood chips." The process by which directly with ammonia concentration (45,46). wood chips (byproducts of the manufacture of On this basis, it was concluded that ammonia particle board, the heels of women's shoes, etc) plays an important role in the pathogenesis of are produced is a marked improvement over murine respiratory mycoplasmosis. that which provides shavings. It is stated that 370 Vol 30, No 2 Rodent Bedding Part II of Two Parts Printed in USA

Quality ce only green logs are used. These are debarked Dust is removed by vacuum aspiration, and the on hisneedsand on l_ prior to production of the chips, thus avoiding product is bagged in four-ply paper bags. Quali- Often,it consistsof the presence of wood preservatives or other sur- ty control is physical: no sharp ends on the ing for the dustine., face contaminants in the final product. Particle shreds. Other laboratories are utilized for mi- Testingisexpensive size is controlled by means of sieves, heat (up to crobiological and toxicological determinations. costof the item. T_ 350°C) is applied for drying, dust is aspirated, currentlybeing don steam is introduced to bring the humidity up to Shipping, Receiving and producer are summ_ about 6-9%, and then it is packaged by means Storage of Bedding cussedina recentart of an augur in three-ply paper bags. These are Depending on the distance involved, thesebe performed? constructed of"Kraft" paper with autoclavable bedding is shipped by railroad box car (long of the various chen glue, requiring no stitching that could result in haul) or truck (short haul), usually to a dealer. animals thatspend t a portal of entry for vermin during transit and There can be considerable friction and jostling tactwith them? The., storage. during transit, and even though the material stillopen fordiscus, In addition to control of particle size, may be dust-free at the source, it can become dust and humidity, vendors or manufacturers dusty before receipt. In terms c also on occasion (frequency not known) submit Because bedding has a long shelf- tailedaccount as to their product to independent laboratories to life, a dealer may store the bedding for a long varioustypes are u test for the presence of microbiological, toxico- time. That being the case, his warehouse another paper. Suffi logical and other chemical agents. In addition, should be protected from vermin, should be dry, expensivebedding i_ particle uniformity, absorptivity, ammonia and the bedding should be palletized. Dating of locally,directfrom evolution and skin irritant properties may be production lots is, to our knowledge, not prac- qualitycontrol.The determined by independent laboratories. ticed, either as to time of manufacture or re- almost any bedding Corncob products (contact): These ceipt at the warehouse. vendor have run oul are the byproducts of both the seed and feed The receiving of bedding at the user requiringa supply t corn industry, animal bedding beintoonly one laboratory often is fraught with the same prob- theholidaysjustbef< of about 70 uses to which they are put. The lems that apply to the receipt of animals, that strike. wood of the cob is separated from the remainder is, there is seldom a special area set aside for Inthesed_ and ground. The granules then pass through those purposes. Sometimes such areas even •ofsome valuetomex screensforsizing,residualdust isaspirated, lack adequate protection from the elements. foundthathe could the granulesare heated to 100-300°C,cooled Add to that the usually inadequately sized stor- by adding an equal and immediately placed in bags similarto age areas provided in an animal care facility, shredderstopinesh those describedabove. Quality controlmay and it becomes important for the vendor (deal- rial was thus pro consistof some or allof the items mentioned er) of bedding to have suitable storage space to seemed improved,m above. maintain sufficient stock to service his custom- tercontrolled.Also, Pelletedpeanut hulls:Once allfor- ers frequently. Delivery from the warehouse is than with shavings eign objectsare removed from the harvested by truck, which should be a closed van. may not yet be the peanuts and the nutsthemselvesremoved,the possiblethatnew be. hullsare recleaned,ground to a smallparticle Bedding Usage: Quality be investigatedforcq size,mixed with bentonite(5%)as a binderand Control by the User: Cost operations. It is axiomatic that all contact bed- pelletedunder steam pressure.The pelletsare Here we cooled,the finesand dust removed by aspira- ding should be sterilized before use, although addition of odor sL tion,and the pelletsare directlybagged. under certain circumstances (with convention- counterproductiveb Quality controlisbased on the ven- al animals, for example, and with a low order of abilitytomonitorh_ dor specificationof granule sizeuniformity, microbiological and other contamination) this omones cannot be eft bulk density,absence of additives,moisture may be judiciously dispensed with. contentof8-12%_ lackofeffecton hepaticmi- Recently, investigators reported on Table 3 crosomalenzyme functionand inabilitytosup- the development of a paper strip containing an port mold growth. Tests by independent indicator sensitive to both heat and steam that Tests of beddi laboratoriesare occasionallyrun (frequency can be inserted into bags of bedding to monitor Chemical properties not known). sterilization (49). It is claimed to be an im- Pesticides and polychlorinat_ Shredded aspen: In general,thisis provement of the indicators that detect heat Aflstoxins derivedfrom treesin Colorado and Wisconsin. alone. The strip is so designed that the integri- Detergent residues The shreds are produced by fracturingthe ty of a barrier system, for example, is not com- Ether extractable substancag Heavy metals wood. They are heat treated at 160-300°C. promised. 371 372 Vo130. No 2 Part II of Two Parts Printed in USA

Quality control by the user depends fertility under such circumstances. Five rodent on his needs and on his ability to afford testing. contact beddings for guinea pigs were studied Often, it consists of nothing more than check- with respect to odor control (52). They were ing for the dustiness of a particular bagful. hardwood chips, pine chips, poplar filaments, Testing is expensive and, therefore, adds to the cellulose pellets and cellulose pellets with cost of the item. The kinds of tests that are chlorophyll. Reduced ammonia levels were currently being done on bedding by user and found with both types of cellulose pellets and producer are summarized in Table 3 and dis- poplar shreds as compared with the others. The cussed in a recent article (50). How often should use of specially designed ventilated cage racks these be performed? What are allowable limits for this purpose has recently been described of the various chemicals and microbiota for (53). The aim was to reduce cage changing from animals that spend their lives in intimate con- once a week to once every 2 weeks. Others tact with them? These are additional questions attempt to solve the problem by more frequent still open for discussion and determination. cage changes or by removing only the soiled In terms of cost effectiveness, a de- portion of the bedding, leaving the clean part tailed account as to the manner in which the for the animals to continue nesting upon. The various types are used could easily comprise problem is not easily solved. Each solution may another paper. Suffice it to say that the least add to cost, all other things being equal, but as expensive bedding is one that can be obtained we know, reduced ammonia levels do result in better animals. locally, direct from a lumber mill without any quality control. The most expensive bedding is almost any bedding that both the user and the Disposal of Soiled vendor have run out of at the same time, thus Bedding requiring a supply to be shipped by air around Within an animal room, cage chang- ing can produce hazardous aerosols. In order to the holidays just before the airlines might go on strike. control these, safety cabinets have been de- vised (54-56). In these days of recycling, it might be Incineration is by far the most ec- of some value to mention that one investigator onomical means of destroying both the bedding found that he could cut his bedding cost in half and the microbiological and toxicological con- by adding an equal volume of paper from paper taminants (radioactive wastes are a separate shredders to pine shavings (51). Nesting mate- problem, of course) (57,58). With regard to dis- rial was thus provided, water absorption posal or re-use of soiled bedding (as a mulch or seemed improved, and odor appeared to be bet- fertilizer), even after sterilization, it should be ter controlled. Also, cages were easier to clean observed that the Resource Conservation and than with shavings alone. Even though this Recovery Act, PL 94-580, as amended in 1978, may not yet be the wave of the future, it is provides that an OffÉce of Solid Waste be estab- possible that new bedding combinations should lished with the Environmental Protection be investigated for cost responsible animal care Agency. Subtitle C relates to hazardous waste, operations. which is defined as ignitable, corrosive, reac- Here we should mention that the tive, toxic, radioactive, teratogenic or addition of odor suppressants to bedding is mutagenic. It might be difficult to prove that cour_terproductive both because of resulting in- soiled bedding, even though it might have been ability to monitor husbandry and because pher- sterilized, would not fall into one of these omones cannot be effective in maintaining high categories.

Table 3

Tests of bedding quality

Chemical properties Physical properties Microbiological properties

Pesticides and pelychlorinated compounts Particle uniformity Standard plate count Aflatoxins Absorptivity Yeasts and molds Detergent residues Ammonia evolution Coliforms and Salmonella Ether extractable substances Visible trauma and irritant potential Pseudomoaas Heavy metals 372 Vo130, No 2 Rodent Bedding Part II of Two Parts Printed in USA

Conclusion 10. Magalhaes, H: Housing, care, and 29. Subcq breeding. In The Golden Hamster. Its Biology and tobiotes, Committ_ We have posed a number of questions Use in Medical Research, p 15-23, Hoffman R A, Laboratory Animal since we initially wondered what happened to Robinson P F, Magalhaes H, Ed, Iowa State Universi- Council: Gnotobiotes Washington, 1970 bedding vendors between 1974 and 1979. Con- ty Press, 1968 11. Poiley S M: Evaluation of require- 30. Smitl sidering the questions that may yet arise from ments for defined laboratory animals in biomedical environment systel ciencies. J Nutr 93:_ the use of the materials currently in fashion, research. Adv Pharmacol Chemother 12:125-184, 1975 31. Mohl perhaps we should be surprised that so many 12. Committee on the Care and Use of plastic system for vendors have remained to serve the field of element studies. LaJ Laboratory Animals, Institute of Laboratory Animal 32. Niel, laboratory animal science. The real task, Resources, National Research Council: Guide for the ofplastic cages for st however, still lies in the future. Taking into Care and Use of Laboratory Animals, National Lab Anita Sci 29:50 Academy of Science, Washington, 1978 account scientific, economic, humane and legal 33. Vesel 13. Committee on Long-term Holding of Environmental and aspects of laboratory animal bedding, is there Laboratory Rodents, Institute of Laboratory Animal spense of laborater agreement that there should be standards for Resources, National Research Council: Long-term 35:1125-1132, 1976 holding of laboratory rodents. ILAR News 19:L1- bedding? Should there be standards covering 34. Pick, L25, 1976 bedding material on more than one species or type of animal (hair- 14. Anonymous: Nonclinical laboratory convulsions in mice. less, HSR, germfree, pregnant, etc)? If so, how studies: Good laboratory practices regulations. Fed 35. Whel Reg 43:59986--60025, 1978 can the promulgation of such standards be ding on the metab 15. Mulder J B: Bedding preferences of sodium by an hepati_ abetted? What are the questions that still re- pregnant laboratory-reared mice. Behav Res Aust J Biol Sci 28:2 quire hard answers before they can be written? Methods Instrum 7:21-22, 1975 36. Burk And who will do the work in order to obtain the 16. Mulder J B: Behavior patterns of rat pup mortality att laboratory animals. Lab Anita (New York) 5:22-28, shavings as beddin_ results on which the standards are to be based? 1976 222, 1978 It isperhaps a "cop-out" to end this discussion !7. Mulder J B: Bedding preferences of 37. Mats_ et al: Influence of po] with so many queries, but it is to be hoped that mice over long-term confinement. Lab Anita Dig 9:54-56, 1974 hematologic values they will stimulate action in the form of in- 30:179-186, 1975 18. Wagner J E: Contact bedding selec- 38. Sabir terest and concern on the part of investigators tion of parturient mice. Lab Anim Dig 9:67-69, 1974 Spontaneous tumor 19. Anderson L T: An analysis of habitat followed by support, both moral and monetary, mice: High incideno reference in mice as a function of prior experience. incidence in Austral from appropriate agencies. ehaviour 47:302--339, 1973 1242, 1973 20. Porter G: Assessment of environmen- 39. Heet, tal influence on the biological responses of the ani- effects of cedar Juni! mal. In Husbandry of Laboratory Animals, p 29-42, and diet on occurrer References Conalty M L, Ed, Academic Press, London, 1967 and hepatomas in C 1. Anonymous: Buyer's guide 1974-1975. 21. Iturrian W B, Fink G B: Comparison Natl Cancer lnst 54:'. Lab Anita (New York), 3:39--40, 1974 of bedding material: Habitat preference of pregnant 40. Schoe 2. Anonymous: Buyer's guide 1978-1979. mice and reproductive performance. Lab Anita Care in the bedding and dl 18:160-164, 1968 Lab Anim (New York), 7:32, 1978 ofspontaneeus tumor 3. Spiegel A, Erichsen S, Heinicke H, et 22. Nolen G A, Alexander J C: Effects of Res 34:2419-2420, 1! al: ICLA recommendations for the specification of the diet and type of nesting material on the reproduction 41. O'Gal animals, the husbandry, and the techniques used in and lactation of the rat. Lab Anita Care 16:327-336, selected plant mater' animal experimentation. Z Versuchstierkd 20:222- 1966 28:2272-2275, 1968 223, 1978 • 23. Eisen E J: Comparison of two cage 42. Aches 4. Porter G: Bedding and nesting mate- rearing regimes on the reproductive performance E,etal:Nasal cancer rials. In The ATA Manual of Laboratory Animal and boay weight of the laboratory mouse. Lab Anita industry.Br Med J Practice and Techniques, p 109-106, Short D J, Care 16:447--453, 1966 43. Reyr Woodnot D P, Ed, Charles C Thomas, Springfield, 24. Hickey T E_ Tompkins E C: Housing Ashburn L L: Multi] 1969 mice in a caging system with automatic flushing, Lab inbredmice exposedt 5. Arrington L R: Introductory Labora- Anita Sci 25:289-291, 1975 oxide.J Natl Cancer 44. Weat] tpry Animal Science, Interstate Printers and Pub- 25. Cline E M, Sojka N J: Mouseproduc- fishers, Danville, 1978 tion: Comparing two caging methods. Lab Anim ammonia fumes by in 6. Simmons M L, Brick J 0: The Labora- (New York) 6:26--29, 1977 81:300--301,1952 tory Mouse. Selection and Management, Prentice- 45. Brode: 26. Ritter M L: Spontaneously hyperten- J E: The role of envil Hall, Englewood Cliffs, 1970 sive rats. Lab Anita (New York) 3:28-29, 1974 7. Anonymous: Charles River Breeding l_V6mycop lasmosiso Laboratories: Bedding for laboratory rodents. 27. Committee on Care and Use of Spon- taneously Hypertensive Rats, Institute of Laboratory 46.Lind_ Charles River Di_. 4:1965 8. Polley S M: Housing requirements. Animal Resources, National Research Council: Spon- Physical,chemical, General considerations. In Handbook of Laboratory taneously hypertensive (SHR) rats: Guidelines for biologicresponse.In Animal Science, Vol 1, p 21-60, Melby E C, Altman N breeding care and use. ILAR News 19:G1--G20, 1976 31--43,Instituteof l H, Ed, CRC Press, Cleveland, 1974 28. Edinger R, Giovanella B C: Current National Academy oI 9. Porter G: Bedding for laboratory ani- knowledge of breedfng and mass production of the 47.Cars_ mals. In Notes for Breeders of Common Laboratory nude mouse. In The Nude Mouse in Experimental and ylamine and dicycl, Animals, p 136--138, Porter G, Lane-Petter W, Ed, Clinical Research, p 15-28, Academic Press, New Chem 29:1247-1251, AcademicPress, New York, 1962 York, 1978

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29.Subcommittee on Standards forGno- 48. Kon S H: Underestimation ofchronic tobiotes,Committee on Standards, Institute of toxicitiesoffoodadditivesand chemicals:thebiasofa Laboratory Animal Resources, National Research phantom rule.Med Hypotheses 4:324-339, 1978 Council:Gnotobiotes,National Academy ofSciences, 49. Zwarun A A, BalishE: Development Washington, 1970 ofa feedand bedding sterilization-pasteurizatlonin- 30. Smith J C, Schwarz K: A controlled dicator.Abstract 72, Publication79-4, American environment system for new trace element defi- AssociationforLaboratory Animal Science,1979 ciencies.J Nutr 93:182-188,1967 50.Weisbroth S H: Chemical contamina- 31. Mohr H E, Hopkins L L Jr: An all tion of lab animal beddings: Problems and recom- plasticsystem for housing small animals in trace mendations,Lab Anita (New York) 8:24-34,1979 element studies.Lab Anita Sci 22:96-98, 1972 51.Nash P: Problem solving:holdingrab- 32.NielsenF H, BaileyB:The fabrication bitsand mice in the small researchlab.Lab Anita ofplasticcages forsuspensioninmass airflowracks. (New York) 6:46--48,1977 Lab Anita Sci 29:502-506 52.Bryant J M, Rezmiarek H: Ammonia E 33. Vesell E S, Lang C M, White W J, et al: controlpotentialoffiverodentcontactbeddings.Ab- nvironmental and genetic factors affecting the re- stract34, Publication79-4, American Association sponse of laboratory animals to drugs. Fed Proc forLaboratory Animal Science,1979 35:1125-1132, 1976 53. Les E P: Reduction of ammonia in 34. Pick J R, Little J M: Effect of type of mouse cages.Abstract 70, Publication79-4, Amer- bedding material on thresholds of pentylenetetrazol ican Association for Laboratory Animal Science, convulsions in mice. Lab Anita Care 15:29-33, 1965 1979 35. Whelan G: The influence of cage bed- 54. Baldwin C L, Sabel F L, Henke C B: ding on the metabolism of sulfobromophthalein Bedding disposalcabinetforcontainment ofaerosols sodium by an hepatic cytosol located enzyme system. generated by animal cage cleaningprocedures.Appl Aust J Biol Sci 28:25-30, 1975 Environ Microbiol 31:322-324, 1976 36. Burkhardt C A, Robinson J L: High 55. Carpenter R H: Biological safety rat pup mortality attributed to the use ofcedar-wood cabinet modified for animal manipulations and bed- shavings as bedding. Lab Anita (London) 12:221- ding disposal. Abstract P 1, Publication 79-4, Amer- 222, 1978 ican Association for Laboratory Animal Science, 37. Matsushita H, Matsumoto H, Ando H 1979 et al: Influence of polypropylene fiber on growth and 56. Rake B W: Microbiological evaluation hematologic values in rats. Bull Azabu Vet Coll of a biological safety cabinet modit]ed for bedding 30:179-186, 1975 disposal. Lab Anita Sci 29:625-632, 1979 38. Sabine J R, Horton B J, Wicks M B: 57. Newberne P M, Fox J G: Chemicals Spontaneous tumors in C3H-A vy and C3H-AvYfB and toxins in the animal facility. In Laboratory mice: High incidence in the United States and low Animal Housing, p 118-138, Institute of Laboratory incidence in Australia. JNatl Cancer Inst 50:1237- Animal Resources, National Academy of Sciences, 1242, 1973 Washington, 1978 39. Heston W E: Testing for possible 58. Darlow H M: Disposal of infective effects of cedar Juniperus virginiana wood shavings laboratory materials. Soc Appl Bacteriol Symp Ser and diet on occurrence of mammary gland tumors 1:281-289, 1971 and hepatomas in C3H-A and C3H-A'_F3 mice. J Natl Cancer Inst 54:1011-1014, 1975 40. Schoental R: Role of podophyllotoxin in the bedding and dietary zearalenone on incidence of spontaneous tumors in laboratory animals. Cancer Res 34:2419-2420, 1974 Questions and Answers 41. O'Gara R W: Biologic screening of selected plant material for carcinogens. CancerRes Question from Robert Whitney Jr, 28:2272-2275, 1968 National Institutes of Health 42. Acheson E D, Cowdell R G, Hadfield Do you feel that all bedding should E, et al: Nasal cancer in woodworkers in the furniture industry. Br Med J 2:587-596, 1968 be sterilized? 43. Reyniers J A, Sacksteder M R, Answer by L M Kraft Ashburn L L: Multiple tumors in female germfree Yes. inbred mice exposed to bedding treated with ethylene oxide. J Natl Cancer In.st 32:1045-1056, 1964 Question from R Whitney 44. Weatherby J H: Chronic toxicity of Do you feel that there may be prod- mm'nonia fumes by inhalation. Proc Soc Exp B iol Med 81:300-301, 1952 ucts or by-products in some ofthe bedding from 45. Bredersen J R, Lindsey J R, Crawford sterilizationitselfthat may be a problem? J E: The role of environmental ammonia in respira- Answer by L M Kraft ltoQr_mycoplasmosis of rats. Am JPatho185:1 I5-130, Yes. There has really been so little 46. Lindsey J R, Conner M W, Baker H J: work done on bedding that we don't know very Physical, chemical, and microbial factors affecting much about it.It'sperfectly true that there are biologic response. In Laboratory Animal Housing, p 31--43, Institute of Laboratory Animal Resources, some beddings on the market, the heat treated National Academy of Sciences, Washington, 1978 ones, that are so low in bacterial count that itis 47. Carswell T S, Morrill H L: Cyclohex- ylamine and dicyclohexylamine. Industrial Eng hard to find the bugs, but there they are. Ijust Chem 29:1247-1251, 1937 think it'san open question.

374 Vol 30, No 2 Part II of Two Parts Rodent Bedding Printed in USA

Comme Comment by Edwin P Les, The There aresome papers on thisas you doubtless know. Here, too,they'renidicolousanimals, versityofWiscons Jackson Laboratory I'dlike I'd like to comment a little bit about and givingthem a cottonwad or an oldcompu- things we see vet. the behavior aspect of bedding. One of the terprintoutthat'sbeen shreddedor anything most prestigious things that at least mice require to lead a nor- likethat,anything that'sinthe form ofa shred mice.Isuspecttha mal life is nesting material, but more than orsomething thatthey'recapableofshredding, that, they have to build nests. They are nest seems to turn them on to nestbuilding. builders, and they need to have material that they can lug around in the cage and build a nest Comment from Gordon W Newell, with. In a study that I was doing with bedding, I National Academy of Sciences was testing a pelletted bedding and comparing Dr Kraft, you mentioned one paper it with white pine shavings. I found that there that we also are interested in but in a different was a bizarre type of behavior going on in the manner, and I bring this up only so that we cage that had the pelletted bedding. This be- don't always jump to too many conclusions. havior consisted of two mice standing head to This is a rather interesting and extensive paper toe so to speak, front to rear, each mouse pick- done in England on the incidence of tumors ing up the tail of the mouse in front of it and among furniture workers, and the kinds of na- walking around in a circle. Not only that, but sal tumors they have developed are described sometimes even a single mouse in a cage would as being very similar to tumors which have pick up its own tail and walk around in a circle. been shown to occur in an on-going experiment I thought that this was so unusual that I con- currently underway in exposing mice and rats tacted our art and photo department to have to formaldehyde. Formaldehyde is also, as you movies made of this, but in the meantime I did know, a component ofmany urea formaldehyde what people should do and looked in the library resins, and there is much interest in this at the and found that this was very well described by moment. Whether or not these particular furni- people who are studying animal behavior. They ture workers are involved with urea formal- ascribed this abnormal behavior to the lack of dehyde resins is something we are going to nest building material and the animal's en- attempt to try to investigate with the people in vironment. They just have to have something England. However, there are these possible to build a nest with. If you deprive them of it, other types of interpretations of information they're not normal any longer. that develops in the laboratory, and I bring it up because life is so complex today, and it's not Comment by L M Kraft easy to jump to a conclusion and be sure that I have not done any work on it,but I one iscorrect. would notbe surprisedtofindthatthefactthat the aspen material was in shreds,ofwhich a nestcan easilybe made, isthe reason the ani- Question from Bob Sediacek, Mas- mals liketheaspenexcelsiormaterialsomuch. sachusetts General Hospital It'seasierforthem to builda nest out of it. I have observed the same thing that Dr Les alluded to with a similar type bedding, Comment by Jim Halkett, New but I have another question. I'd appreciate your England Nuclear comments on the use of antibiotics in bedding. Many years ago, we were breeding This seems like a two-edged sword. We've some mice, and to each cage we added one sheet already got more medication resistant bugs of kleenex, and the mice had a ball. They just than we know what to do with now. shredded the thing up and got their young in amongst it, and it was just terrific. Probably Answer by L M Kraft most of you have tried this, but it is a 100 The people with whom I speak don't percenter. want it.The only way they would have itis under a suspended Wire cage in the cardboard Comment by L M Kraft substance.But in the contactbedding, most One of the things that I did not men- peoplewith whom I have spoken don'twant it tion because I didn't want to go overtime was at allbecause itusuallyinterfereswith their trying to breed wild rodents in the laboratory. experiments. 376 375 Vol 30, No 2 Part II of Two Parts Printed in USA

Comment by Ralph Anslow, Uni- the source. As you heard what Dr Kraft said versity of Wisconsin about storage of shavings from furniture fac- I'd like to comment on one of the tories, most are blown out of the facility where things we see very commonly in some of our they may become nesting spots for wild rodents most prestigious areas and that is mites on as well as birds. I think this is a logical explana- mice. I suspect that bedding may very likely be tion of the source of mites.

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