The in vitro enzyme-inducing and cytotoxicproperties of South African laboratory animalcontact bedding and nesting materials

F. J. Potgieter1, R. Torronen2 & P. I. Wilke3

'Animal Unit G20, Faculty of Medicine, University of the Orange Free State, PO Box 339, 9300Bloemfontein, Republic of South Africa, 20epartment of Physiology, University of Kuopio,PO Box 1627, FIN-70211 Kuopio, Finland and 30epartment of Animal Science, University of theOrange Free State, South Africa

SummaryEnzyme-inducing and cytotoxic effects of South African bedding materials wereinvestigated using a mouse hepatoma cell line, Hepa-1, cell culture system. This cellculture system is a convenient and sensitive method for the screening of beddingmaterials for the presence of compounds that could be potentially harmful to animals andthus the experimental outcome. Cells were exposed to acetone extracts of the differentmaterials or their components. Corn cobs displayed very little or no CYP1A1-inducing orcytotoxic effects, whilst vermiculite and unbleached pulp from pine and eucalyptusshowed greater induction and cytotoxic properties. The latter properties were lower thanthose produced by the different recycled paper extracts. Pine shavings (Pinus elliottii) andthe different wood components making up industrial sawdust expressed the highestcytotoxic and CYP1A1-inducing properties.

Keywords Laboratory animals; direct or contact bedding material; wood shavings; sawdust;paper; com cobs; vermiculite; cytotoxicity; enzyme induction; CYP1A1; cytochrome P450

Information obtained on the type oflaboratory animal bedding and nestingmaterials used by South African animalfacilities (Potgieter & Wilke 1993) showedlittle knowledge of the composition of thematerials and their possible effect onexperimental animals and thus experimentaloutcome.

This problem is not limited to South Africa[SAl. Deficiency in knowledge of beddingis also illustrated by the vague specificationsand requirements set by laboratory animalregulatory organizations (Guide forLaboratory Animal Facilities 1968, NIHSTD No.4 1983, Canadian Council onAnimal Care 1984, NIH-13-107j 1984,European Convention 1985L resultingin the continued incorporation ofpotentially harmful materials in theexperimental design. It is well-documentedAccepted 21 April 1994

that hydrocarbons present in wood-derived bedding materials can inducecytochrome P450-dependent microsomalenzyme systems of animals (Pick & Little1965, Ferguson 1966, Veselll967, Fujiiet al. 1968, Wade et al. 1968, Sabine 1975,Cunliffe-Beamer et al. 1981, Weichbrodet al. 1988, T6rr6nen et al. 1989), resultingin altered drug metabolism.

The latter predicament is probably theresult of the absence of a single beddingmaterial that conforms to all desirablecriteria set for rodent contact beddingmaterial (Kraft 1980L one that couldonly be eradicated if the lack of dataon the biological influences of differentbedding materials is addressed. Thusa more suitable bedding material couldbe selected, improving its compatibilitywith the experimental objective.

Laboratory Animals (1995) 29, 163-171

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Since very little was known, a studywas conducted to find out more aboutthe properties of South African contactbedding and its effect on laboratoryanimals. Enzyme-inducing and cytotoxiceffects of several bedding and nestingmaterials were evaluated using the invitro method described by Torronenet 01. (1989). Cultures of the mousehepatoma cell line Hepa-l were exposedto acetone extracts of the bedding andnesting materials and their components.After the exposure, cytotoxicity [effectson the viability of cells) and CYPIAI(cytochrome P4501Al) induction weremeasured. The Hepa-l cell line was usedbecause it exhibits high CYPIAIinducibility by several model chemicals(Karenlampi et 01. 1989, Karenlampi &Torronen 1990). CYPIAI is the only formof cytochrome P450 known to be expressedin this cell line. Although representingonly a minute fraction of the overallcytochrome P450-dependent drugmetabolism, the Ah receptor-mediatedCYPIAI enzyme activity has been shownto have an important role in toxicity,carcinogenesis, mutagenesis andteratogenesis caused by environmentalchemicals (Nebert 1989).

Materials and methods

Bedding materialsBedding materials specified by users from aprevious survey (potgieter & Wilke 1993)were investigated. These were: exfoliatedvermiculite, corn cobs, wood shavingsblpurpose manufactured bedding from virginpine (Slash pine, Pinus elliotti)), 4 kindsof used paper (printed paper (n = 2),plotter paper (n = 1) and photocopy paperIn = 1)), and industrial sawdust [a mixtureof wood shavings and sawdust from acombination of different tree speciesutilized in the building and furnitureindustries).

"VicafilVermiculite™, Mandoval Vermiculite(PTY)LTD, PO Box4114, 1450Alrode, RSA.bByproductDevelopment Services, PO Box 549,4320 Umhlanga Rocks, RSA.

Potgieter, Torronen & Wilke

The shredded computer print wasincluded in this study as 11% of thefacilities mentioned in the previous survey(Potgieter & Wilke 1993) used it fornesting purposes. The computer-associatedpapers included were: white continuousprinter paperd (PWP), blue bar continuousprinter papere (PBP), photocopy paper! (PHP)and plotter paperg (PLP). Before testing, thematerials were soaked in hot water [60°C),mechanically pulped, excess water removed,washed twice with hot water, granulatedby hand and allowed to dry in an autoclave.Two additional samplesc, namely unbleachedpine pulp [consisting of Pinus elliottii,P. patula and P. taeda) and eucalyptus pulp(predominantly Eucalyptus grandis) werealso investigated. This was because non-bleached paper has been shown to be lessinductive than bleached paper, whilerecycled paper towels expressed strongerinducing properties than their bleachedcounterparts (Karenlampi & Torronen 1990).

Industrial sawdust which was included as21% of the contact bedding used in SouthAfrica comes from wood destined primarilyfor the building industry. These materialsare preserved against fungi, termites andbeetles, and thus contain additionalpoisonous compounds. The main types ofwood present in industrial sawdust areMeranti or Philippine mahogany (Shoreaspp.) and SA Pine, but types may varyaccording to the demand of the buildingindustry. Wood shavings and sawdustoriginating from major timber distributorsmay, therefore, also include traces ofYellow tongue, 1mbuia (Ocotio sp.) andSaligna (Eucalyptus saligna). It is doubtfulif wood designated as Saligna comes onlyfrom this species because at least 14different Eucalyptus species are used forafforestation in SA. Bison board and Supawood are manufactured from wood chipsand fibres from a variety of trees, whilst

CSappiKraft (PTY)LTD, 1209PO NgodwanaRSA.dparagonBusiness Forms, Roodepoort,RSA.cBarlanForms, Port Elizabeth, RSA.fRotatrim, Mondi Paper, PO Box 62290, 2107Marshalltown, RSA.gPerformPlot, Hi Performance Systems, HewlettPackard, RSA.

Enzyme-inducing and cycotoxic properties of bedding and nesting materials 165

SA Pine may come from Pinus elliottii,P. patula, P. pinaster, P. radiata, orP. taeda. Samples of these different treatedwood types were obtained from acommercial retailerh. It was impossible toidentify which pine species were involved.

Cottonwooli was included as it was usedas a control bedding material underneathwire floor inserts.

Experimental approachPreliminary experiments were carried out todetermine suitable assay concentrations forthe different samples. In the final experimentsfive concentrations of each sample werestudied in order to show that the responseis dose-dependent. Due to the largenumbers of samples In = 18) andconcentrations tested, replicate assayscould not be run.

It was not possible, using this testprocedure, to make accurate and statisticallyrelevant comparisons between the individualsamples. Instead this series of experimentsrather serves as a screening procedure toshow the presence of cytotoxic andenzyme-inducing compounds in thesamples tested. Only rough estimates aboutthe cytotoxic and enzyme-inducingpotencies of the samples are obtained. Suchresults can be used, e.g. for the eliminationof the most toxic materials. For accurateranking of the toxic potencies of 2 or morebedding materials a different experimentaldesign should be used.

MethodsThe methods described by Torronen et al.(1989) for the in vitro evaluation andcomparison of bedding materials for cytotoxicand CYPIAl-inducing properties were used.Acetone extracts corresponding to 5 or 10 gof the original bedding material in 1mlsolution were prepared.

For the cytotoxicity assay 1: 1 dilutionseries was made on a 24-well culture plateand incubated with Hepa-l cells (25,000

hFT Building Supplies, PO Box 872, 9300Bloemfontein, RSA.iJohnson & Johnson, East London, RSA.

cells in a final volume of 1ml in eachwell) at 37°C for 72 h.

Survival of the cells was estimated byassaying the total protein content in eachwell, using the method of Bradford (1976).iThe results are expressed as percentages ofthose of the control wells. Approximatemedian effective doses (EDso), indicatingthe sample concentrations that killed 50%of the cells, were calculated from the dose-response curves. 2,4-Dinitrophenol wasused as a reference compound in thecytotoxicity assays.

Cytotoxicity data were used to select theconcentration ranges for the enzymeinduction assays which cannot beperformed at toxic sample concentrations.The CYPIAI induction assays (one sampleof each bedding type at the pre-determinedconcentrations was used) were carried outas described by Torronen et al. (1989).Indcction of CYPIAI was detected as anincrease of aryl hydrocarbon hydroxylase(AHH) activity.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin(TCDD) was used as as reference (or model)inducer. Hepa-l cell cultures were exposedto InM TCDD, producing maximal AHHinduction [Karenlampi &. Torronen 1990).

ResultsExposure of Hepa-l cultures to extracts ofthe bedding and nesting materials resultedin 2 types of findings. First, the extractscaused cell death, i.e. they were cytotoxic.Secondly, they elicited an increase inCYPIAl-mediated AHH activity.

For the preliminary experiments acetoneextracts, corresponding to 10 g of theoriginal material in 1ml of solution, wereprepared. When subjected to thecytotoxicity assay 2 of the samples, corncobs and vermiculite, showed lowcytotoxicity with their EDso value higherthan 50 mg/ml (the highest concentrationpossible in this assay for extracts of 10 mg/ml).The EDso for unbleached pulps wasapproximately 40 mg/ml. For the rest of thesamples EDso was smaller than 15 mg/ml;

iBio-RadAssay, Bio-Rad Laboratories, Richmond,CA, USA.

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for the most toxic samples (wood shavingsand wood types present in industrialsawdust) the EDsocould not be properlydetermined. Since the extracts of 10mg/mlappeared to be too concentrated for themajority of the samples, extractscorresponding to 5g of the original materialper ml were prepared for the finalcytotoxicity assays. These results arepresented in Table 1. According to theresults of the preliminary and finalexperiments, the approximate EDsovaluesobtained for the cytotoxic properties of thedifferent bedding types divided thematerials into three groups: Group I withlow cytotoxicity (EDso>25 mg/ml)j GroupII with intermediate cytotoxicity (EDso= 5to 15mg/ml) and Group ill with highcytotoxicity IEDso<5 mg/ml).

In Group I, corn cobs showed practicallyno cytotoxicity over the concentrationrange studied. Vermiculite and theunbleached pulps were somewhat moretoxic than corn cobs. In Group II, therecycled paper products PWP and PBPappeared to be slightly more cytotoxic thanPLP and PHP. Preserved wood typespresent in industrial sawdust were clearlymore cytotoxic than the pulp and recycledpaper products, Supa wood and Yellowtongue (Group II) being less toxic than therest (Group ill). Wood shavings,manufactured primarily for use aslaboratory animal bedding, were amongstthe most toxic materials. The EDsovalueobtained for 2, 4-dinitrophenol, whichserved as a positive control, was46±6JLg/ml (n=6).

During the preliminary experiments theAHH-inducing effects of vermiculite, theunbleached pulps and the recycled paperproducts were determined twice. The finalsample concentrations used in these assayswere 5, 10, 25, 35 and 50mg/ml and I, 5,10, 15 and 35 mg/ml. The increasing orderof the AHH-inducing potency of thesesamples was vermiculite, pine pulp, PBP,eucalyptus pulp, PLP, PWP and PHP.However, the recycled paper products werefound cytotoxic during the induction assayat concentrations higher than 10mg/ml.Since cytotoxicity interferes with the

Potgieter, Terrenen & Wilke

Table 1 Cytotoxicity is expressed as percentagesurvival of Hepa-1 cells after exposure to differentconcentrations (mg original material in one mltissue culture medium) of the samples. Beddingand nesting material is arranged from the top ofthe table according to increasing cytotoxicity

Concentration (mg/ml)Beddi~~/nesting

0 1.6 3.1 6.2 12.5 25materia

Control (Solvent) 100 102 100 10B 110 9B

Group 1[EDso>25 mg/ml]Corn cobs 100 109 107 105 111 89Vermiculite 100 88 91 89 72Eucalyptus pulp 100 94 83 82 63Pine pulp 100 100 90 77 54

Group II[EDso=5-15 mg/ml]

PLpa 100 69 64 60 43 23PHpa 100 71 55 51 35 12Cotton wool 100 69 57 42 29 3PBpa 100 75 64 40 26 2PWpa 100 70 62 44 21 1Supa woodb 100 102 106 84 40 0Yellow tongue 100 102 100 76 24 0

Group III

[EDso<5 mg/ml]

Sterilized wood 100 68 40 12 0shavings

Merantib 100 82 56 6 0SA Pineb 100 78 46 2 0Imbuiab 100 67 24 0Bison board 100 66 24 0Eucalyptusb 100 23 0Non-sterilized 100 20 0wood shavings

aRecycledpaper productsbpreserved wood types that could be present in industrialsawdust

determination of the enzyme-inducingpotency, the final AHH-induction assays werecarried out at non-toxic concentrations, i.e.different concentrations were used forGroup I, Group II and Group ill materials.

For the AHH induction studies of GroupI materials, sample concentrations up to50 mg/ml could be used. A concentration-dependent increase in AHH activity wasseen for all the materials (Table 21. Theinduction effect measured was the smallestfor corn cobs, whilst eucalyptus pulpproduced higher AHH activities than pinepulp and vermiculite.

Enzyme-inducing and cycotoxic properties of bedding and nesting materials 167

Table 2 Effect of Group I bedding and nestingmaterials on the AHHactivity of Hepa-1 cellcultures. Concentration is expressed as mg originalbedding or nesting material in one ml tissueculture medium, AHHactivity as pmoles 3-0H-benzo(a)pyrene formed/min/mg protein

Bedding/nesting Concentration (mg/ml)materialGroup I 0.5 10 25 35 50

Control (Solvent) 1.6 1.7 2.1 2.2 2.1Corn cobs 3.8 3.5 6.4 9.5Vermiculite 3.3 4.2 5.4 12.3 13.7Pine pulp 4.4 5.7 8.2 14.9 16.7Eucalyptuspulp 5.0 8.8 17.4 19.9 25.2

Sample concentrations as high as thoseused for the Group I materials could not beused for the Group IT and ill materials dueto higher cytotoxicity. The recycled paperproducts PBP and PLP produced almost noinduction of AHH activity when concentrationsup to 10 mg/ml were studied, whilst PWPand especially PHP were more inductive(Table 3). Supa wood, Yellow tongue, Imbuiaand Bison board, some of the preserved woodtypes present in industrial sawdust, had amore pronounced AHH-inducing effect(Tables 3 and 4) than that evoked with therest of Group III materials lonly a verysmall effect could be measuredllTable 4).The latter were probably due to the lowsample concentrations used.

The maximal AHB activity produced bylnM TCDD was 46.l±2.7(n=6) pmolesof 3-0B-benzo [a] pyrene formed/min/mgprotein.

Table 3 Effect of Group II bedding and nestingmaterials on the AHHactivity of Hepa-1 cellcultures. See the legend in Table 2

Bedding/nesting Concentration (mg/ml)materialsGroup II 0.5 2.5 5 10

PBP' 1.6 2.4 2.8 3.7 4.5PLP' 2.0 2.4 3.8 2.7 5.3Cotton wool 2.2 1.9 3.5 4.5 6.5PWP' 1.8 2.8 6.5 6.3 9.7Supa woodb 2.9 3.6 8.0 15.8 19.9PHP' 1.2 7.1 10.9 18.4Yellowtongue 3.0 4.4 10.6 21.9

"Recycledpaper productsbpreservedwood typesthat couldbe present in industrialsawdust

be a useful technique for eliminating thosematerials posing a threat to experimentalresults, especially as researchers try toeliminate as many external variables fromtheir experimental design as possible.

Norris and Adams (1976) found a higherrat pup mortality when paper tissues (53% Irather than woodwool (13%) was suppliedas nesting material. They attributed thisdecline in the number of animals weanedto differences in nest-building and nestmaintenance activities, the latterdecreasing 5 days post-partum. This declinein animals might also be a consequentialcytotoxic effect, especially as Burkhart &Robinson (1978) found similar deviationswith rats raised on different wood derived

Table 4 Effect of Group IIIbedding and nestingmaterials on the AHHactivity of Hepa-1 cellcultures. See the legend in Table 2

Discussion Bedding/nesting Concentration (mg/ml)

Due to the difference between in vivo and materialGroup III 0.5 1.5 2 2.5

in vitro systems, in vitro cytotoxicity andenzyme induction tests might be considered Merantia 1.4 1.1 1.5 1.4 1.5as excessively sensitive, resulting in the Eucalyptus' 1.4 1.4 1.1 1.6 1.6supposition that results found in vitro may SAPine" 0.9 2.1 2.7 3.4 3.9

Sterilizedwood 2.2 2.6 3.4 5.4 4.4not occur in the in vivo system. It could shavingsthen be argued that in vitro methods could Non-sterilizedwood 1.8 3.2 3.3 4.4 5.5not be used to set criteria for animal shavingsbedding and nesting materials. Imbuia" 3.8 8.0

While this may be so, very little is Bisonboard" 3.7 11 10.0 17.6 19.6known about the in vivo cytotoxic effects 'Preservedwood typesthat couldbe present in industrialof bedding materials, in vitro methods may sawdust

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bedding materials. They demonstrated a60% mortality rate for rat pups raised oncedar shavings compared with less than 3%for those raised on corn cobs (here found tobe the least cytotoxic) and aspen. Nomention was made on whether nestingmaterials were supplied. The growth rate ofthe cedar raised pups up to 3 weeks of agewas 80% less than those of the other 2bedding types. According to the latterauthors 'the reduced weight gain andincreased mortality of pups raised on cedar-wood bedding is not known. It appears thatthe pups were ingesting or inhaling acompound which was toxic to them. Thepossibility exists that it may have beentransferred in the dams' milk'. If this istrue, the use of in vitro methods toevaluate the cytotoxic effect of beddingmaterials is potentially informative.

Previous (Torronen et a1. 1989) andpresent in vitro enzyme induction resultsconfirm earlier in vivo results (Pick &Little 1965, Ferguson 1966, Vessell 1967,Fuji et a1. 1968, Wade et a1. 1968,Schoental1973, Sabine 1975, Cunliffe-Beamer et ai. 1981, Weichbrod et a1. 1988)that wood derived bedding materials mayact as enzyme-inducing agents.

The Hepa-1 cell culture method useddetects the Ah receptor-mediated inductionof CYP1Al. However, other drugmetabolizing enzymes such as CYP1A2(cytochrome P4501A21,NAD(PlH:menadioneoxidoreductase, UDP-glucuronosyltransferase and glutathione transferase,may be co-induced with CYP1A1 in animaltissues (Nebert 1989). Therefore, the invitro AHH induction-elicited bylaboratory animal bedding-detected inthe Hepa-1 test system can be regarded asan indication of potential in vivo inductionof other enzymes of the [Ah] genebattery.

Vermiculite and 3 of the plant-relatedproducts (corn cobs and the 2 unbleachedpulps) were the more superior of theproducts tested having the lowestcytotoxicity and inducibility.

Compared with the unbleached pulpsamples investigated, the recycledcomputer paper products (PLP, PHP, PBP

Potgieter, T6rr6nen & Wilke

and PWPJ, all of bleached origin,demonstrated higher cytotoxicity.Unfortunately the enzyme-inducingproperties of these materials could not beproperly compared as their cytotoxicproperties required sample concentrationssmaller than those used for Group Imaterials. PWP and especially PHP,however, appeared to be stronger inducersthan the unbleached pulps. Thiscorresponds with the enzyme inductionstudies of Karenlampi and T6rr6nen (19901,who found that bleached or recycledconsumer paper products were moreinductive than unbleached papers. At theconcentration range tested, the AHH-inducing properties of PBP and PLP seemto be comparable with those of pine pulp.Of the recycled paper products tested PLPappears to be the least effective beddingmaterial, that is as far as cytotoxicity andAHH induction are concerned. It is ofinterest to note that cottonwool showedcytotoxic and enzyme-inducing propertiessimilar to those of the bleached paperproducts.

The Slash Pine [(Pinus elliottiiEngelmannl synonyms: P. beteIOpbylla[Sudw.] i P. caribaea [auct. pro parte. llonMorelet] may contain organic compoundssuch as O!- and l3-pinene (toxic to testbacteria (Kurttio et al. 1990)), camphene,limonene, terpinolene, myrcene, 13-phelandrene, turpentine, rosin and resinacids (Potgieter & Wilke 1992). Any ofthese compounds may result in the adverseenzyme-inducing and cytotoxic propertiesfound with pine shavings. Sterilization ofthe wood shavings considerably reducedtheir cytotoxicity. However, sterilizationappears to have little effect on the enzyme-induction properties of this material. Thefinding agrees with those of Cunliffe-Beamer et a1. (1981). They concluded thatsteam sterilization, (3 pre-vacuum steampulses at 65mm Hg followed by 15min at121°C and a drying cycle of 15 to 20 min)does not significantly alter pentobarbitalsleep times and liver body weight ratios inmice for sterilized mixed hardwood, Whitepine, White spruce and Red cedar beddingmaterials, compared with non-sterilized

Enzyme-inducing and cycotoxic properties of bedding and nesting materials 169

materials of the same source. The differencein cytotoxicity observed between sterilizedand non-sterilized wood shavings is probablythe result of a removal of certain morevolatile chemical compounds throughevaporation [heat and vacuum). Enzymeinduction studies showed no major differencesbetween the values obtained for the sterilizedand non-sterilized wood-shaving samples. Itmay be concluded that the cytotoxicity andenzyme induction evoked result fromdifferent chemicals. The large difference incytotoxicity of wood shavings comparedwith unbleached pine pulp (containingwood derived from P. elliottii), is probablybecause of the pulping process whichremoved most of the volatile hydrocarbons.

Most of the wood types contributing tothe composition of industrial sawdustillustrate very high cytotoxic and enzyme-inducing properties. The South AfricanBureau of Standards (SABSI (1980) specifiesthat industrial timber should be preserved,or protected, against destruction by fungi,insects and marine borers. Theseprotectants and preservatives include woodpreserving creosote, tributylin oxide-lindane, pentachlorophenol,pentachlorophenol-zinc naphtenate,mixtures of copper-chromium-arseniccompounds and boron timber preservatives.The latter is non-toxic to humans and isthe only preservative recommended for usewith wood that will be in direct contactwith feed for livestock. It readily leachedout of wood so it has a very limitedapplication as a preservative and is onlyused for interior wood applications. Thelatter practice is further diminishedbecause boron preservative does not protectwood against subterranean termites. Theuse of coal-tar creosote and woodpreservatives with a coal-tar creosote basis,mixtures of copper-chromium-arseniccompounds, or pentachlorophenol or itssalts as a preservative for wood that will bein continuous direct contact with animalsis not recommended. Industrial sawdustcould, therefore, contain any concentrationor combination of the latter poisonouspreservatives, harmful to the animal.However, it is questionable whether the

wood preservatives used, were the onlyfactor resulting in the cytotoxic nature ofthese materials because no virgin material,except the wood shavings originating fromP. elliottii, were available for comparison.The latter, compared with SA Pine,demonstrated more cytotoxic properties,but it could be that another pine specieswith less cytotoxic properties was involved.These high cytotoxic and enzyme-inducingproperties of industrial sawdust, plus theconstant changes in composition, make theuse of this undefined material for beddingapplications, undesirable.

Conclusion

It is advisable to use those bedding andnesting materials evoking the leastcytotoxic and enzyme-inducing effects toobtain the most reliable results possible forany experimental animal study.

The results clearly demonstrate thatsome bedding materials currently used inSouth Africa should be eliminated. Industrialsawdust and purpose-manufactured woodshavings from P. e1liottii (Group IIImaterials) are highly cytotoxic and mayresult in enzyme induction greater thanfound with Group I bedding materials.Shredded recycled paper (Group IImaterials), with intermediate activity, mayalso cause adverse effects on theexperimental animal, including theviability of their litters.

Group I materials are the best source ofbedding and nesting materials based uponenzyme-inducing and cytotoxic properties.Corn cobs show the lowest effect, makingit the best bedding material currently used.It has, however, seasonal availability andbecause of mechanization of the agriculturalsector with this type of combine harvesters,the availability of this product is verylimited. It may also become contaminatedby pesticides used in the agricultural sector.

Vermiculite, the second best material inthis study may become an animal houseenvironmental pollutant because of its highdust content and inclinations towardsfurther dust formation due to the animals'action on it during direct bedding applications.

170

Hastings (1967) reported a reduction inlitter size and histological lung changesin mice maintained on this bedding.This material may be detrimentalto the health of both animal andtechnician.

Unbleached pulps may, therefore, be thechoice for direct (contact) beddingapplications. It should, however, beinvestigated further before finally decidingon its suitability as a direct beddingmaterial.

Acknowledgments The skilful technical assistanceof Ms Ecva-Liisa Palkispiiii is gratefullyacknowledged. We are also indebted to theDepartment of Physiology, University of Kuopioand the Department of Animal Science, Universityof thc Orange Free State for financially supportingthis study. We would also like to thank thepcrsonnel of the Ngodwana paper and pulp mill(Sappi Kraft (PTYI LTD) for their co-operationin providing the unbleached pulps for thisstudy.

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