Disposition of [Ring-U-14C]styrene in Rats and Mice Exposedby Recirculating Nose-Only Inhalation

P. J. Boogaard,*,1 K. P. de Kloe,* S. C. J. Sumner,† P. A. van Elburg,* and B. A. Wong†

*Department of Molecular Toxicology, Shell Research and Technology Center, Amsterdam, Shell International Chemicals B.V., P.O. Box 1030BN Amsterdam, The Netherlands; and†Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709

Received April 19, 2000; accepted July 18, 2000

The disposition of styrene was studied in a group of 12 SpragueDawley rats and two groups of 30 CD1 mice exposed separately to160 ppm [ring-U-14C]styrene of high specific radioactivity of 1.92TBq 3 mol–1 (52 Ci 3 mol–1) for 6 h. A nose-only exposure systemwas successfully adapted to (1) recirculate a portion of the flow tolimit the amount of 14C-styrene required, and (2) avoid any poly-merization of the compound. The mean uptake of styrene in ratswas 113 6 7 mmol 3 kg–1 3 h–1 and stable over time. The meanuptake in mice was higher, 189 6 53 and 183 6 76 mmol 3 kg–1

3 h–1, for the first and second mouse inhalation experiment, butdecreased steadily over time. Some of the mice, but none of therats, showed signs of overt toxicity. The overall excretion of sty-rene and its metabolites was quantitatively similar in rats andmice. Urinary excretion was the primary route of excretion whilefecal excretion accounted for only a very small part of the radio-activity. There was, however, a significant difference between miceand rats in the exhalation of 14CO2, which must have resulted fromopening and subsequent breakdown of the aromatic ring. In micethe exhalation of 14CO2 accounted for 6.4 6 1.0 and 8.0 6 0.5% ofthe styrene retained during the first and second mouse inhalationexperiment. In rats, exhalation of 14CO2 accounted for only 2.0 60.7% of the retained styrene. Together with the results from thequantitative whole-body autoradiography (showing significantlyhigher binding in mouse lung and nasal passages compared to rat)the larger production of 14CO2 might be indicative of the formationof reactive ring-opened metabolites in the mouse lung, which, inturn, might be related to the observed development of bronchioal-veolar tumors and nasal effects in mice exposed to styrene.

Key Words: styrene; inhalation exposure; disposition; autora-diography; Sprague Dawley rats; CD1 mice; metabolism; CO2;urine; feces.

Styrene is one of the most widely used monomers in thepolymer production industry. Its major use is in the productionof polystyrene, resins, paints, and synthetic rubbers and in thereinforced plastic industry (Milleret al., 1994). Human expo-sure occurs mainly in the workforce handling styrene mono-mer. The highest occupational exposures have been measured

in reinforced plastic industries. Styrene is an irritant that maydepress the peripheral and central nervous systems and causehepatotoxicity and pneumotoxicity in experimental animals(Bond, 1989; Gadberryet al., 1996; Sumneret al., 1997).

One of the most important pathways in the mammalianmetabolism of styrene is oxidation to styrene-7,8-oxide (SO),an established bacterial and mammalian mutagen. Despite thefact that SO is rapidly detoxified by epoxide hydrolase cata-lyzed hydrolysis and glutathione-S-transferase catalyzed con-jugation, the genotoxicity of SO has raised concern about thepotential carcinogenicity of styrene (IARC, 1994a,b). A criticalreview of 11 long-term carcinogenicity studies in animalsrevealed a number of adverse effects in the mouse but not inthe rat, and the reviewers concluded that the evidence forstyrene carcinogenicity was, at best, equivocal (McConnell andSwenberg, 1994). Some cases of leukemia and lymphoma instyrene workers were reported, but invariably these workershad also been exposed to chemicals that are suspect or provenleukemogens, such as butadiene and benzene. Several cohortstudies were conducted in workers from a variety of styreneindustries, but increased incidences of lymphatic and hemato-poietic cancers were not significant or not correlated to styreneexposure levels. IARC evaluated these studies as inadequate indetermining the carcinogenic effect of styrene in humans.Although the same review concluded that only limited evi-dence in experimental animals existed for the carcinogenicityof styrene, styrene was classified as a possible human carcin-ogen (2B), because it is metabolized to SO (IARC, 1994a).

In recent bioassays in Sprague Dawley rats and CD1 mice,nasal toxicity and an increased incidence of masses in thebronchioalveolar region in lungs of mice were reported, butthere were no increases in tumor incidence in rats exposed toup to 1000 ppm styrene (Cruzanet al., 1998; 2000). However,the tumorigenicity of styrene was not related simply to SOconcentrations in blood. SO concentrations were lower in miceexposed to 160 ppm styrene than in rats exposed to 200–1000ppm styrene. However, at a concentration of 160 ppm, styreneis toxic in mice but not in rats.

Clearly, a detailed understanding of the fate and effects ofstyrene is an essential component in the risk assessment of

1 To whom correspondence should be addressed at P.O. Box 162, 2501 ANThe Hague, The Netherlands. Fax:131 70 377 6380. E-mail:peter.j.boogaard@si.shell.com.

TOXICOLOGICAL SCIENCES58, 161–172 (2000)Copyright © 2000 by the Society of Toxicology

161

styrene. Therefore, a series of studies was set up to obtain amore complete and detailed picture of the disposition, metab-olism, and genotoxic potency of styrene in rats and mice duringand following inhalation exposure. The present report de-scribes the inhalation exposure and the overall disposition ofstyrene in rats and mice. In a companion paper (Boogaardetal., 2000), the DNA binding of styrene in liver, lung, andisolated lung cells is reported. A third report, on the metabo-lism of styrene, is being prepared as a separate publication.

[Ring-14C]styrene was used at a high specific radioactivity ofapproximately 1.85 TBq3 mol–1 (50 Ci 3 mol–1) to enable asingle inhalation exposure to a relatively low concentration of160 ppm. Chronic exposure to this concentration led to thedevelopment of bronchioalveolar tumors and signs of toxicityin male mice, although no adverse effects were observed inmale rats.

MATERIALS AND METHODS

Radiochemical synthesis. [Ring-U-14C]styrene was prepared in three stepsfrom [ring-U-14C]benzoic acid (Fig. 1). Initially, a 1.85-GBq (50 mCi) aliquotof [ring-U-14C]styrene with a specific radioactivity of 2.04 TBq3 mol–1 (55.2Ci 3 mol–1), a radiopurity of 96% (major contaminant 4%14C-benzaldehyde),and a chemical purity of 90% (major contaminant 5% benzene) was synthe-sized. This material was tested for its stability under various conditions. Whenstored in capillary tubes at 4°C, rapid polymerization occurred, which wasprobably catalyzed by active hydroxyl groups on the large glass surface.DMSO solutions of the14C-styrene were also unstable. Stored in glass vials inthe dark as a neat liquid in the presence of 100–200 ppm 4-tert-butylcatechol

(pTBC), the14C-styrene was stable for a period of at least 3 weeks with highrecovery of styrene monomer (94%) and no indication of polymerization.Subsequent storage at room temperature for 1 day did not cause any loss (VanElburg, 1998). Based on the results of this stability test, 28.1 GBq (759 mCi)of [ring-U-14C]-styrene was synthesized (Chemsyn Laboratories, Lenexa, KA,USA), in two batches of 19.94 GBq (539 mCi) and 8.14 GBq (220 mCi). Thespecific radioactivity was 1.92 TBq3 mol–1 (52 Ci 3 mol–1) as determined byGC and densitometric analyses (Van Elburg, 1998). The first batch had aradiochemical purity, as determined by high-performance liquid chromatogra-phy (HPLC), of 96.8% with 3.0%14C-benzaldehyde as the major contaminant.The chemical purity, as determined by HPLC and GC, was 96.5%. This batchwas used in two portions of 11.80 GBq and 8.14 GBq, for the rat and firstmouse exposure, respectively. The second batch of14C-styrene had a radio-chemical purity of 96.7%, with 3.2% U-14C-benzaldehyde as the major con-taminant. The chemical purity was 96.4%. This batch of 8.14 GBq was usedfor the second mouse exposure.

Chemicals. Unless stated otherwise, chemicals were of the highest purityavailable. Tetraethyl ammonium hydroxide was purchased as a 20% aqueoussolution from Merck (Darmstadt, Germany). Nitrogen (zero gas,. 99.998%)was obtained from Sunox (Cary, NC, USA) and oxygen (USP grade,. 99.9%)from Holox (Norcross, GA, USA). Styrene for calibration (159 ppm styrene inN2, custom prepared) was purchased from Praxair (Danbury, CT, USA).

Animals. Male Sprague Dawley rats and CD1 mice were purchased at theage of 9–10 weeks from Charles River Co. (Raleigh, NC, USA). Followingacclimatization, the rats were used at an age of approximately 12 weeks (;360g) and the mice at an age of approximately 10–12 weeks (;36 g). Animalswere housed according to standard animal care procedures with free access tofood (NIH-07; Zeigler Brothers, Gardners, PA, USA) and purified water in aclimate-controlled (relative humidity 55%, temperature 226 2°C) room on a12-h light-dark cycle. This study was approved by the Institutional AnimalCare and Use Committee and was performed in accordance with the Declara-tion of Helsinki and the Guide for the Care and Use of Laboratory Animals asadopted and promulgated by the U.S. National Institutes of Health. Theinhalation exposures were short term and were performed in the absence offood and water.

Generation and control of exposure atmosphere.The required atmo-sphere of 160 ppm14C-styrene was generated using a stainless steel pressurevessel system (38.3 l) as shown schematically in Figure 2. After flushing thepressure vessel with nitrogen (zero grade), the14C-styrene was transferred ontothe bottom of the vessel, which was subsequently closed. The styrene was thenallowed to evaporate and diffuse through the pressure vessel overnight. Priorto the exposure, the vessel was pressurized to a predetermined value byaddition of nitrogen (300 l for the rat exposure and 209 l for the mouseexposures). To begin exposure, the nitrogen-styrene mixture was metered intothe nose-only chamber inlet line and mixed with oxygen (USP grade) anddilution air prior to mixing with the recirculating flow and entering thenose-only chamber. The oxygen flow was monitored with a rotameter and thedilution air with a mass flow controller. An IR spectrophotometer (MIRAN1A, Foxboro, MA, USA) served as both a mixing chamber and as a styreneconcentration monitor (vide infra). A portion of the outlet atmosphere wasrecirculated back into the chamber inlet using a stainless steel diaphragm pump(Model MB-41, Metal Bellows Co., Sharon, MA, USA) to push the air flowthrough the recirculation line. A valve upstream of this pump was used tocontrol the flow rate. A condenser was placed in the recirculation line. Chilledwater was passed through the condenser jacket to condense moisture from therecirculating atmosphere. The condensate was collected in a trap in the bottomof the condenser. The recirculated atmosphere was also passed through a trapcontaining SodaSorb to absorb CO2 and through a 60-mm filter to removeparticles. Styrene is not adsorbed to SodaSorb (Leavenset al., 1996). Theportion of the flow that was not recirculated was passed through KOH traps toremove CO2 and through sets of charcoal filters to absorb styrene. Duringexposure, this exhaust flow was adjusted to maintain the static pressure insidethe nose-only chamber at a level approximately equal to or slightly less thanambient atmosphere pressure.

FIG. 1. [Ring-U-14C]-styrene was synthesized from [ring-U-14C]-benzoicacid. The [ring-U-14C]-benzoic acid was reduced with lithium aluminumhydride to [ring-U-14C]-benzyl alcohol with 79% yield. The [ring-U-14C]-benzyl alcohol was subsequently oxidized to [ring-U-14C]-benzaldehyde usingmanganese(IV) oxide with 80% yield. Finally, the [ring-U-14C]-benzaldehydewas converted to [ring-U-14C]-styrene with triphenylphosphonium iodide inbenzene in the presence of aqueous sodium hydroxide, and a trace of 4-tert-butylcatechol to prevent polymerization, with 40% yield.

162 BOOGAARD ET AL.

The styrene concentration in the system was monitored by two independenttechniques. The primary monitoring was by IR spectrophotometry (Sumneretal., 1997). Prior to exposure, the IR spectrophotometer was calibrated, over therange of 0 to 302 ppm using a closed loop technique. In this technique, astainless steel diaphragm pump connected to the inlet and outlet of the IRspectrophotometer circulates the contents of the IR spectrophotometer. Knownvolumes of styrene were injected into the known volume of the recirculatingatmosphere, and the response of the IR spectrophotometer was recorded. Thesecondary monitoring was by gas chromatography. A gas chromatograph (GC)(HP5890 Series II, Hewlett Packard, equipped with a 7 ft3 1/8-inch stainlesssteel 35/60 Tenax column and a flame ionization detector kept at 250°C, elutedwith helium at 20.3 ml3 min–1) equipped with a multiport sampling valve wascalibrated with standards prepared using Tedlart bags filled with a knownvolume of nitrogen into which a known volume of styrene was injected. Inaddition, commercially prepared gas reference standards were used. The GCwas used to monitor the concentration of styrene at the inlet (Fig. 2, 1) and theoutlet (Fig. 2, 2) of the chamber and also after the first charcoal filter (Fig. 2,3) to check for eventual breakthrough. The sensor of an oxygen monitor wasplaced in the outlet stream of the chamber to measure the oxygen. The monitorwas calibrated against room oxygen content, which was assumed to be 21%,prior to each exposure. During the course of the exposure, styrene and oxygenconcentrations were continuously recorded on a strip chart recorder. Thestyrene and oxygen flows were adjusted periodically to maintain 160 ppm ofstyrene and approximately 21% oxygen (Table 1). All flow settings and styrene

and oxygen concentration readings were recorded approximately every 30 minover the course of the exposure.

Inhalation exposure of rats and mice. Exposures were conducted inCannon-type nose-only exposure towers (Cannonet al., 1983) constructedfrom inert materials (stainless steel with Teflon delivery lines) with 52 posi-tions (Lab Products, Maywood, NJ, USA). The tubes were blocked at unusedexposure positions. Prior to starting the exposure, the recirculating pump wasturned on and allowed to run for about 30 min to warm up. The recirculatingflow was measured with a flow monitor and adjusted to give the desiredrecirculation. Oxygen flow was started prior to loading the animals, and allflows except for the pressure vessel flow were set to their predeterminedvalues. Once the animals were installed in the exposure tower, the pressurevessel flow was started. As that flow increased, the dilution air was decreasedproportionally. The exposure began when the styrene flow started. The expo-sure was ended by shutting off the pressure vessel flow and increasing thedilution air flow to maintain the pressure balance in the chamber. The flowswere maintained until the styrene concentration dropped to zero. After theconcentration reached nearly zero, the animals were removed from the expo-sure tower. The exposure groups consisted of 12 rats or 30 mice. Two separateinhalation experiments were conducted on mice (i.e., 60 mice total). Theselarge numbers of animals were required to obtain sufficient material for DNAadduct analysis (Boogaardet al., 2000). In addition, 2 rats and 5 mice servedas controls and did not receive treatment.

Disposition studies. Immediately after cessation of exposure, half theanimals (6 rats and 15 mice in each exposure experiment) were killed for

FIG. 2. Schematic representation of the recirculating nose-only exposuresystem.

TABLE 1Exposure Conditions and Rate of Uptake

(values are means 6 SE)

Study 1 Study 2 Study 3

Species and number 12 rats 30 mice 30 miceAnimal weight (g) 3656 9 37.46 1.3 36.36 1.1Concentration14C-styrene (ppm) 1596 3 1606 3 1586 5Average inlet flow (ml3 min21) 9806 66 5106 114 5996 133Average outlet flow (ml3 min21) 9556 48 5656 51 6136 139Recirculating flow (l3 min21) 4.08 4.0 4.0Starting pressure (psig) 100 65 65Ending pressure (psig) 10 22 11Uptake of styrenea

(mmol 3 kg21 3 h21)1136 7 1896 53 1836 76

Oxygen at inlet (%) 22.66 0.6 22.36 0.4 21.56 0.5Oxygen at outlet (%) 20.26 0.8 20.66 0.8 20.26 0.6Oxygen consumption

(ml O2 3 kg21 3 s21)0.436 0.06 1.136 0.40 0.866 0.31

Note.Values are means6 SE. The inlet concentration, Cin, may be calcu-lated from the inlet flows (Qpv, the flow from the pressure vessel, Qda, thedilution air flow, and Qox, the oxygen flow) and the concentration in thepressure vessel (Cpv): Cin 5 (Qpv 3 Cpv)/(Qpv 1 Qda 1 Qox). The outletconcentration may be calculated from the styrene concentration measured inthe infrared spectrophotometer, CIR, the styrene concentration in the pressurevessel, and the flow rates through the system: Cout 5 {C IR 3 (Qpv 1 Qox 1Qda 1 QR) 2 (Qpv 3 Cpv)}/Q R, where QR is the recirculating flow. The oxygenuptake was calculated similarly.

a A nose-only exposure chamber equipped with a recirculating loop can bemodeled as a well mixed system. At steady state the following equationapplies: Q3 (Cout 2 Cin) 5 2V 3 k 3 Cout, where Cout is the outletconcentration, Cin is the inlet concentration, k is the reaction rate, V is thevolume of the reactor, and Q is the flow through the reactor. The disappearanceof reactant in the chamber,2V 3 k 3 Cout, is the uptake of styrene by theanimals, thus: Uptake5 Q 3 (Cout 2 Cin).

163DISPOSITION OF INHALED 14C-STYRENE IN RATS AND MICE

collection of tissues for macromolecular binding studies (Boogaardet al.,2000). Of the remaining animals, 4 rats and 8 mice (4 in each exposureexperiment) were transferred to individual gas-tight metabolism cages. Fecesand urine were collected over ice for 0–6, 6–24, and 24–42 h followingexposure. Radioactivity in samples was determined by scintillation counting asdescribed below. The expired air from each cage was drawn by a vacuumpump at a rate of 500 ml3 min–1 for rats and 200 ml3 min–1 for mice througha series of three charcoal filters (Supelco ORBO, 0.9 g) for the collection ofexhaled organic volatiles (14C-styrene and14C-metabolites) and subsequentlyserially through two 1 M KOH solutions (500 ml each for rats and 250 ml eachfor mice) for determination of CO2. Traps for volatiles and CO2 were changedat intervals, 1, 3, 6, 24, and 42 h after cessation of exposure, and analyzed asdescribed below. For the collection of larger urine volumes necessary formetabolite isolation and identification, 2 additional rats were housed individ-ually and 22 mice (11 for each exposure experiment) in groups of 3 or 4 micein polycarbonate metabolism cages. At the end of the collection period, allanimals received a lethal dose of pentobarbital and were processed for auto-radiography or DNA analysis (Boogaardet al., 2000). The cages were washedwith water, and the measured aliquots of the washings were collected andcounted by liquid scintillation counting (LSC).

Urine, feces, and charcoal traps for exhaled volatiles were transferred toglass tubes, sealed, and stored individually at –80°C until transport and furtheranalysis.

Quantitative whole-body autoradiography.One rat and 2 mice (one fromeach inhalation experiment) received a lethal dose of pentobarbital 42 h aftertermination of exposure. Following confirmed death, the carcasses werestretched and mounted on a plastic support. The tails were clipped off, the eyeswere moistened with 2% (w/v) aqueous carboxymethyl cellulose solution, andthe carcasses were then frozen in a mixture ofn-hexane and excess solid CO2.The frozen carcasses were stored at –80°C until dispatch on solid CO2 toCovance Laboratories, Harrogate, UK, for analysis. The frozen carcasses wereset in blocks of 2% (w/v) aqueous carboxymethylcellulose and mounted ontothe stage of a cryomicrotome (MPV 450 MP, Thermometric Ltd, Northwich,UK) maintained at –20°C. Sagittal sections (nominally 30mm thickness) wereobtained from five different levels to expose a range of smaller tissues thatcould not easily be dissected, such as nasal passages, central nervous system,and glandular organs. The sections were mounted on Syrom 90 tape (MilnesPackaging Group, Brighouse, UK), lyophilized (Lyolab B, Life SciencesLaboratories Ltd, Luton, UK), placed into contact with imaging plates (Fujitype BAS IIIs, Raytek Scientific Ltd, Sheffield, UK), and stored in a refrig-erated lead-lined exposure box for 3 days. The distribution of radioactivity inthe sections was measured using a radioluminography system (Fuji 1500,Raytek Scientific Ltd). Quantitation was obtained by comparison with a rangeof 14C-blood standards, which were included with each autoradiogram, using aPC-based image analysis package (Seescan Densitometric Software, LablogicLtd, Sheffield, UK). For all calculations, the density and quench characteristicsof the tissues analyzed were assumed to be similar to those of blood.

Quantitation of radioactivity. Liquid samples were taken up in UltimaGold scintillation cocktail (Canberra-Packard, Groningen, The Netherlands) inantistatic scintillation vials and measured by LSC using TriCarb 2200 CAcounters (Canberra Packard). The machines were calibrated using a commer-cial 14C internal standard kit for organic solvents (Wallac, Turku, Finland). Thecalibration was checked daily by counting a set of quenched standards com-mercially prepared in sealed glass vials. Counting efficiency was determinedusing the spectral index of the internal standard (SIE), and cpm values wereautomatically transformed to dpm. Samples were corrected for background.

The radioactivity in the KOH trap solutions containing exhaled14CO2 wasmeasured by LSC. Duplicate aliquots of 1 ml were added to preweighed 20-mlantistatic scintillation vials containing 3 ml purified water to which 16 mlscintillation cocktail was added before counting. In some cases, 1-ml aliquotsof the KOH solution were evaporated to dryness to remove possibly dissolvedstyrene. The dry residue was subsequently taken up in 1 ml purified water and19 ml scintillation cocktail was added before counting. The urine samples werediluted 100 times with purified water and duplicate 10-ml aliquots were

counted by LSC. Cage washings were pooled for each cage, and a 1-ml aliquotwas counted by LSC.

Charcoal filters containing exhaled volatiles were analyzed for radioactivityby emptying the charcoal from the filter into a conical centrifuge tube andadding 2 ml formamide. After extraction, the charcoal was pelleted by cen-trifugation (15003 g, 5 min) and the supernatant removed. Aliquots of thesupernatant were analyzed by LSC.

Feces were pulverized in liquid N2 using a hammer mill (6700 Freezer/Mill,Glen Creston Inc., Stanmore, UK), and aliquots (approximately 100 mg) weretransferred to glass tubes with 1.0 ml tetraethyl ammonium hydroxide andclosed. The samples were kept for 48 h at 60°C until dissolved. The sampleswere centrifuged for 5 min at 25003 g, and the supernatants counted by LSC.Colored samples were treated with hydrogen peroxide prior to addition of thescintillation cocktail.

RESULTS

Nose-Only Inhalation Exposures

The exposure concentrations generated with the recirculat-ing nose-only exposure system were within 93% of the targetconcentration of 160 ppm throughout the course of the expo-sures (Fig. 3). The concentrations of14C-styrene averaged1596 3 (SE) ppm for rats and 1606 3 and 1586 5 ppm forthe first and second mouse exposure, respectively. In rats, theuptake of 14C-styrene was stable during the 6-h period andaveraged 1136 7 mmol 3 kg–1 3 h–1 (Table 1). In contrast tothe rats, the uptake rate of styrene in mice decreased steadilyover time (Fig. 4). The average14C-styrene uptake was 189653 and 1836 76 mmol 3 kg–1 3 h–1, for the first and secondmouse exposure, respectively. The oxygen uptake was pro-portionally related to the styrene uptake in both rats and mice(Fig. 5).

Animal Observations

No signs of toxicity were observed in rats during or after theexposure to14C-styrene. Animals were damp after the inhala-tion exposure, either from urine or humidity in the air, butappeared otherwise well. Rats that were held in metabolismcages for 42 h after cessation of exposure appeared healthy andwere eating and drinking normally. In contrast to the rats, someof the exposed mice showed signs of toxicity, such as ahunched and unkempt appearance, at the end of exposure.These symptoms persisted in some of the mice during the 42-hholding period in metabolism cages.

Excretion of Radioactivity

A summary of the excretion of radioactivity is given inTable 2. From the radioactivity that was estimated to be takenup by the rats, 796 7% was accounted for by excreta (urine,feces, CO2, and exhaled volatiles). For the two mouse expo-sures, 846 10% and 776 10% of the amount estimated to betaken up by the animals was accounted for by excreta.

Radioactivity in the urine accounted for 756 7% of thestyrene retained by rats. Of the radioactivity that was taken upduring exposure, 206 5% was excreted in the urine during

164 BOOGAARD ET AL.

exposure and 556 6% in the period from 0 to 42 h aftercessation of exposure. The small volumes of urine produced bythe mice during exposure (tube urine) could not be collected,but 6 6 2% of the retained radioactivity in the first mouseexperiment could be washed from the exposure tubes. Thisradioactivity is considered to be mostly due to urine contami-nation of the tubes. The radioactivity in the urine collectedfrom mice in the period from 0 to 42 h after cessation ofexposure was 636 9%. The urinary elimination was virtuallycomplete 42 h after termination of exposure, with the greatestproportion excreted in the first 6 h after cessation of exposure.In rats, the excretion of styrene metabolites in urine was fasterthan in mice. In rats, 376 4% of the radioactivity taken upduring exposure was excreted in the urine in the first 6 h aftertermination of exposure, and 156 3% and 36 1% in thesubsequent two periods of 18 h, respectively. In mice, 34611% of the retained radioactivity was excreted in the urine in

the first 6 h after cessation of exposure but still 256 4% and4 6 1% in the subsequent two periods of 18 h.

Small amounts of radioactivity were excreted in the feces.For the rats, the total amount of radioactivity excreted throughthe feces was 1.06 0.6% of the retained radioactivity. In thesecond mouse experiment, a similar value was found, 1.360.2%. In the first mouse experiment, a higher average value of8.5 6 5.6% was found, which was almost completely due tothe higher value measured in a single feces sample collectedbetween 6 and 24 h after cessation of exposure. Althoughcontamination of this sample with urine cannot be completelyruled out, it is unlikely that such a contamination would in-crease the value to the extent measured. Although the produc-tion of 14CO2 was only a minor part of the metabolism, a majordifference was seen between mice and rats in the amount of14CO2 that was being exhaled. Mice exhaled 3 to 4 times more14CO2 than rats. Virtually all of the14CO2 was produced duringthe exposure. In rats, the average amount of14CO2 measuredfor the period of 24–42 h after exposure was relatively high,

FIG. 3. 14C-styrene concentrations in the nose-only exposure chambersduring the inhalation studies. The target concentration (160 ppm) is indicatedby the dotted line.

FIG. 4. Styrene uptake in rats and mice nose-only exposed to 160 ppm14C-styrene.

165DISPOSITION OF INHALED 14C-STYRENE IN RATS AND MICE

and higher than during the two previous periods. However, thisvalue was not statistically different from the previous timepoints due to the large standard deviation associated with thisvalue. At the end of the holding period in the metabolismcages, only a small percentage of the styrene metabolized bythe animals was associated with the tissues (Table 3).

Quantitative Whole-Body Autoradiography

The concentrations of radioactivity measured in most tissueswere below those present in blood for both rat and mouse(Tables 3 and 4). In addition, the qualitative distribution ofradioactivity in the various tissues was similar when comparingspecies, although mice tended to contain significantly higherlevels. High levels of radioactivity were noted in the livers ofrat and mice (Fig. 6–8). However, the levels were more ele-vated in mice (approximately 4.5 times those found in bloodwhen compared with 1.5 times in the rat). High levels werealso present in the kidney cortex of each species. Concentra-tions were about 1.5 times that measured in rat blood but about6- to 7-fold higher than the concentration in mouse blood.

Concentrations in the renal medulla were more comparablebetween the species and much lower than in the renal cortex (infact, lower than in the blood in both species). Special attentionwas paid to the distribution of radioactivity in the nasal pas-sages, major airways, and lungs (Fig. 9). The level of radio-activity in the lungs of rats was lower than in the blood (about0.6 times), whereas the levels were clearly higher in lungs ofmice compared to blood (more than 2-fold higher). The radio-activity was mainly located in discrete regions of the lungs,presumably in the bronchi. The levels in the nasal mucosa weremuch higher than in the blood (more than 3-fold in the rat and2- to 13-fold in the mice), and the radioactivity appeared toreside mainly in the olfactory mucosa as opposed to the respi-ratory mucosa (Fig. 10).

DISCUSSION

The high specific radioactivity of the styrene at;1.85TBq 3 mol–1 (50 Ci 3 mol–1) that was needed to attain thedesired limit of detection for subsequent macromolecular bind-ing studies (Boogaardet al., 2000) gave rise to two challengesin setting up the nose-only exposure. The first was the gener-ation of the styrene atmosphere. A common approach is tointroduce styrene into a stream of clean air using a syringepump (Sumneret al, 1997). Because this requires heating ofthe styrene at the injection point to aid vaporization, thismethod could not be used because the risk of polymerization ofthe styrene was substantial. The alternative, to aid vaporizationby spreading the styrene over glass wool, was not possibleeither, as the stability tests suggested that large glass surfacesalso catalyzed polymerization. We therefore decided to use avapor system in which the neat styrene was transferred into apressure vessel at ambient temperature. The vessel was sealedand the styrene allowed to completely vaporize overnight.Prior to the exposure, the vessel was pressurized, resulting in astyrene and nitrogen mixture that was metered into the expo-sure chamber air stream to obtain the desired exposure con-centration. The second challenge to overcome was due to thetotal amount of radioactivity required. For nose-only exposureof 12 rats to 160 ppm styrene during 6 h, more than 20 GBq(0.54 Ci) of 14C-styrene was needed. Because such a largeamount of radioactivity could not be accommodated within theinstitutional license, the system was adapted to allow recircu-lation of a portion of the14C-styrene. The designed system wassuccessful in that no signs of polymerization were observed. Inaddition, a constant flow close to the target concentration of160 ppm styrene could be maintained in the Cannon-tower forthe full 6 h of theexposures, while the partial recirculationreduced the amount of styrene normally needed for a nose-onlyexposure of rats to 60%.

The uptake of styrene in mice was on average 186mmol 3kg–1 3 h–1, but a steady decrease in uptake to about a quarterof the initial uptake was measured during the 6 h ofexposure.This reduction was caused partly by a reduction in the venti-

FIG. 5. Oxygen uptake in rats and mice nose-only exposed to 160 ppm14C-styrene.

166 BOOGAARD ET AL.

lation rate and partly by reduction of the overall metabolism, asoxygen consumption by the mice was reduced to about half ofthe initial value in the first h of exposure and remained at thislow level during the rest of the exposure period. Some of themice showed signs of toxicity at the end of the exposureperiod. The uptake in rats was lower than in mice (113mmol 3kg–1 3 h–1) and virtually constant over the entire period of 6 h.There was no change in oxygen consumption by the rats, norwere any signs of toxicity observed.

The data from quantitative whole-body autoradiographyshow a slightly larger retention of radioactivity in mice, withan average concentration in blood in the two mice about 30%higher than in the single rat that was analyzed. Most organs hadradioactivity levels that were equal to or less than the blood inboth rats and mice. A notable exception was the nasal mucosa,in which higher levels of radioactivity than in any other tissuewere measured in both rat and mouse. The rather large differ-ence between the two mice is probably due to inconsistenciesin the sectioning rather than a true reflection of animal varia-

tion. The disposition of radiolabeled styrene has been studiedonly once in rats (Carlsson, 1981) and a few times in mice(Ghantouset al., 1990; Kishiet al., 1989; Lof et al., 1984).Accumulation of styrene in the nasal mucosa was also ob-served, but not quantified, in male C57BL mice 8 h after a10-min inhalation exposure to an undefined concentration of[8-14C]-styrene (Ghantouset al., 1990) and in pregnant CD-1mice exposed to 2.3mmol [8-14C]-styrene by tail vein injection(Kishi et al., 1989). Kishi and coworkers proposed that thehigh concentrations of radioactivity in the nasal passages andlung might originate partly from14CO2 through metabolicdecarboxylation of the sidechain. However, our present resultsindicate that metabolism is virtually complete 45 h after ces-sation of exposure, which excludes the possibility that theradioactivity comes from14CO2. In both the present study andthe study by Kishiet al., the radioactivity more likely origi-nates from tissue binding of styrene metabolites generatedinsitu. This would imply that rats and mice both form reactivemetabolites from styrene in the nasal mucosa, but only mice

TABLE 2Disposition of Radioactivity in kBq

During exposure 0–6 h after exposure 6–24 h after exposure 24–42 h after exposure Total

Rat exposure

Urine 93,6536 22,406 (19.8) 175,7696 19.370 (37.3) 70,8916 13,286 (15.0) 12,3106 6,212 (2.6) 352,6236 33,050 (74.7)Feces ND 2,8226 2,118 (0.60) 4866 376 (0.10) 1,4726 915 (0.31) 4,7806 2,712 (1.0)CO2 8,8126 3,548a (1.9) 136 4 (0.003) 306 10 (0.006) 7546 652 (0.16) 9,6096 3,607 (2.0)Volatiles ND 2226 27 (0.05) 196 8 (0.004) 356 16 (0.007) 2766 29 (0.06)Tubesb 3,3416 1,505 (0.71) — — — 3,3416 1,505 (0.71)Cagesb — — — — 4,0866 1,437 (0.87)Total 105,8066 22,735 (22.4) 178,8266 19,485 (37.9) 71,4266 13,292 (15.1) 14,5716 6,313 (3.1) 374,7156 33,226 (79.4)

Mouse exposure 1

Urine ND 26,6236 6,988 (33.4) 19,9946 2,890 (25.1) 3,1276 356 (3.9) 49,7446 6,171 (62.3)Feces ND 4026 464 (0.50) 6,0446 4,582 (7.5) 3946 154 (0.49) 6,8396 4,481 (8.5)CO2 5,0746 827a (6.4) 2.56 0.6 (0.003) 4.06 1.1 (0.005) 1.96 0.8 (0.002) 5,0826 827 (6.4)Volatiles ND 316 14 (0.04) 2.86 0.4 (0.004) 1.76 0.5 (0.002) 366 14 (0.05)Tubesb 4,4296 1,108 (5.6) — — — 4,4296 1,108 (5.6)Cagesb — — — — 2,2366 288 (2.8)Total 9,5036 1,383 (11.9) 27,0596 7,003 (33.9) 26,0456 5,417 (32.2) 3,5256 388 (4.4) 68,3666 7,756 (84.5)

Mouse exposure 2

Urine ND 26,8006 9,789 (35.3) 19,4266 4,040 (25.6) 3,7606 521 (4.9) 49,9866 7,845 (65.8)Feces ND 966 42 (0.13) 7976 216 (1.0) 1026 41 (0.13) 9956 227 (1.3)CO2 6,0476 345a (8.0) 2.86 0.3 (0.004) 3.26 0.7 (0.004) 2.36 0.6 (0.003) 6,0556 345 (8.0)Volatiles ND 376 10 (0.05) 2.66 0.3 (0.003) 1.56 0.3 (0.002) 416 10 (0.05)Tubesb 2906 277 (0.38) — — — 2906 277 (0.38)Cagesb — — — — 9166 308 (1.2)Total 6,3376 442 (8.3) 26,9366 9,789 (35.4) 20,2296 4,046 (26.6) 3,8666 523 (5.1) 58,2836 7,867 (76.7)

Note.% retention (shown in parentheses) was calculated from the total uptake (1136 7, 1896 53, and 1836 76 mmol 3 kg21 3 h21 for the rat, first, andsecond mouse exposure, respectively), body weight (3656 9, 37.46 1.3, and 36.36 1.1 g, for the rat, first, and second mouse exposure, respectively), durationof exposure (6 h), and the specific radioactivity of the14C-styrene (1.92 TBq3 mol21). ND: not determined; exhaled organic volatiles could not be determinedduring exposure since they could not be distinguished from the14C-styrene in the recirculating atmosphere.

a Values were obtained after evaporation of KOH solution to dryness.b Values represent radioactivity washed out of the nose-only tubes (Tubes) and of the metabolism cages (Cages).

167DISPOSITION OF INHALED 14C-STYRENE IN RATS AND MICE

form substantial amounts of reactive metabolites from circu-lating styrene in the lung; the tissue concentration of radioac-tivity in rat lung is well below the concentration in blood(Table 3). This formation of reactive intermediates would beconsistent with the recent observations in CD-1 mice andCD rats upon chronic exposure to styrene (Cruzanet al.,1998; 2000). Styrene-related non-neoplastic histopathologicalchanges, mainly respiratory metaplasia of the olfactory epithe-lium with changes in the underlying Bowman’s gland, wereobserved in the nasal passages of both mice and rats. Theseverity of these changes increased with styrene concentrationand duration of exposure, but occurred in mice at much lowerstyrene concentrations than in rats. In mice, the incidence ofbronchioalveolar adenomas was significantly increased after 2years of exposure, but in rat lungs no histopathological changeswere observed (Cruzanet al., 1998; 2000).

In liver and kidney cortex, again, higher concentrations ofradioactivity were found in mice compared to rats. The con-centration of radioactivity in mouse liver was 4.5 times theconcentration in blood, whereas the concentration in rat liverwas only slightly higher than in blood. In the kidney cortex ofmice, 6.7 times the concentration in blood was measured,whereas the concentration in the kidney cortex of rats was only1.4 times higher than in blood. In a previous study in Sprague-Dawley rats exposed to 43.5 and 240 ppm of [7-14C]styrene for1 to 8 h, high concentrations of14C were found in kidneys,liver, and subcutaneous fat. At all time points (i.e., up to 6 hpostexposure) concentrations in kidneys were 3 to 14 timeshigher than in subcutaneous fat or liver (Carlsson, 1981).However, this high level of radioactivity in the rat kidney is atransient phenomenon and most likely related to the fact that

TABLE 3Tissue Concentrations of Radioactivity after Administration

by Inhalation of 14C-Styrene to the Male Rat and Mouse

Sacrifice time after dosingAnimal number and sex

Tissue

KBq of radioactivity3 g21 of tissue

45 hRat 1

44 hMouse 1

42 hMouse 2

Vascular/lymphaticBlood 2.846 3.193 4.178Aorta 1.500 2.415 1.836Mandibular lymph nodes 1.240 1.248 1.508

Excretory/metabolicKidney cortex 3.897 22.58 26.39Kidney medulla 1.570 1.708 2.840Liver 4.035 13.73 19.29

Central nervous systemBrain 0.810 0.864 1.184Pineal body 0.952 1.333 NSSpinal cord 0.696 0.667 0.900

EndocrineAdrenal 1.943 2.124 3.341Pituitary 0.866 2.819 2.117Thymus 0.896 1.302 1.663Thyroid 1.621 1.972 2.177

SecretoryExorbital lachrymal gland NS 1.049 NSHarderian gland 8.207 1.791 1.779Intraorbital lachrymal gland 2.869 12.08 5.506

Salivary glands 1.159 3.460 3.195FattyBrown fat 2.634 2.025 2.263White fat 2.345 0.478 2.325

GonadsBulbourethral gland 1.655 NS 4.019Epididymis 0.729 2.074 1.849Preputial gland 2.390 NS 2.786Prostate 1.600 NS NSSeminal vesicles 0.709 1.003 0.982Testis 0.700 3.978 1.518

MuscularMuscle 0.704 0.554 1.109Myocardium 1.444 1.642 2.454Tongue 1.040 1.710 NS

OcularLens 3.718 2.477 4.327Uveal tract 1.123 3.752 5.684

UnclassifiedBone marrow 0.636 0.864 1.348Lung 1.762 7.358 8.714Nasal mucosa 9.156 42.57 9.166Pancreas 1.187 1.544 1.747Skin 4.341 5.191 4.874Spleen 1.660 1.777 2.338Tooth pulp 1.099 1.197 NS

GastrointestinalStomach mucosa 1.675 1.656 3.073Small intestine mucosa 8.557 1.856 5.854Caecum mucosa 3.416 3.063 2.098Large intestine mucosa 2.847 2.555 NSRectum mucosa NS NS NS

Note.For all animals, the upper limit of quantification5 82.77 kBq3 g21;the lower limit of quantification5 0.195 kBq3 g21. NS, tissue not sectioned.

TABLE 4Relative Tissue Concentrations of Radioactivity after Ad-

ministration by Inhalation of 14C-styrene to the Male Rat andMouse

Animal number and sexSacrifice time after dosing

MR00545 h

MM10544 h

MM20542 h

Bile ducts 1 NA NAGall bladder NA 1 1Kidney pyramid 2 2 2Bladder 2 3 3Esophagus 3 3 3Stomach contents 2 2 2Small intestine contents 2 2 2Cecum contents 2 2 2Large intestine contents 2 2 NSRectum contents NS NS NS

Note. NS, tissue not sectioned; NA, not applicable. Assessment of therelative levels of radioactivity in the autoradiograms was made by visualinspection of the printed electronic autoradiograms. Relative levels of radio-activity in the tissues were then recorded in a simple scoring code: 35concentration described as high; 25 concentration described as intermediate;1 5 concentration described as low.

168 BOOGAARD ET AL.

almost all the retained radiolabeled styrene is cleared throughthe kidney. At the time points chosen in this study, renalexcretion is at its maximum. Similarly, the high concentrationsof styrene in adipose tissue reported by Carlsson (1981) werealso transient. We measured levels of radioactivity in fat, 45 hafter cessation of exposure, that were equal or slightly less thanin blood, indicating that styrene is stored in fat during exposurebut swiftly released after termination of exposure. Differencesobserved in distribution appear to be related to the timing ofsampling rather than to the different ways of administration of

the 14C-styrene as suggested previously (Sumneret al., 1995).Observed differences between the present study and earlierstudies may also be explained, at least partly, by the position of theradiolabel: [8-14C]styrene will easily lose its label through meta-bolic decarboxylation within a short period after the end of expo-sure and become undetectable in (auto-)radiography, whereas[ring-U-14C]styrene will not release14CO2 through simple decar-boxylation and may be detected in the tissues for a longer period.

The position of14C-label in the ring in the present studyimplies that exhaled14CO2 is a result of styrene metabolisminvolving opening and degradation of the aromatic ring. Ringopening is probably preceded by ringoxidation (Blaesdaleet

FIG. 6. Whole-body autoradiograms of a male Sprague-Dawley rat 45 hafter nose-only inhalation exposure to14C-styrene (animal number MR005).Abbreviations: ad, adrenal; ao, aorta; bd, bile ducts; bdr, bladder; bl, blood;bm, bone marrow; br, brain; bf, brown fat; bug, bulbo-urethral gland; ca,caecum; ed, epididymis; elg, exorbital lachrymal gland; gb, gall bladder; Hd,harderian gland; ilg, intra-orbital lachrymal gland; kdc, kidney cortex; kdm,kidney medulla; kdp, kidney pyramid; li, large intestine; le, lens; lv, liver; lu,lung; lma, mandibular lymph nodes; mu, muscle; my, myocardium; nm, nasalmucosa; oe, oesophagus; pa, pancreas; pb, pineal body; pt, pituitary; pg,preputial gland; pr, prostate; re, rectum; sg, salivary glands; sv, seminal vesicles;sk, skin; si, small intestine; sc, spinal cord; sp, spleen; st, stomach; ts, testis; th,thymus; ty, thyroid; to, tongue; tp, tooth pulp; uvt, uveal tract; fa, white fat.

FIG. 7. Whole-body autoradiograms of a male CD1 mouse 44 h afternose-only inhalation exposure to14C-styrene (animal number MM105). Forabbreviations, see Figure 6 caption.

169DISPOSITION OF INHALED 14C-STYRENE IN RATS AND MICE

al., 1996). Evidence of ringoxidation of styrene was found inthe formation of 4-vinylphenol, which was reported as a uri-nary metabolite of styrene (Bakke and Scheline, 1970; Pfa¨ffliet al., 1981). Other evidence for ring-opened metabolites ofstyrene was recently reported (Sumneret al., 1995). Formationof reactive metabolites through ringoxidation of styrene (orSO) and subsequent ring opening might be catalyzed by spe-cific isoforms of cytochrome P450 present in specific celltypes. Formation of14CO2 was limited during exposure andalmost absent after cessation of exposure in both rats and mice.Nevertheless, there was a substantial difference between ratsand mice, with mice producing 3–4 times more14CO2 than

rats, accounting for 6.46 1.0 and 8.06 0.5% of the totalstyrene retained during the first and second mouse exposure,respectively. This larger production of14CO2 in mice than inrats, in combination with the substantially higher tissue bindingobserved in the lungs, might be indicative of the formation ofreactive ring-opened metabolites in the mouse lung, which in turnmight be related to the observed development of bronchioloalveo-lar tumors and nasal effects in mice exposed to styrene.

One of the major metabolic pathways of styrene is thecytochrome P450-dependent oxidation to SO, which is rapidlydetoxified by enzyme-catalyzed epoxide hydrolysis or gluta-thione (GSH) conjugation. High concentrations of cytochrome

FIG. 8. Whole-body autoradiograms of a male CD1 mouse 42 h afternose-only inhalation exposure to14C-styrene (animal number MM205). Forabbreviations, see Figure 6 caption.

FIG. 9. Enlargement of whole-body autoradiograms of a male Sprague-Dawley rat (animal RM005, top), and two CD1 mice (MR105, middle;MR205, bottom) mouse showing the lung 42–45 h after nose-only inhalationexposure to14C-styrene.

170 BOOGAARD ET AL.

P450 are found in liver, lung, and renal cortex. The extensivecytochrome P450-dependent oxidation of styrene to SO andring-opened metabolites, which also are likely to be detoxifiedfor a substantial part through GSH conjugation, might lead tolocal depletion of GSH. Such a GSH depletion would not onlyexplain the observed extensive tissue binding in liver, kidney,and lungs, but also the toxicity observed in mice, as moreoxidative metabolism occurs in mice than in rats. Despite the

high tissue binding in mouse lung, the DNA binding was verylow (; 1 adduct per 108 nucleotides) and not significantlydifferent from the DNA binding in rat lung (Boogaardet al.,2000). This suggests that a nongenotoxic mechanism, possiblycaused by a cytotoxic metabolite, underlies the observed bron-chioalveolar tumor formation in mice.

ACKNOWLEDGMENTS

The authors thank John E. Murphy, Tim Moore, and Rod Snyder fortechnical assistance and the CIIT animal care staff for their assistance in thestudy. This study was sponsored in part by the Styrene Information andResearch Center.

REFERENCES

Bakke, O. M., and Scheline, R. R. (1970). Hydroxylation of aromatic hydro-carbons in the rat.Toxicol. Appl. Pharmacol.16, 691–700.

Blaesdale, C., Kennedy, G., MacGregor, J. O., Nieschalk, J., Pearce, K.,Watson, W. P., and Golding, B. T. (1996). Chemistry of muconaldehydes ofpossible relevance to the toxicology of benzene.Environ. Health Perspect.104(S6), 1201–1209.

Bond, J. A. (1989). Review of the toxicology of styrene.CRC Crit. Rev.Toxicol.19, 227–249.

Boogaard, P. J., De Kloe, K. P., Wong, B. A., Sumner, S. C. J., Watson, W. P.,and Van Sittert, N. J. (2000). Quantification of DNA adducts formed in liver,lungs, and isolated lung cells of rats and mice exposed to14C-styrene bynose-only exposure.Toxicol. Sci.57, 203–216.

Cannon, W. C., Blanton, E. F., and McDonald, K. E. (1983). The flow pastchamber: an improved nose-only exposure system for rodents.Am. Ind. Hyg.Assoc. J.44, 923–928.

Carlsson, A. Distribution and elimination of14C-styrene in rat. (1981).Scand.J. Work Environ. Health7, 45–50.

Cruzan, G., Cushman, J. R., Andrews, L. S., Granville, G. C., Johnson, K. A.,Hardy, C. J., Coombs, D. W., Mullins, P. A., and Brown, W. R. (1998).Chronic toxicity/oncogenicity study of styrene in CD rats by inhalationexposure for 104 weeks.Toxicol. Sci.46, 266–281.

Cruzan, G., Cushman, J. R., Andrews, L. S., Granville, G. C., Johnson, K. A.,Bevan, C., Hardy, C. J., Coombs, D. W., Mullins, P. A. and Brown, W. R.(2000). Chronic toxicity/oncogenicity study of styrene in CD-1 mice byinhalation exposure for 104 weeks.J. Appl. Toxicol.,(in press).

Gadberry, M. G., DeNicola, D. B., and Carlson, G. P. (1996). Pneumotoxicityand hepatotoxicity of styrene and styrene oxide.J. Toxicol. Environ. Health48, 273–294.

Ghantous, H., Dencker, L., Gabrielsson, J., Danielsson, B. R. G., and Bergman,K. (1990). Accumulation and turnover of metabolites of toluene and xylenein nasal mucosa and olfactory bulb in the mouse.Pharmacol. Toxicol.66,87–92.

IARC (1994a). Styrene.IARC Monographs on the evaluation of carcinogenicrisks to humans60, 233–320.

IARC (1994b). Styrene-7,8-oxide.IARC Monographs on the evaluation ofcarcinogenic risks to humans60, 321–346.

Kishi, R., Katakura, Y., Okui, T., Ogaea, H., Ikeda, T. and Miyake, H. (1989).Placental transfer and tissue distribution of14C-styrene: an autoradiographicstudy in mice.Br. J. Ind. Med.46, 376–383.

Leavens, T. L., Moss, O. R., and Bond, J. A. (1996). A dynamic inhalationsystem for individual whole-body exposure of mice to volatile organicchemicals.Inhal. Toxicol.8, 655–677.

FIG. 10. Enlargement of whole-body autoradiograms of a male Sprague-Dawley rat (animal RM005, top), and two CD1 mice (MR105, middle;MR205, bottom) mouse showing the nasal cavity 42–45 h after nose-onlyinhalation exposure to14C-styrene.

171DISPOSITION OF INHALED 14C-STYRENE IN RATS AND MICE

Lof, A., Gullstrand, E., and Byfa¨lt-Nordqvist, M. (1984). Tissue distribution ofstyrene, styrene glycol and more polar styrene metabolites in the mouse.Scand. J. Work Environ. Health9, 419–430

McConnell, E. E., and Swenberg, J. A. (1994). Review of styrene and styreneoxide long term animal studies.Crit. Rev. Toxicol.24, S49–S55.

Miller, R. R., Newhook, R., and Poole, A. (1994). Styrene production, use, andhuman exposure.Crit. Rev. Toxicol.24, S1–S10.

Pfaffli, P., Hesso, A., Vainio, H., and Hyvo¨nen, M. (1981). 4-Vinylphenolexcretion suggestive of arene oxide formation in workers occupationallyexposed to styrene.Toxicol. Appl. Pharmacol.60, 85–90.

Sumner, S. C., Asgharian, B., Moss, O., Cattley, R. C., and Fennel, T. R.(1995). Correlating styrene metabolism and distribution with hepatotoxicity.The Toxicologist. 15, 4.

Sumner, S. C. J., Cattley, R. C., Asgharian, B., Janszen, D. B., and Fennell,T. R. (1997). Evaluation of the metabolism and hepatotoxicity of styrene inF344 rats, B6C3F1 mice, and CD-1 mice following single and repeatedinhalation exposures.Chem. Biol. Interact.106,47–65.

Van Elburg, P.A. (1998). Synthesis and stability of [ring-U-14C]-labelledstyrene to be used in animal exposure experiments. Report CA.98.20479.Shell International Chemicals BV, SRTCA, Amsterdam.

172 BOOGAARD ET AL.