age-related percutaneous penetration of 2-sec-butyl-4,6-dinitrophenol in rats

8/12/2019 Age-Related Percutaneous Penetration of 2-Sec-Butyl-4,6-Dinitrophenol in Rats

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FUNDAMENTAL AND APPLIED TOXICOLOGY 19, 2 58 -2 67 (19 9 2)

Age-Related Percutaneous Penetrationof 2-sec-Butyl-4,6-dinitrophenol(Dinoseb) in Rats1L A R R Y L. H A L L , * H E N R Y L. F I S H E R , * M A R T H A R. SuMLER,t M I C H A E L F. H U G H E S , A N DP. V. S H A H $

*Environmental Toxicology D ivision, Health Effects Research Laboratory. US Environmental Protection Agency, Research Triangle Park,North Carolina 27711, \ManTech Environmental Technology Inc, PO. Box12313, Research Triangle Park, North C arolina 27709,

an d Hoffmann-La Roche Inc., 340 Kingsland Street, Nulley, New Jersey 07110Received August 27 . 1991: accepted March 6. 1992

Age-Related Percutaneous Penetration of 2-jec-Butyl-4,6-di-nitrophenol (Dinoseb) in Rats. H A L L , L.L ., F ISHER , H. L.,S U M L E R , M. R., H U G H E S , M. F., A N D S H A H , P. V. (1992).Fundam. Appl. Toxicol. 19, 258-267.

[14C] Dinoseb was appliedtopreviously clipped back skin of33-and 82-day-old female Fischer 344 rats at a dosage range of210-2680 nmol/cm 2 . Radioactivity in the treated skin, tissues,urine, and feces was determinedat 1,6, 24, 48, 72, and 120 hrfollowing dermal application. In vitro dermal absorptionof[ l4 C]d inose b was also measured in rats of the same age by staticand flow-through methods.Invivo dermal absorption inbothyoung and ad ults appeared biphasic with 55.6 and 82.7% of therecovered dose, respectively, penetrating in 72 hr. In vitromea-surements of skin absorptionat72hrwith static cells showedhigher values in young and lower values in the adult comparedto in vivo dermal absorption values.Invitro flow-through mea-surements at 72 hr gave lower dermal absorption values for bothyoung and adult rats, compared to in vivovalues. Following invivoapplication, adults excreted about 70% ofthetotal recovereddose in urine, 16% in feces, and retained 7% in the body at 120hr. HPLC analysisofurine collectedat24hrfrom adults ad-ministered [ l4C]dinoseb showed extensive metabolism of parent.Excretion and retention resultsfor young were about 80%ofthe adult values, which also was the young to adult ratio of dermalpenetration. Blood had the highest concentrationofdinoseb-derived radioactivity of the tissues e xam ined. The kidney to bloodratio averaged 0.60 in young and 0.41 in adults, while the liverand carcass to blood ratio averaged 0.18inyoung and 0.11inadult. Dermal absorption inyoung rats w as slightly less thanthatinadults, and the subsequent kinetics of retention and ex-cretion appeared different. Invitrodermal penetration of dinosebwas usually lower than invivoabsorption, t,1992SocietyofT0*icoiog>

Durham and Wolfe (1962) have shown that dermal ratherthan inhalat ion or oral exposure topesticidesisthe major1 The research described inthis article has been reviewed by the H ealthEffects Research Laboratory. U.S. Environmental Protection Agency, andapproved for publication. Approval does not signify that the contents nec-essarily reflect the views and policiesofthe agency, nor doe s mentionoft r ade names orcomm ercial prod ucts cons t i tute endorsement or recom-mendat ion for use.

route of concern for agricultural wo rkers. Skin perme abilityis therefore important in therisk assessme nt ofchem icalsused in agriculture. Many factors that influence percutaneouspenetration ofchem icals have been identified a nd includespecies variation, site of application, occlusion, age, dosage,and others (Franklin etal., 1989).Concern over the effect of ageinpesticide toxicity stem sfrom the exemption to work granted to prepubescent childrenand adolescents (EPA, 1980) in agriculture. Only a few stud-ies have been reported that address the age issue. Behl et al(1985) reviewed the li teratureonage-related derm al pene-tration. Behletal. (1984) found invitro mouse skin per-meability to aseriesof alcohols increased as a functionofageto ama x i mu m at25 daysofage then decreasedby 6days and subsequently rem ained cons tant. K naak et al.(1984) reported that, based on radioactivity, triadimefon waslost more rapidly from the skin of young than from the skinof adult rats. McCorm ack etal. (1982) reported no differencein the permeabilityofprem ature, full term infant oradul thuman skinto a series of alcohols while fatty acids showedage dependence. Westeret al. (1977) found no difference inthe dermal absorption of testosterone between newborn andadult rhesus monkeys. Shah eta l. (1987b) comparedthedermal penetration of pesticides in young and adult Fischerrats and reported age-dependent skin absorptionin 11of14compounds studied with four chemicals having greater pen-etration in the young rat than the adult.Invitropercutaneousabsorption oftriclocarban was greater inhuma n newbornforeskin (2.5%) compared tohum an adul t abdominal skin(0.23%) (Wester and Maibach, 1985).Few studies have examined the effect ofdosageonskinpenetration. Wester and Noonan (1980) observed dose-de-pendent dermal absorption of testosterone and benzoic acidin both rhesus monkeys and man. Incontras t , F eldmannand Maibach (1974) observed constant fractional penetra-tion of parathion through skin. Shahet al.(1987b) examinedthe effect ofdosageonskin ingress with 14 pesticides andreported dose-dependent dermal absorption in 11 of th echemicals.In vitromethod s of measuring skin absorption are of greatinterest because of the ethical and practical limitations of in

0272-0590/92 $5.00Copyright 1992 by the Society of Toxicology.All rights of reproductioninany form reserved.25 8


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DERMAL ABS ORP TION OF DINOS EB 259vivostudies in m an. Franz ( 1978 ) reported good rank o rdercorrelation between in vitroand in vivomeasurements ofskin absorption in man. Hawkins and Reifenrath (1984)found a significant correlation of percutaneous penetrationbetweeninvitroporcine skin andinvivoresults in man. K aoel al.(1985) demonstrated that skin permeation can be ac-companied by extensive "first pass" metabolism. For somechemicals such as benzo[a]pyrene (Kao el al., 1985), invitroderm al p enetration is significantly influenced by itsmetabolism, requiring the presence of viable skin. Meth-odology is now routinely used to maintain skin viability invitro(Kaoelal. 1985; Collierelal. 1989).In the research reported here, the effect of age and dosageon the percutaneous absorption and disposition of dinoseb(2-^c-butyl-4,6-dinitrophenol), a contact herbicide used forpre- and postemergent weed control and as a preharvest des-sicant, was investigated. The results are compared with theaid of a preliminary physiological com partm ental model. Inaddition, the dermal absorption determined using two invitromethods are compared to the invivo results.

MATERIALS AND METHODS[ I4 C]Din oseb (uniformly r ing labeled, sp act 4 .29 mC i/m mo l) was pur-chased from Pathfinder Labs., Inc. (St. Louis, MO). Radiochemical purityof the compound was greater than 98% as determined by HPLC. Unlabeleddinoseb was obtained from the U.S. EPA R epository for Toxic and H azardousMaterial (Research Triangle Park, NC). Minimum essential media, Eagle'sformula with Earl's salts and L-glutamine and without phenol red and sodiumbicarbonate, Hepes (A'-2-hydroxyethylpiperazine-A''-2-ethanesulfonic acid),fetal bovine serum, sodium pyruvate, L-serine, and L-aspartic acid were pur-chased from GIBCO (Grand Is land, NY). Anhydrous sodium carbonate ,sodium hydroxide, ethyl ether (anhydrous) and acetone (reagent grade)were purchased from Fisher Scientific Co. (Fairlawn, NJ). Emulphor EL-

620 was from GAF Corp. (New York, NY).Guaranteed time-pregnant Fischer 344 rats were purchased from theCharles River Breeding Farm (Kingston, NY). Male pups were culled fromthe litters because of the dorsal skin thickening that occurs at sexual maturity.The female pups were randomized and replaced with the dams (eight pups/dam). At weaning, the females were again randomized and divided intotwo groups, using rank ordered weights. The first group was used for thestudies in young animals and the second group was used for the adult studies.Thirty-three-day-old (DO) animals were used for the young group becausethe restraints used to protect the application site did not retard growth sig-nificantly as they did in younger animals and vaginal opening, a hormone-dependent event, starts at approximate ly 4 0 days of age. This provided a 7-day period to perform the studies before puberty began. Eighty-two-DOadult female rats were chosen for logistical reasons. The an imals were m ain-tained according to the National Institutes of Health Guide for the Careand Use of Laboratory Animals. Food (Purina Rat Chow, No. 5001, St.Louis, MO) and water were available ad libitum. The animals were main-tained on a 12-hr light/dark cycle at constant temperature and humidity.Twenty-four hr prior toin vivoand in vitroexperiments, animals were lightlyanesthetized with ethyl ether and hair on the backs of the animals was re-moved with electric clippers (Oste r Corp ., Model A2, M ilwaukee, WI) usinga size 40 blade. The clipped area was then washed with acetone to removedirt and sebaceous gland secretions.In vivo experiments. The dose-effect on percutane ous penetration of[14C]dinoseb was studied a t 250 (60) , 536 (129) , and 2680 nmol/ cm 2 (644

j ig /cm 2) in the young ra t and 210 (52), 536 (129), and 2680 nmol/cm 2(644 Mg/cm 2) in the adult rat. The serial termination studies were performed

a t 285 nmol /c m 2 (68 Mg/cm2) in both age groups. Thr ee anim als were usedfor each dose per age group.

Animals weights were recorded, the animals were lightly anesthetized withethyl ether, and the treatment area in the middorsal region was marked witha template. The treatment area was 2.8 cm 2 in young and 5.6 cm 2 in adultswhich represents approximately 2.3%of the total body surface area. Dino sebin acetone (100 /A young. 200 ^1 adult ) was applied in sm all drops with aHamilton microliter syringe within the marked area. The treated area wasprotected by gluing a 5-ml perforated plastic disposable beaker (Fisher Scientific Co., Raleigh, NC) on the young animals and a perforated plasticblister from a Cathavex single-use filter (Millipore Co rp., Bedford, M A) onthe adult anim als with a cyanoacrylate adhesive (Pro duct 472, Loctite Corp.,Newington, CT). In addition, the treated area and plastic blister and beakerwere protected by restraining the animal w ith a collar of latex rubber tubing(1.12 cm o.d.) secured with 20 gauge bus wire beh ind t he forelegs as describedby Bartek el al. (1972). The treated animals were mainta ined in Nalgenemetabo lism cages (Nalge Co., Rochester, NY ) for urine and feces collectionand were provided with food (Dustless Precision Pellets, 45 mg rod ent cho wformula, Bio Serve, Inc., Frenchtown, NJ) and water ad libitum.

Animals were lightly anesthetized with ethyl ether and terminated byexsanguination at specified time points. The dose-effect animals were ter-mina ted at 72 hr and the serially terminated an imals at 1, 6, 24, 48 , 72, and120 hr. The plastic blisters and beakers along with treated skin were removedfirst to prevent contam ination . The plastic blisters and beakers, treated skin,urine, feces, liver, kidney, an untreated back skin sample adjacent to theapplication site, and carcass were analyzed for radioactivity as described byShahel al (19 85) . Aliquots of urine were directly analyzed by mixing withInstagel (Packard Instrument Co., Downers Grove, IL). All other sampleswere oxidized in a Packard Tri-Carb sample oxidizer (Model B-306) priorto counting in a Packard Tn-Carb 2660 liquid scintillation system.

Metabolism study. Twenty-four-ho ur urine sample s were collected fromadult rats treated with [I4C]dinoseb either dermally or via the tail vein. Thedermal-treated animals (N = 3) received 285 nmol/cm 2 of compound inacetone over a 5.6-cm 2 area. The iv-treated animals (N = 3) received anequivalent dose (1 .6 ^mol) administered in 95% ethanol/Emulphor/water(1 /1 /3 ) . The dosed animals were then handled as described previouslyThe urine was kept cold during collection with U-Tek refrigerant packs(Polyfoam Packers Corp., Wheeling, IL). After collection (24 hr), the urinewas kept at -20C until analyzed.Urine from the iv-treated animals was filtered (0.2 jim, Rainin InstrumentsWoburn, MA) and directly analyzed by HPLC. Urine from the dermaltreated animals was concentrated mvacuo,reconstituted in a minim al volum eof 20 mM sodium phosphate (pH 5.5)/m ethanol (80 /20 ) and f i l tered priorto HPLC analysis. Recovery of radioactivity in the concentrated sampleswas 80%. The chrom atography system consisted of a Hewlett- Pack ard 1090liquid chromatograph (Palo Alto, CA) and a Waters Resolve C,8 column(5/urn, 3.9 mm X 15 cm; Millipore Corp .. Milford, M A) conne cted in serieto a Radiomatic F lo-one/Beta radiochromatography detector (Model 280,Meridan, CT). Packard Flo-scint II was used as the scintillant. Initial con-ditions of the system were 80% 20 mM sodium phosphate (pH 5.5, solventA) and 20% methan ol (solvent B, Burdick and Jackson, Mu skegon, M I),flowing at a rate of 0.7 ml/min. After injection, the solvent mixture waschanged linearly over 12 min to 50% solvent B followed by a 2 min g radientto 100% B. which was held for 6 min. Initial conditions were then reestab-lished.

In vitro experiments. Penetration through excised rat dorsal skin wadeterm ined using both the static Franz cells and the flow-through diffusioncells described by Bronaugh and Stewart (1985). The in vitro receptor fluidused in both the static and flow-through systems was minimum essentialmedium containing 4% fetal bovine serum, aspartic acid (0.2 mM), serine(0 .2 mM). sodium pyruvate (1 .0 mM), and gentamicin (100 mg/l i ter)(El-more and Swift. 1977). The pH of the me dium was adjusted to 7.3 withNAOH and filter sterilized (0.2 (im. Nalgene disposable filterware). Theme dium reservoir for the flow-through diffusion cells was gassed with 100%oxygen. In preliminary studies in our laboratory using histopathologic as


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260 HALL ET AL.sessment, no significant morphological changes were noted in skin diskscultured with this medium for up to 120 hr.

The eq uipm ent used in the static diffusion cell studies included the Franzdiffusion cells (9 mm diam FDC-100, Crown Glass Co., Sommerville, NJ),and Franz diffusion cell drive console for eight cells (Crown Glass Co .). Aconstant temperature c ircula t ing bath (Haake-B uchler , M odel D16, F isherScientific Co.) was used to contro l the tem pera ture o f the diffusion cells at32 C.

The equipment used in the flow-through studies included Teflon flow-through diffusion cells (Crown Glass Co.), a diffusion cell heating block(Posibloc, Crown Glass), a peristaltic multichannel cassette pum p (M anostat,Fisher Scientific Co.), an ISCO fraction collector (Model 328,1SCO, Inc.,Lincoln, NE) with a 10X time multiplier modification, and a constant tem-perature bath (Haake-Buchler, Model D16). Tygon tubing (55 inch i.d.,R-3603, Fisher Scientific Co.) was used to transport medium from the res-ervoir to the flow cells.

All glassware was autoclaved and plasticware was chemically sterilizedwith Alcide (Alcide Corp., Westport, C T) and 70% ethyl alcohol p riorto use.

Animals were terminated by CO 2asphyxiation a nd the previously clippedskin was excised and immediately floated on culture medium to permitrelaxation of the sample. Approximately 350-Mm-thick skin sections wereremoved with a dermatome (Padgett Dermatome, Kansas City , MO) andrefloated on culture m edium until disks of skin were punched using 0.5 and1.0 inch diameter bow punches. The exposure area was 0.64 and 0.32 cm 2for the Franz and flow-through cells, respectively. Each skin disk was treatedwith 285 nmol/cm 2 of [I4 C] dinoseb in acetone (36 / t l /cm 2 ). The flow rateof the flow-through cell system was maintained at 3 ml /h r and the sam pleinterval was 182 min. The total receptor fluid from the Franz cells wasremoved hourly for the first 5 hr and then four times per day.At 72 hr following skin application, the cell tops, o-rings, and treated skinwere washed three times with 1 ml of70 %ethyl alcohol to remove unabsorbedchemical. The treated skin was then combusted in the Packard sample ox-idizer to measure the radioactivity in the skin. All other samples were mixedwith 10 ml of Insta-Gel for scintillation counting.

Data analysis and modeling Statistical comparisons to determine theeffect of age on skin penetration of dinoseb were made using the StatisticalAnalysis System (SAS, SAS Institute Inc., 1985) with age and age X timeas class variables in the general linear model (G L M ). Because differencesin absorption were found as a function of age, statistical analyses were alsoperformed on the content and concentration data after normalizing by ab-sorbed dose. Reciprocal variance was used for weighting. The significanceof the laboratory m ethod used (flow-through, static,invivo) was determ inedwith SAS using age, method, age X method, and age X method X time asclass variables in the GLM with reciprocal variance for weighting.

A preliminary flow-limited physiological pharmacokinetic model (Fig.1) was developed similar to that used by Bischoff el al (1971). The modeldescribes the disposition of chemical-derived radioactivity, assuming in-stantaneous mixing within a compartment and first-order kinetics. It thussimulates the disposition of the paren t plus any metabolites or transformationproducts that contain the radiolabel. This model and also the laboratorydata will give an upper bound to the parent as well as any single or com-bination of transformation products containing the radiolabel. This modelsimulates the data with a limited number of parameters. Temporal aspectsof the exposure conditions different from those used in the laboratory canalso be simulated. The model can be a useful means of comparing resultsfrom different lab oratories with different experim ental c onditions if a com-mon ground of model compartments and ra te constants are produced byeach labora tory. Fo r these reasons, as well as the ability to determin e dif-ferences in compartmental rate constants between young and adult animals,a model that traces the radiolabel can be useful. This model has not beenvalidated with other data, at different doses, with multiple species, or forother routes of administration, w hich again shows the initial and preliminarynature of it.

DermalDose

Blood

Kidney

H I2

Liver

Un-treatedSkin

Muscle

\

\

3

* TUrine

4

* Feces

FIG. 1. Schematic of physiological com partm ental m odel for dinosedermal absorption and disposition in the rat.

Two c ompa rtments for treated skin were found necessary to fit the dermaabsorption results. For amountsy, an d y2 in compartment 1 and 2, respetively, the amount remaining at the treated skin site is y, + y2 and thdifferential equations are-^=-(kl+k 3)y l, y , ( 0 ) = 1,d\>2 , , = k3y, - A'2.1'2,

where ki, k2, k3 are rate constants. The amounts y, in o ther compartment(/) are determined from

dy 3at


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DERMAL ABS ORP TION OF DINOS EB 261whereQ,is the blood flow rate to the organ comp artm ent; V,is the volumeof organ compartment; R, is the equilibrium tissue to blood ratio for organcom partme nt; / is kidney, liver, untreated skin, or muscle;yB, VBare valuesfor the blood compartment;y} an dy4 are amounts in compartments 3 fromkidney and 4 from liver; kK, k^,, kU2 , kL, kFI, &R are rate constants forkidney, urine, liver, and feces. The volumes (weights) of kidney, liver, andbody were determined directly while those for blood, muscle, and skin werecalculated as a percentage of the body mass(Lutz el ai, 1977). The bloodflow rates were scaled linearly with com partm ental volumes (Bu ngay el ai,1981). Experimental data on blood, kidney, liver, skin, and body were fitusing CONSAM (Berman et ai, 1983). The parameters determined werethe equilibrium tissue/blood ratios, RT, the dermal rate constants k,, k2,an d k}, the kidney and urine rate constants kK, kvl, km, and the liver andfeces rate constants kL an d kF1 an d k^.

RESULTSThe results of the studies of the effect of age and dose onthe percutaneous absorption, distribution, and excretion ofdinoseb are shown in Tables 1 and 2. Mean recoveries rangedfrom 87.5 to 105.9% in young and 87.9 to 102% in adultrats.Dermal absorption of dinoseb appeared to be biphasic. In6 hr the skin penetration was about 44% in both young andadult while at 120 hr 75.9% was absorbed in young a nd 92.5%in adults.The maximum body burden of dinoseb-derived radioac-tivity observed in young was 41 % and occurred at 6 hr, butby 120 hr the body burden had dropped to 5.8%. In adultsa maxim um observed body burden of 52% occurred at 24

hr and subsequently the body burden declined to 6.8% at120 hr. Dinoseb appeared, in general, to be distributedthroughout the body as evidenced by the carcass containingmost of the body burden (Tables 1 and 2 ). The highest con-centration of dinoseb-derived radioactivity was usually seenin the blood (Table 2) followed by the kidney.The effect of dose on penetration and distribution wasalso examined at 72 hr in young and adult rats (Tables 1and 2). Constant fractional dermal penetration was seen inboth young and adult with greater absorption occurring inadults. Blood and kidney showed the highest concen trations.Except for kidney, tissue content and concentration tendedto increase with increasing dosage. Kidney content and con-centration appeared to decrease with increasing dosage. Urinecontent and concentrat ion appeared constant .

The primary route of excretion of dinoseb-derived radio-activity was urinary. By 120 hr the young excreted about 4.6and the ad ult 4.3 times as much in urine as in feces. Urin aryexcretion was 57.7 and 69.5% of the recovered dose in you ngand adults, respectively (Table 1).HPLC analysis of 24-hr urine samples collected fromadults treated with dinoseb showed extensive metabolism ofthis pesticide. The metabolite profile of radioactivity excretedin urine was similar for the iv (Fig. 2A) and dermal (Fig.2B) treated anim als. Detection of parent in the urine samplesfrom the iv- and dermal-treated animals was minimal.The yo un g/a du lt ratio of dinoseb-derived radioactivity intissues and excreta are seen in Table 3. Since absorption was

TABLE 1Distribution and Deposition of [ l4ClDinoseb-Derived Radioactivity (Percentage of Recovered Dose) in Youngand Adult Fischer 344 Rats after Dermal Application

Dose*(nmol/cm2)Young28 5

25 053 52680

Adult285

25 053 52680

Time(hr)

1624487212072727216244872120727272

B o d /burden

1 1 2 :4 0 . 8 :3 8 0 :2 0 . 2 :8.3 d5.8d14.4d14.8d22.6d21.1 d

t 2.2t 5.4t 1 8b 3.2b 0.4b 0.4b 0.8b 1.1b 0.8: 6.6

43.1 10.052.4 3.826.8d23.0d: 0.8: 1.96.8 0.222.9 1.124.4d28.1 i: 0.8: 3.5

Kidneys

0 20 0.041 00 0.081.57 0.040.67 0 050.20 0 020.28 0.011.11 0070.93 0.030.83 0.09

0.24 0 02 (0.77 0 211.06 0.030.55 0 030.43 0 070.17 0.02 (1.09 0.060.87 0.040.70 0.02

Liver

3.78 0.142.66 0.472.53 0.061.24 0.173.63 0.043.36 0.03.03 0.07.15 0.13.72 0.15J.98 0.09>.62 0.84).O8 0.08.58 0.13.38 0.073.44 0.02.37 0.07.56 0.02.69 0.15

Percentage ofRecovered Dose

Carcass

9.5 1 93 3.1 4 530.2 1.216.5 2 87.0 0.34.8 0 311.2 0.711.5 0.918.3 0 418.8 6 637.7 8.745.5 3.72 3.1 0 720.0 1.75 .8 0 219.7 1.021.1 0 724.6 3.3

Unne

0 . 59 0112 12 0.7525.7 1 835 6 6.440.7 1 357.7 3 052.8 4 455.0 1 747.8 1.90.032 0 0181.04 0.1029.7 2.151.5 0.851.8 3 069.5 1.650.1 0.652.3 1.050.7 2.2

Feces

0 007 :0 07 :3 88 :5 33 :6.59 :12.5 :10.5 :11.7 :12.4 :

t 0.001tO.OltO 37t0.87tO 18t 1.0t 0 . 7t O1t 1 1

0.007 0.0010.063 :b0.0384.49 0 929.79 0.117.81 0 6616.2 :b0.9213.3 0.213.8 0 914.4 1.7

Absorbed

11 . 8 :4 3 . 0 :6 7 . 6 :61 1 :5 5 . 6 :75 9 :

t 2.2t 6.0t 3.4t 10.5t 1.5b 1 777 7 5.9"8 1 . 5 :8 2 . 9 :21.1 d44.2d86.6d88.1 i8 2 . 7 :92.5d

b 2.8"b 1 0"b 6.6b 10.1b 0.8t 0.4b 1.4b 0.886.4 1.1"90.5 1.1"93.2d 0.6"

Treated

88 2 57.0 32.4 38 9 44.4 24.1 22.3 18.5 17.1 78.9 55.8 13.4 11.917 3 it7.5 13.6 +9.5 6.8

skin

2.26.03.410.51.51.75.92.81 0

6.610.10 80. 41.40. 81.11.10.6

Recovery (% ofapplied dose)

94.3 0.190.0 0.893.3 0.490 .4 0 3100.1 :87.5 :94.9 :96.2 :105 9 :

1003 :9 8 . 0 :98 7 :9 6 6 :102 . 0 :98.7 :87.9 :91.5 :

b1 7fc 8.1t O 7t 1.4t 3 . 0b8.5t 3.3t2 . 2t2 . 2t 2 . 6t 0 . 5b 1.8t 0 . 690.4 0.7

" Average of three animals SEM .'Tr eatm ent area was 2 .8 cm2 in young (33 DO) and 5.6 cm 2 in adult (82 DO) rats.' Body burden equals the sum of content in liver, kidney, carcass, and blood sample." S h a h a / . . ( 1 9 8 6 b ) .


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262 HALL ET AL.TABLE2Concentrationof[uC]Dinoseb-Derived RadioactivityinYoungandAdult Fischer344Rats following Dermal Dosing"

Dose'(nmol/cm2)Young

28 5

25 053 52680

Adult28 5

21 053 52680

Time(hr)

1624

4872120727272

16244872

120727272

Body

63.159.663.469.866.076.468.059.461.3

153161155145167153156148153

weight

+++++

2.11.00.70.41.41.32.31.62.2

5452146344

Kidneys

0.29 0.071.73 0.112.26 0.040.890.060.280.020.300.021.35 0.051.47 0.131.27 0.07

0.19 0.010.68 0.090.78 0.020.41 0.030.32 0.040.12 0.010.84 0.030.76 0.060.57 0.05

Liver

0.31 0.061.07 0.130.81 0.020.36 0.040.18 0.010.08 0.010.33 0.010.40 0.040.62 0.06

0.17 0.010.53 0.110.46 0.020.23 0.010.22 0.010.07 0.010.21 0.010.25 0.010.28 0.02

Carcass

0.17 0.040.63 0.070.54 0.020.26 0.040.11 0.010.06 0.0040.19 0.010.23 0.020.34 0.1

0.14 0.050.23 0.070.33 0.030.18 0.010.13 0.0030.04 0.0020.14 0.0060.16 0.0050.19 0.023

Concentration*Untreated skin

0.19 0.041.04 0.140.68 0.050.37 0.040.13 0.080.12 0.060.39 0.030.61 0.090.55 0.03

0.11 0.020.37 0.120.47 0.040.23 0.010.17 0.010.06 0.0020.18 0.010.21 0.020.23 0.03

Blood

1.26 0.243.49 0.272.79 0.171.38 0.180.52 0.030.30 0.040.89 0.021.17 0.061.50 0.04

0.70 0.061.56 0.211.74 0.160.92 0.010.66 0.040.22 0.010.69 0.040.72 0.051.02 0.08

Urine

5.96 1.121.2 7.512.8 0.86.80 1.92.20 0.23.70 0.64.10 0.54.60 0.44.00 0.6

(0.93 0.65) X 10 36.80 3.903.60 0.202.60 0.501.68 0.031.36 0.111.80 0.102.30 0.202.20 0.10

Feces

0.011 0.000.12 0.034.00 0.22.30 0.31.28 0.072.30 0.133.41 0.903.90 0.173.50 0.70

0.018 0.000.066 0.021.82 0.141.84 0.131.25 0.141.34 0.052.90 0.203.40 0.304.60 0.70

Mean of three animals SEM.*Percentage of recovered dose/g tissue wet weight.cTreatment area was 2.8 cm2in young (33 DO) and 5.6 cm2in adult (82 DO) rats.

higherinadults ,theratiowassignificantly less thanone forall tissues except kidney . Differencesinkinet ics (age X t im e)were notedforkidney, carcass, urine,andfeces.Areanalysesof the fractional co ntent data normalized for absorpt ionshows differences as a function ofa gein body, kidney,car-cass,ur ine ,and feces. Also noted were differencesinkineticsas a function of age in body, kidney, carcass, and urine(Table3).

6 x1 2

0 002.50 x1CH

0.000 00 10.00Timemin) 20 00F I G . 2. HP LC rad ioch roma togram of 24 hr urine collected from ratsadministered dinoseb in travenously (A) ordermally(B).Also shown is aradiochromatogram ofd inoseb(C).

Table4showstheyoung/ad ul t ra t iosofconcentrat ionodinoseb-derived radioactivity in tissuesand excreta.Significant differences were seen for liver, blood, untreated skincarcass,and feces. Kinetic differences were seen for kidneand feces. When normalized for absorption, only carcasand urine showed overall ageeffects. K idne y, carc ass,anfeces showed kinetic(age X time) differences as a functioof age.

A comparison of the three methods used to determindermal penetrat ion ofdinosebisshown inTable 5. Theinvivo resultsat 72 hr suggest greater absorption in the adul(84.3%) than in young (55.7%)aswellasdifferences in thkineticsof penetrat ion. The flow-through m etho d showedmuch lower absorpt ion in adults (20.6%)and a reversalithe young/adult rat io. The static system detected no agdifference between youngand adult exceptat the 1-h rsampling point,but did show kinetic differences as a functioof age.The fastest rateof ingress occurred withinthefirsthr with both invitromethods (Fig.3).Evidenceofreceptofluid saturationwasseen,forexample,as an increasein thrateof absorption between 10 and 20 hr after dosing witthe static cells.

A prel iminary physiological compartmental model(Fig1)was fittedto thedata.The results from themodelcomparedto thedataforyoungandadult ratsareshownin Fig4and theparametersaregiveninTable6. Thetreated skin


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DERMAL ABSORPTION OF DINOSEB 263TABLE3RatiosofAverage YoungtoAdult Tissue Contentsof[l4C]Dinoseb-Derived Radioactivity with Statistical Significance"*

Time(hr)1624

4872120MeanAg eTimeAgeXTime

Body0.53**0.950.72*0.760.36***0.850.72*-**

*.***.****

Kidneys0.821.29***1.49***1.22**0.47*1.701.22*-**

*,***,**

Liver0.791.020.820.780.450.830.81*

*NSC

Carcass0.50**0.88**0.66***0.710.35*-**0.820.67***

*.****

Urine18.19**2.040.860.69*0.78*'**0.83*0.80***

*.***.**

Feces0.991.160.870.54*-**0.84**0.77*0.74*

**

Absorbed0.560.970.78*0.69*0.67*0.82*0.76*

**

N S

Treated Skin1.12**1.022.42*3.26*2.56*3.21*1.54***

NS Model: fractional content= age,time,age X time.bApplied dermal dosage285nmol/cm2with exposure areaof2 .8cm2inyoung(33 DO) and 5.6 cm2inadult(82 DO).' N S = / 7 > O . O 5 .*p


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264 HALL ET AL.T A B L E 5Comparison of Skin Absorpt ion of [ l4 C]Dinoseb-Derived Radioactivity in Young

and Adult Fischer 344 Rats Determined by Three Methods"

Time(hr)1624

4872Anova'All

AgeAgeXTime

Young6.4 29.0 +41.0 45.0 47.3

1.03.53.73.83.9

Flow system

Adult2.411.215.718.620.6

0.62.2 2 . 5 3 . 0 3 . 5

In

Ratio*2.64*2.61*2.62*2.42*2.29*2.47**

*

Vitro

Young3.020.759.170.974.9

0.2 2.0 7.48.4 8.0

Static system

Adult1.517.052.366.270.8

0.1 1.13.13.83.8

Ratio*2.00*1.221.131.071.061.10*

*

Young11.138.863.055.355 7

2.1 5 . 7 2 . 9 9 . 7 2.4

In Vivo

Adult22.3 8 . 843.1 9 . 485.5 1.885.1 1.984.3 2.7

Ratio0.500.900.740.650.660.70*

'Percentageofapplied dose, mean SEM.* Young/adult.cAnalysiso fvariance*p


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DERMAL ABS ORP TION OF DINOS EB 265

0.0

co

1.00.80.60.40.20.0

*n yJo

B

Absorption

s_i0.70.6

c 0.5 0 . 4 0.3"" 0.20.10.0

Body 0.70.6c 0.59 0.4n 0.3

4 0.20.10.025 50 75 100 125 25 50 75 100 12 5

0.0180.0160.014g 0.012 0.010S 0.008i 0.0060.0040.0020.000

KidneyX / ^ i *P ^ ^ - i

0.0450.0400.035g 0.030S 0.025 0.020it 0.0150.0100.0050.000

Liver 0.040I 0.0355 0.0305 0.025a 0.020o 0.015Z 0.010E 0.0050.00025 50 75 100 125 25 50 75 100 125

Feces

Carcass

50 75Time (hours)

100 125

25 50 75 100 125Blood

25 50 75 100 1250.014i 0.012f 0.010 . 0.008g 0.006^ 0.0042 0.002" o.ooo

Skin A way

V ~ ~ ~ - ^ . ~25 50 75 100

Time (hours)12 5

F IG. 4 . In vivo skin absorption and disposition of dinoseb-derived radioactivity in young (33 DO) and adult (82 DO) Fischer 344 rats and modelresults: young observed (X ); adult observed value (D ): model prediction for young ()i model prediction for adult (- ) . T hree anim als were used foreach time point. Dosage was 285 nmol/cm 2 .

and Guthrie (1983) on carbaryl skin penetration suggests abiphasic process. Guy et ah (1985) have proposed a bio-physically based kinetic model of skin absorption that ismult iexponential . The mechanism for this phenomenon isbased on recognized cutaneous biology and penetrant mo-

lecular weight and lipophilicity. It is interesting thou gh thatin the present study a difference was noted in compartmentalsizes as a function of age.The effect of dosage of dinoseb on skin penetration wasexamined and scaled for the larger body size of the adults

TABLE 6Parameters for Compartmental Physiological Model of[' CIDinoseb-Derived Radioactivity Retentionin Young (33 DO) and Adult (82 DO) Fischer 344 Rats

Organ compartmentBloodKidneyLiverMuscleSkinRate constants

VT (ml)"14.0 W1.336.2578.0 (50)25.0 (16)fe, = 0 . l 2 0 f

fe K= 1.038feL = 0.0889

Adult (body wt, 156.0 g) 2 B (ml/min)*

38.95.8510.04.683.I3

fe 2 = 0 . 0 0 3 0 6 kfeu,=0.l4l kfeF, = 0 . 0 2 8 1 /

0.440.290.I80.25

:3 = 0.0204cU2 = 0.102c R = 0 .0l3

VT (ml)"5.97 (9)0.7283.21

33.2 (50)10.6 (16)fe, = 0.126feK = 0.661fe L = 0.0765

Young (body wt, 66.4 g)


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266 HALL ET AL.by increasing the area ofskin exposure. Constant fractionalpene tration , th e classical diffusional behavior, was observedwith this compound over the dosage range studied. Othercompounds that show this behavior include parathion andthe methylated arsenicals (Feldman and Maibach, 1974;Shah etal. 1987b). However, dose dependence on skin ab-sorption has been reported with other chemicals, includingtestosterone, benzoic acid, captan, and kepone (Wester andNo onan, 1980; Shah etal. 1987b).Dinoseb is excreted from the body in significant amountsand is extensively metabolized after exposure. The primaryroute of excretion is via the urine. We observed 57.7 and69.5% ofthedose excreted by this route in young and adultrats by 120 hr. Bandal and Casida (1972) reported that 60%of a po dose of dinoseb is excreted in the urine and 30% inthe feces by rats in 74 hr. We observed lower amounts offecal excretion with 12.5 and 16.2% in young and adult rats,respectively, after dermal exposure. Bandal and Casida re-ported over18m etabolites of dinoseb were excreted in urinealong with 1 of parent. Our results showed extensive me-tabolism of dinoseb after dermal and iv administration. Sev-eral metabolites were excreted in urine and m inimal a mou ntsof parent were detected. The radiochromatograms of theurine from the iv- and dermal-treated rats were similar. Ifskin is able to metabolize dinoseb is not known, because itmay penetrate unmetabolized and be transported to otherorgans with m etabolic capabilities prior to urinary excretion.Despite the detection of metabolites of dinoseb in urine,blood appears to contain only parent after feeding in ratsand pre gnant mice (Bandal an d Casida, 1972; Gibson andRao, 1973). However, liver and kidney radioactivity wasapproximately 50% parent in the mice (Gibson and Rao,1973). Liver/blood ratios of dinoseb-derived radioactivityafter oral dosing in rats and pregnant mice were approxi-mately 0.8 and kidney/blood ratio in mice was 0.69. Thefitted steady-state tissue/blood ratios (RT) from the physi-ological comp artmen tal model for kidney, liver, muscle, andskin were 0.68, 0.28, 0.14, and 0.30 in the young and 0.44,0.29, 0.18, and 0.25 in adult. Thus results reported here aresimilar to those reported in the literature.Much interest exists in the useofin vitromethods to de-termine dermal penetration because of ethical and safetyreasons. Two invitrodermal absorption methods, the clas-sical static Franz and the flow-through cell methods, wereused in this study. Good agreement between these in vitromethods using tritiated water, benzoic acid, and cortisonein absorption profiles and extent of absorption in additionto predictinginvivoresults have been reported by Bronaughand Stewart (1985). Modification ofthereceptor fluid withPEG-20 oleyl ether was necessary in their study with themore lipophilic compound cinnamyl anthranilate in orderto improve correlation with invivoresults. The results of thein vitroexperim ents were in general agreement with theinvivoresults, providing derm al pe netration values four or lesstimes the expected values. The results, however, were not

precise enough to detect the age difference identified withthe invivo study. As the in vitrostudies were run consecutively, the possibility exists that concurrent measurementswould be more powerful in terms of detecting the age dif-ferences found.A preliminary physiological compartmental model wasused to integrate and simulate the pharmacokinetics of di-noseb-derived radioactivity and d etermine the temporal pat-tern of absorption, the excretory rate constan ts, and the tissueto blood ratios at steady state. The results (Fig. 4 and Table6) ofthesimulation provide a good fit to the data, give rea-sonable estimates ofthe tissue to blood ratios, and identifydifferences in the excretory rate constants consistent withthe statistical analysis. In add ition, a biphasic pattern of skinabsorption was identified through the simulations. A lthoughthe results are derived from radioactivity measurem ents, themodel should still provide utility in the extrapolation of bodyburden and tissue concentrations following dermal exposure.Since a significant portion of the radioactivity excreted inurine by 24 hr were metabolites of dinoseb, the model thatis based on dinoseb-derived radioactivity is most likely de-scribing primarily these metabolites.The effect ofage,dose, and method of dermal penetrationassessment on the percutaneous penetration ofdinosebhasbeen determined in the Fischer rat. Dermal absorption wasshown to be dependent on age and independent of dose. I nvitro measurement of dermal absorption was found to produce results quantitatively similar to invivoresults, but thmethod was not sensitive enough to detect small differencesin skin absorption. Through the use ofapreliminary phys-iological compartmental model, kinetic differences in thedisposition of dinoseb in young and adult rats were identifiedand quantitated.

ACKNOWLEDGMENTSThe authors thank Nina J. Month, John H. Goodwin, Brenda C. Edwardsand Ja net J. Diliberto for their excellent technical assistance and M rs. NancyC. Sawaya and Ms. Nina Pittman for typing the manuscript.

REFERENCESBandal, S. K., and Casida, J. E. (1972). Metabolism and photoalteration

of 2-jec-butyl-4.6-dinitrophenol (DNBP herbicide) and its isopropyl car-bonate der ivat ive (dinobu ton acar icide) . J Agric. FoodChem 20 , 12351245.Bartek, M.J. . LaBudde. J. A., and Maibach, H. I. (1972). Skin permeabilityin vivo Co mparison in rat. rabbit, pig and m an. J Invest Dermaiol 58114-123.Behl. C. R.. Flynn. G. L.. Kunhara. T.. Smith. W. M . Bellantone. N. H..

Gatmaitan. O.. Pierson. C. L. Higuchi. W. I. , and Ho. N. F. H. (1984)Age and anatomical site influences on perm eation of skin of male hairlessmous e . J Soc. Cosmel Chem 35 , 237-252 .

Behl, C. R.. Bellantone. N. H.. and Flynn, G. L. (1985). Influence of ageon percutaneous absorption of drug substances. In Percutaneous Absorption Mechanisms-Methodology-Drug Delivery (R. L. Bronaugh and H. IMaibach, Eds.), Chap. 14. pp. 183-212. Dekker. New York.


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DERMAL ABS ORP TION OF DINOS EB 267Berman, M., Beltz, W. F.,Greif,P. C, Chabay, R., and Boston, R. C. (1983 ).

CONSAM User's Guide National Cancer Inst i tu te , DHHS.Bischoff, K. B., Dedrick, R. L., Zaha rko, D . S., and Longstreth, J. A. (197 1).Methotrexate pharmacokinetics. J. Pharm. Sci. 60, 1129-1133.Bronaugh, R. L., and Stewart, R. F. (19 85). M ethods forinvitropercutaneousabsorption studies. IV. The flow-through diffusion cell.J Pharm Sa. 74 ,64 -67 .Bungay, P. M., Dedrick. R. L, and Matthews, H.B .(1981) . Enteric transportof chlordecone (K epon e) in the rat.J. Pharmacokinet. Biopharm. 9 , 3 0 9 -

341.Collier, S. W., Sheikh, N. M., Sakr, A., Lichtin, J. L., Stewart, R. F., andBronaugh, R. L. (1989). Maintenance of skin viability during in vitropercutaneous absorption/metabolism studies . Toxicol. Appl. Pharmacol99 , 522 -533 .Durham, W. F.. and Wolfe, H. R. (1962). Measurement of exposure ofworkers to pesticides. Bull WHO 26 , 75-91 .Elmore, E., and Swift, M. (1977). Growth of human skin fibroblasts indialyzed fetal bovine serum. In Vitro 13, 837-842.Environmental Protection Agency (EPA) (1980). Interagency Agreement

between Department of Labor Employment Standards Administration andEnvironmental Protection Agency O ffice of Pesticide Programs. EPA-IAGNo. AR-16-F-0-038-0.

Feldman. R. J.. and Maibach, H. I. (1974). Systemic absorption of pesticidesthrough the skin of man. In Occupational Exposure to Pesticides. Reportto the Federal Working Group on Pest Managem ent from the Task G roupon Occupation Exposure to Pesticides, Appendix B, pp. 120-127.

Franklin, C. A., Somers, D. A., and Chu, I. (1989). Use of percutaneousabsorption data in risk assessment. J. Am. Coll. Toxicol 8 , 815-827.

Franz, T. J. (1 978 ). The finite dose technique as a valid in vitro model forthe study of percutaneous absorption in man. Curr Probl. Dermatol. 7,58-68.Gaines, T. B., and Linder, R. E. (1986 ). Acute toxicity of pesticides in adult

and weanling rats.Fundam. Appl. Toxicol. 1, 299 -308 .Gibson, J. E., and Rao, K. S. (1973). Disposition of 2-.s>butyI-4,6-dini-trophenol (Dinoseb) in pregnant mice. Food Cosmet. Toxicol. 11 , 45 -

52 .Guy, R. H., Hadgraft, J., and Maibach, H. I. (1985). Percutaneous absorptionin man: A kinetic approach. Toxicol Appl. Pharmacol. 78, 123-129.

Hawkins, G. S., Jr. , and Reifenrath, W. C. (1984). Development of an invitromodel for determining the fate of chemicals applied to skin. Fundam.Appl Toxicol. 4 , S133-S144.

Kao, J., Patterson, F. K.., and Hall, J. (1985). Skin penetration and metab-olism of topically applied chemicals in six mamm alian species, includingman: An in vitro s tudy with benzo(a)pyrene and testosterone. Toxicol.Appl. Pharmacol. 81 , 502 -516 .

Knaak, J. B., Yee, K., Ackerman, C. R., Zweig, G., and Wilson, B. W.(1984). Percutaneous absorption of Triadimefon in the adult and youngmale and female rat. Toxicol. Appl. Pharmacol. 72, 406-416.

Lutz, R. J., Dedrick, R. L., Matthews, H. B., Eling, T. E., and Anderson.M. W. (1977). A preliminary pharmacokinetic model for several chlo-rinated biphenyls in the rat. Drug. Metab. Dispos. 5 , 386 -396 .

McCormack. J. J. . Boisits, E. K., and Fisher. L. B. (1982). An in vitrocompariso n of the permeability of adult versus neonatal skin. In NeonatalSkin, Structure and Function (H. I. Maibach and E. K. Boisits, Eds.),Chap. 11, pp. 149 -164. Dekker, New York.

SAS Institute, Inc. (1985). SAS User's Guide: Statistics, version 5 SAS In-stitute, Inc., Cary, NC.Shah, P. V., and Guthrie, F. E. (1983). Percutaneous penetration of three

insecticides in rats: A comparison of two methods for in vivo determi-nation. J Invest. Dermatol. 80 , 291 -293 .Shah, P. V., Sumler, M. R., Ioannou, Y. M., Fisher, H. L., and Hall, L. L.

(1985). Dermal absorption and disposition of 1,4-Diphenylguanidine inrats.J. Toxicol. Environ Health 15 , 623 -63 3 .Shah, P. V., Fisher, H. L., Month, N. J., Sumler, M. R., and Hall, L. L.(198 7a). Derm al penetration of carbofuran in young and adult Fischer344 rats. J. Toxicol. Environ. Health22 , 207 -223 .Shah, P. V., Fisher, H. L., Sumler, M. R., Monroe, R. J.,Chernoff, N., and

Hall, L. L. (19 87b ). Com parison of the penetra tion of 14 pesticides throughthe skin of young and adult rats. J. Toxicol. Environ. Health 2 1 , 3 5 3 -366.

Wester, R. C , and Maibach, H. I. (198 5). Animal models for perc utaneou sabsorption. In Models in Dermatology (H. I. Maibach and Lowe, Eds.),Vol. 2, pp. 159-169. Karger, Basel.Wester, R. C , and N oona n, P. K. (19 80) . Relevance of animal models forpercutaneous absorption. Int. J Pharm. 7 , 99-110.Wester, R. C, Noo nan, P. K.., Cole, M. P., and M aibach, H. I. (197 7).Percutaneous absorption of testosterone in the newborn rhesus monkey:Comparison to the adult. Pediatr. Res 11 , 737 -739 .

age-related percutaneous penetration of 2-sec-butyl-4,6-dinitrophenol in rats

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