Percutaneous Absorption, Metabolism, and Hemolytic Activity of n-Butoxyethanol

Percutaneous Absorption, Metabolism, and Hemolytic Activityof n-Butoxyethanol. BARTNIK, F. G., REDDY, A. K., KLECAK, G.,ZIMMERMANN, V., HOSTYNEK, J. J., and KUNSTLER, K. (1987). Fundam.Appl. Toxicol. 8, 59–70. A series of studies was conductedto examine the percutaneous absorption, distribution, excretion,and hemolytic activity of n-butoxyethanol (BE). Rats receivinga subcutaneous dose of ¹⁴C-labeled BE excreted the radioactivityin the urine (79%), expired air (10%), and feces (0.5%) within72 hr. Of the organs analyzed, thymus and spleen showed elevatedspecific radioactivities as compared with blood. A percutaneousapplication of BE on rats, under nonocclusive conditions, showed25–29% absorption within 48 hr. Peak blook levels of BEoccurred at 2 hr after application; butoxyacetic acid (BAA)was found to be the major metabolite. Comparison of in vitroskin penetration data showed the following absorption patternof BE: hairless rat >> pig > human skin. Hemolysisand associated hematological changes were noted in the ratswhich received single dermal applications of 260–500 mg/kgof BE. In vitro, BAA showed markedly greater hemolytic abilityon rat erythrocytes than did BE. Human erythrocytes showed nohemolysis when incubated with BE or BAA at concentrations thatare hemolytic to rat erythrocytes. An intravenous dose of 62.5mg/kg of BE does not result in hemolysis or hemoglobinuria inthe rat. The rat may be an animal model with increased susceptibilityto the effects of BE compared with humans because of its rapidpercutaneous absorptive ability and its greater hemolytic sensitivity.     

Identification of Stage-Specific Changes in Protein Secretion by Isolated Seminiferous Tubules from the Rat Following Exposure to Either m-Dinitrobenzene or Nitrobenzene

The objective of this study was to identify effects of two knownSertoli cell toxicants on the secretion of proteins by seminiferoustubules (ST) isolated from adult rats at different stages ofthe spermatogenic cycle and cultured in vitro for 24 hr with[³⁵S]methionine Adult rats received a single oral dose of 50mg/kg metadinitrobenzene (m-DNB) or 300 mg/kg nitrobenzene (NB).Long lengths of ST at stages II–V, VI–VII or IX–XIIwere then isolated from control and treated rats at 1 or 3 dayspost-treatment; selection of stages was based on the stage specificityof the early (24–72 hr) adverse effects of m-DNB and NBon spermatogenesis in vivo. In addition, ST at the same stageswere isolated from untreated rats and cultured in the presenceor absence of m-DNB or NB (10^–4 M). Incorporation of [³⁵S]methionneinto secreted proteins was assessed and the pattern of proteinsecretion evaluated using two-dimensional sodium dodecyl sulfate-polyacrylamidegel electrophoresis (2-D SDS-PAGE). ST isolated from rats pretreated24 hr earlier with NB in vivo showed a significant decreasein the overall incorporation of [³⁵S]methionne into secretedproteins at stages VI–VIII and IX–XII, whereas STat stages II–V showed no such change; comparable proteinchanges were observed when 10^–4M NB was added in vitrofor 24 hr to ST isolated at the same stages from untreated rats.Similar results were obtained when ST protein secretion wasevaluated 72 hr after treatment with NB in vitro or after theaddition of NB in vitro to isolated ST from untreated rats for24 or 72 hr. In general, the results obtained after exposureto m-DNB were comparable to those observed for NB, with theexception that the incorporation of [³⁵S] into secreted proteinsby ST at stages II–V tended to be increased by m-DNB exposure.Analysis of ST-secreted proteins by 2-D SDS-PAGE identified6 "marker proteins" (MP) which showed major reproducible changesin secretion following exposure to either m-DNB or NB. In mostcases (MP–1, –2, –3, –5, and –6)their secretion was reduced markedly; two of these proteins(MP–2 and MP–3) are secreted almost exclusivelyat stages VI–VIII and correspond in molecular size andpI to two recently reported androgen-regulated proteins whichare thought to be products of the Sertoli cell. Exposure toeither NB or m-DNB also resulted in the appearance of one protein(MP–4) which was not secreted in detectable amounts byST from control animals. Previous data has shown that by 24hr after administration of either m-DNB or NB to rats thereis massive loss/degeneration of pachytene spermato cytes atstages VI–XII, whereas stages II–V of the spermatogeniccycle are not affected until 72 hr. The presently observed proteinchanges therefore either precede (stages II–V) or accompany(stages VI–XII) germ cell degeneration. This suggeststhat they may have potential use as markers of early toxicant-induceddamage and/or that changes in these proteins may mediate someof the adverse testicular effects of m-DNB and NB.     

Further Validation of an in Vitro Method to Reduce the Need for in Vivo Studies for Measuring the Absorption of Chemicals through Rat Skin

Current requirements for the registration of agrochemicals,particularly in the U.S.A., often require the provision of dermalabsorption data. In this process the rat is often used and complexin vivo studies, using large numbers of animals, are performed.We have compared the data obtained from in vivo and in vitrodermal absorption studies using eight pesticides with a rangeof physicochemical properties. Measurements were made of the¹⁴C-labeled pesticides which could be washed from the skin,were associated with (on/in) skin, or absorbed through the skinfollowing dermal applications in vivo and in vitro at varioustime points over a 24-hr exposure period. Good agreement wasfound between the amounts washed from and associated with theskin in vivo and in vitro. Over the time period 4–24 hrafter application the in vitro experiments predicted the invivo absorption within a factor of 2–3. These resultsshow that, with a range of pesticide molecules, the in vitromethod accurately predicted in vivo absorption supporting theutilization of the in vitro method for risk assessment fromexposure to pesticides and other chemicals.     

In Vivo and in Vitro Percutaneous Absorption and Skin Evaporation of Isofenphos in Man

Studies were done to determine the percutaneous absorption ofisofenphos in human volunteers from whom informed consent hadbeen obtained. In vivo absorption in man was 3.6±3.6%of applied dose for 24-hr exposure and 3.6±0.5% for 72-hrexposure. Skin wash recovery data show that isofenphos evaporatesfrom in vivo skin during the absorption process; the surfacedose is minimal (<1%) by 24 hr. Skin stripping showed noresidual isofenphos in stratum corneum. This explains the similarabsorption for 24 and 72-hr dose prewash exposures. Skin surfacerecovery in vivo with soap and water was 61.4±10.4 forthe first dosing time (15 mm). Time-recovery response declinedwith time to 0.5±0.2% at 24 hr. In vitro absorption utilizingflow-through diffusion methodology with human cadaver skin andhuman plasma receptor fluid gave 2.5±2.0% dose absorbed,an amount similar to In vivo studies. An additional 6.5±24%was recovered in the skin samples (total of 9%). Skin surfacewash at 24 hr recovered 79.7±2.2% and skin content was6.5±2.4% (total dose accountability of 88.7±4.6%).Thus, isofenphos was available for absorption during the wholedosing period. Neither in vitro absorption nor in vitro evaporationstudies predicted the potential skin evaporation of isofenphos.Published dermal studies in the rat had predicted isofenphosabsorption at 47% of applied dose (12-fold greater than actualin man). Subsequent toxicokinetic modeling predicted possibleconcern with the use of isofenphos. This is an example wherethe choice of the rat produced a nonrelevant absorption prediction.In vivo studies in human volunteers seem more relevant for predictingpercutaneous absorption in man.     

In Vivo and in Vitro Percutaneous Absorption and Skin Decontamination of Arsenic from Water and Soil

The objective was to determine the percutaneous absorption ofarsenic-73 as H₃A_sO₄ from water and soil. Soil (Yolo County65-California-57-8) was passed through 10-, 20-, and 48-meshsieves. Soil retained by 80 mesh was mixed with radioactivearsenic-73 at a low (trace) level of 0.0004 µg/cm² (microgramsarsenic per square centimeter skin surface area) and a higherdose of 0.6 µg/cm². Water solutions of arsenic-73 at alow (trace) level of 0.000024 µg/cm² and a higher doseof 2.1 µg/cm² were prepared for comparative analysis.In vivo in Rhesus monkey a total of 80.1 ± 6.7% (SD)intravenous arsenic-73 dose was recovered in urine over 7 days;the majority of the dose was excreted in the first day. Withtopical administration for 24 hr, absorption of the low dosefrom water was 6.4 ± 3.9% and 2.0 ± 1.2% fromthe high dose. In vitro percutaneous absorption of the low dosefrom water with human skin resulted in 24-hr receptor fluid(phosphate-buffered saline) accumulation of 0.93 ± 1.1%dose and skin concentration (after washing) of 0.98 ±0.96%. Combining receptor fluid accumulation and skin concentrationgave a combined amount of 1.9%, a value less than that in vivo(6.4%) in the Rhesus monkey. From soil, receptor fluid accumulationwas 0.43 ± 0.54% and skin concentration was 0.33 ±0.25%. Combining receptor fluid plus skin concentrations gavean absorption value of 0.8%, an amount less than that with invivo absorption (4.5%) in the Rhesus. These absorption valuesdid not match current EPA default assumptions. Washing withsoap and water readily removed residual skin surface arsenic,both in vitro and in vivo. The partition coefficient of arsenicin water to powdered human stratum corneum was 1.1 x 10⁴andfrom water to soil it was 2.5 x 10⁴. This relative similarityin arsenic binding to powdered human stratum corneum and soilmay indicate why arsenic absorption was similar from water andsoil. This powdered human stratum corneum partition coefficientmodel may provide a facile method for such predictions.     

The Predictive Accuracy of in Vitro Measurements for the Dermal Absorption of a Lipophilic Penetrant (Fluazifop-Butyl) through Rat and Human Skin

The predictive accuracy of in vitro measurements in estimatingdermal absorption has been evaluated in rat and human skin usingfluazifop-butyl (FB), a lipophilic model compound, at dosagerates of 2.5, 25, and 250 µg/cm² In vitro studies usedrat and human epidermal membranes mounted in static diffusioncells with radiolabeled FB and receptor fluids of 50% aqueousethanol (Aq Et), 6% polyethylene glycol 20 oleyl ether in saline(PEG), or tissue culture medium (TCM). In vivo rat studies withradiolabeled FB were carried out to parallel previously publishedhuman volunteer studies. For rat skin, in vitro measurementswith all types of receptor fluid provide an adequate prediction(generally within a factor of 3) of in vivo absorption. Absorptiondata for human epidermal membranes with a receptor fluid ofAq Et were adequately predictive of the in vivo absorption.In contrast, membranes with PEG or TCM significantly underestimatedthe in vivo absorption. The results support the conclusion thatin vitro studies are useful to predict in vivo dermal absorptionin rat and man, when appropriate receptor fluids are used.     

Absorption, Metabolism, and Excretion of N,N-Diethyl-m-toluamide Following Dermal Application to Human Volunteers

The absorption, metabolism, and excretion of N,N-diethyl-m-toluamide(DEET) in male human volunteers following dermal applicationof |¹⁴C|DEET was studied. DEET was applied to two groups ofsix volunteers either as the undiluted technical grade materialor as a 15% solution in ethanol. The material was applied overa 4 x 6-cm area on the volar surface of the forearm and wasleft in contact with the skin for 8 hr, then rinsed off theskin. Application sites also were tape stripped at 1, 23, and45 hr after rinsing. Serial blood samples and all urine andfeces were collected for 5 days after application. Aliquotsof these materials were analyzed for total radioactivity inorder to define absorption and excretion patterns. Urine samplesalso were analyzed by HPLC to characterize the metabolic profileand/or to identify metabolites. Absorption of DEET as evidencedby plasma radioactivity occurred within 2 hr after dose application.Elimination of radioactivity from plasma was rapid and quantifiablelevels of radioactivity were observed in plasma for only 4 hrafter the end of the 8-hr exposure period. Urine was the principalroute of excretion of radioactivity and accounted for an averageof 5.61 and 8.33/ of the applied dose in the undiluted DEETand 15/ DEET in ethanol groups, respectively. Excretion of radioactivityin the feces was less than 0.08/ of the applied dose in bothgroups. DEET did not accumulate in the superficial layers ofthe skin as evidenced by low amounts of radioactivity in thetape strippings. The major fraction of the applied radioactivitywas recovered in the skin rinses. Absorbed DEET was completelymetabolized and six major metabolites were observed in urine.Two major urinary metabolites tenta tively were identified.Based upon the percentage of applied dose recovered in the excreta,dermal absorption of DEET ranged from 3 to 8% with a mean of5.6/ in the volunteers applied undiluted technical grade DEET.The corresponding values for the volunteers applied 15/ DEETin ethanol were 4 to 14/ and 8.4/, respectively.     

Vitamin A Potentiation of Vinylidene Chloride Hepatotoxicity in Rats and Precision-Cut Rat Liver Slices

Pretreatment of large doses of vitamin A (VA) is known to potentiatethe hepatotoxicity of carbon tetrachloride. Therefore the effectsof 1-day VA pretreatment on VDC hepatotoxicity was examinedboth in vivo and in an in vitro system of precision-cut ratliver slices. Male Sprague-Dawley rats were pretreated with250,000 IU/kg VA by oral gavage. After 24 hr rats were administered50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices wereprepared from VA pretreated rats 24 hr later and the liver sliceswere exposed for 2–8 hr to 0.025–1.0 µl VDCevaporated into the gas phase of the incubation vials. VA pretreatmentresulted in an enhancement of VDC toxicity, both in vivo andin vitro. There was a dose-dependent increase in plasma ALT24 hr after VDC treatment of rats and an increase in K⁺ leakagefrom liver slices after VDC exposure. Histological analysisof the liver or the liver slices revealed that VA+VDC treatmentresulted in centrilobular necrosis of the liver. When GdCl₃(10 mg/kg iv) was administered just before VA pretreatment ofrats, VDC toxicity was partially reversed as observed by a decreasein ALT in vivo and a decrease in the loss of K⁺ in vitro. Theseresults indicated that Kupffer cells, the resident macrophagesof the liver, were partially responsible for the VA-potentiatedVDC hepatotoxicity. One-day pretreatment of VA induced cytochromeP450IIE1 protein content as well as its enzymatic activity asmeasured by p-nitrophenol hydroxylation. Because VDC is bioactivatedby cytochrome P450IIE1, the increase in VDC hepatotoxicity afterVA may be due to an increased bioactivation of VDC in the liverand in precision-cut liver slices. Thus, more than one mechanismmay be involved in the VA enhancement of VDC hepatotoxicity.     

Percutaneous Absorption of [14C]DDT and [14C]Benzo[a]pyrene from Soil

Percutaneous Absorption of [¹⁴C]DDT and [¹⁴C]Benzo[a]pyrenefrom Soil. WESTER, R. C, MAIBACH, H. I., BUCKS, D. A. W., SEDIK,L., MELENDRES, J., LIAO, C, AND DIZIO, S. (1990). Fundam. Appl.Toxicol. 15, 510–516. The objective was to determine percutaneousabsorption of DDT and benzo[a]pyrene in vitro and in vivo fromsoil into and through skin. Soil (Yolo County 65-California-57-8;26% sand, 26% clay, 48% silt) was passed through 10-, 20-, and48-mesh sieves. Soil then retained by 80-mesh was mixed with[¹⁴C]-labeled chemical at 10 ppm. Acetone solutions at 10 ppmwere prepared for comparative analysis. Human cadaver skin wasdermatomed to 500 µm and used in glass diffusion cellswith human plasma as the receptor fluid (3 ml/hr flow rate)for a 24-hr skin application time. With acetone vehicle, DDT(18.1 ± 13.4%) readily penetrated into human skin. Significantlyless DDT (1.0 ± 0.7%) penetrated into human skin fromsoil. DDT would not partition from human skin into human plasmain the receptor phase (<0.1%). With acetone vehicle, benzo[a]pyrene(23.7 ± 9.7%) readily penetrated into human skin. Significantlyless benzo[a]pyrene (1.4 ± 0.9%) penetrated into humanskin from soil. Benzo[a]pyrene would not partition from humanskin into human plasma in the receptor phase (<0.1 %). Substantivity(skin retention) was investigated by applying ¹⁴C-labeled chemicalto human skin in vitro for only 25 min. After soap and waterwash, 16.7 ± 13.2% of DDT applied in acetone remainedabsorbed to skin. With soil only 0.25 ±0.11% of DDT remainedabsorbed to skin. After soap and water wash 5.1 ±2.1%of benzo[a]pyrene applied in acetone remained absorbed to skin.With soil only 0.14 ±0.13% of benzo[a]pyrene remainedabsorbed to skin. In vivo percutaneous absorption of DDT inrhesus monkey was significantly less (p < 0.02) from soil(3.3 ± 0.5%) than from acetone solution (18.9 ±9.4%). DDT in vitro skin penetration values into human skinwere similar to in vivo absorption values in the rhesus monkey.In vivo absorption in the rhesus was not statistically differentfrom published in vivo absorption in man (10.4 ± 3.6%).In vivo percutaneous absorption of benzo[a]pyrene in rhesusmonkey was significantly less (p < 0.015) from soil (13.2± 3.4%) than from acetone solution (51.0 ± 13.2%).Thus, with in vitro and animal in vivo systems relevant to man,skin absorption of DDT and benzo[a]pyrene from soil was significantlyless than when the chemicals were applied to skin in acetoneSolvent.     

Comparison of the penetration of 14 pesticides through the skin of young and adult rats

In vivo percutaneous absorption of 14 pesticides was studied in young (33-d-old) and adult (82-d-old) female Fischer 344 rats, at three different dose levels. Carbon-14-labeled pesticides in acetone were applied to previously clipped middorsal skin. The treatment area was 2-3% of the body surface area. Penetration of the pesticides during a 72-h period ranged from approximately 1%-90%, depending on compound, dose, and age of animal. No clear age-related pattern of dermal absorption among compounds was found. Only chlordecone, folpet, and permethrin did not show significant age-dependent differences in skin penetration. Atrazine, carbaryl, chlorpyrifos, and hexachloro-biphenyl had greater absorption in the young, while carbofuran, captan, dinoseb, DSMA, MSMA nicotine, and parathion displayed greater absorption in the adult. The majority of the compounds showed dose-dependent penetration. The dose-response curves for penetration were not parallel for 8 of the 14 compounds studied.     

Acute, Distribution, and Subchronic Toxicological Studies of Succinate Tartrates

The estimated single-dose oral toxicity (50% lethality) of succinatetartrates (ST) was 2–3 g/kg in rats. ST produced minimalto moderate dermal irritation but no evidence of systemic toxicityin a standard acute percutaneous toxicity test in rabbits. STwas not an eye irritant in a standard rabbit low-volume eyeirritation test ST was not genotoxic in a series of six genotoxicitytests. A 14-day oral gavage study in rats at a dose range of0.05–1.0 g ST/kg/day produced only gastric irritation.The no-observed-effect level (NOEL) for gastric irritation was0.1 g/kg for males and 0.05 g/kg for females. A 28-day percutaneoustoxicity study in rabbits produced minimal to moderate dermalirritation and no adverse systemic effects at a high dose of450 mg ST/kg/day. Single-dose absorption, distribution, andelimination (ADE) studies in male rats showed that 10–15%of an oral dose and 1–3% of a dermal dose were absorbed.Approximately 98% of the orally administered ST was eliminatedas ¹⁴C in urine, feces, or expired CO₂ after 72 hr. Approximately80% of the dermally absorbed ¹⁴C dose was eliminated in urine,feces, or expired CO₂ after 72 hr. In conclusion, no adverseeffects were noted in acute toxicity, genotoxicity, or subchronictoxicity studies conducted with ST.     

Dermal penetration of [14C]captan in young and adult rats.

Age dependence in dermal absorption has been a major concern in risk assessment. Captan, a chloroalkyl thio heterocyclic fungicide, was selected for study of age dependence as representative of this class of pesticides. Dermal penetration of [14C]captan applied at 0.286 mumol/cm2 was determined in young (33-d-old) and adult (82-d-old) female Fischer 344 rats in vivo and by two in vitro methods. Dermal penetration in vivo at 72 h was about 9% of the recovered dose in both young and adult rats. The percentage penetration was found to increase as dosage (0.1, 0.5, 2.7 mumol/cm2) decreased. Two in vitro methods gave variable dermal penetration values compared with in vivo results. A static system yielded twofold higher dermal penetration values compared with in vivo results for both young and adult rats. A flow system yielded higher dermal penetration values in young rats and lower penetration values in adults compared with in vivo results. Concentration in body, kidney, and liver was less in young than in adult rats given the same absorbed dosage. A physiological pharmacokinetic model was developed having a dual compartment for the treated skin and appeared to describe dermal absorption and disposition well. From this model, tissue/blood ratios of captan-derived radioactivity for organs were found to range from 0.35 to 3.4, indicating no large uptake or binding preferences by any organ. This preliminary pharmacokinetic model summarizes the experimental findings and could provide impetus for more complex and realistic models.     

Dermal penetration of carbofuran in young and adult Fischer 344 rats

Dermal penetration of carbofuran was determined in young (33 d) and adult (82 d) female Fischer 344 rats employing in vivo and in vitro methods. In vivo dermal penetration at 120 h was 43% for young and 18% for adult rats. The half-time for carbofuran skin penetration (in vivo) was 128 h for the young and 400 h for the adults. The young to adult ratio of dermal penetration was greater than 1 at all time points (average 2.9) and had a maximum of 4.2 at 24 h. Cumulative urinary excretion approached about 95% of the absorbed dose in both the young and adult animals at 120 h. Whole-body retention was slightly higher in adults. Kidney showed the highest tissue-to-blood concentration ratio (4.6 in adult, 2.3 in young). The ratio for the carcass was 2.8 in the adult and 2.4 in the young. The urine/blood concentration ratio was high, 435 in the adult and 573 in the young. The feces/blood ratio was 44 in the adult and 65 in the young. Skin absorption by the in vitro continuous-flow system was 41% for the young and 11% for the adult at 72 h, compared to 36% and 13% by the in vivo method. The static in vitro method gave consistently lower skin penetration values of 12% for the young and 8.8% for the adult. Differences in the kinetics of retention and excretion were observed between the young and adult animals.     

Nonclinical Toxicology Studies with Zidovudine: Genetic Toxicity Tests and Carcinogenicity Bioassays in Mice and Rats

Zidovudine (ZDV), an antiviral drug active in the treatmentof acquired immunodeficiency syndrome (recommended human dose,100 mg every 4 hr while awake), was evaluated for mutagenicand carcinogenic potential in a battery of short-term in vitroand in vivo assays and in lifetime studies in mice and rats.In L5178Y mouse lymphoma cells (tk^+/– locus), a weak positiveresult was obtained only at the highest concentrations tested(4000 to 5000 µg/ml) in the absence of metabolic activation.In the presence of metabolic activation, the drug was weaklymutagenic at concentrations of 1000 µg/ml and higher.Following 24 hr treatment in the absence of metabolic activation,ZDV was moderately mutagenic at concentrations up to 600 µg/ml;dose-related structural chromosomal alterations were seen atconcentrations of 3 µg/ml and higher in cultured humanlymphocytes. Such effects were not noted at the two lowest concentrationstested, 0.3 and 1 µg/ml, and BALB/c-3T3 cells were transformedat concentrations of 0.5 µg/ml and higher. No effectswere seen in the Ames Salmonella plate incorporation and preincubationmodification assays (possibly due to bacteriocidal activityof ZDV at low concentrations) at concentrations ranging from0.01 to 10 µg/plate or in a single-dose intravenous bonemarrow cytogenetic assay in CD rats. In multidose micronucleusstudies, increases in micronucleated erythrocytes were seenin mice at doses of 100 to 1000 mg/kg/day. Similar results wereseen in rats and mice after 4 or 7 days of dosing at 500 mg/kg/day.In carcinogenicity bioassays, adjusted doses of 20, 30, or 40mg/kg/day and 80, 220, and 300 mg/kg/day were given to CD-1mice and CD rats, respectively, for up to 22 months in miceand 24 months in rats. ZDV caused a macrocytic, normochromicanemia in both species. No evidence of carcinogenicity was seenin male mice or rats. In female mice, five malignant and twobenign vaginal epithelial neoplasms occurred in animals given40 mg/kg/day. A single benign vaginal epithelial tumor was seenin a mouse given 30 mg/kg/day. In rats, two malignant vaginalepithelial neoplasms were seen in animals given 300 mg/kg/day.In a 7-day study in mice, ZDV was shown to be devoid of estrogenicactivity. In an oral pharmacokinetics study, the AUC was 17and 144 µg/ml hr in female mice and rats given 40 or 300mg/kg of ZDV, respectively. In contrast, the average steady-stateconcentration in humans at the recommended daily dose is 0.62µg/ml. Twenty-four hour urine concentrations were 1245and 4417 µg/ml in female mice and rats given 40 or 300mg/kg of ZDV, respectively. These values were approximately26-and 136-fold higher than the human urine concentration atthe recommended daily dose. In a one- to three-day study withintravenously administered sodium fluoroscein in rats and mice,retrograde flow of urine into the vagina was demonstrated. Ina subsequent lifetime carcinogenicity bioassay in mice in whichZDV was given intravaginally at concentrations of 5 or 20 mgZDV/ml in saline, 13 vaginal squamous cell carcinomas were seenat the highest concentration tested. It was concluded that thevaginal tumors seen in the oral carcinogenicity studies werethe result of chronic local exposure of the vaginal epitheliumto high urine concentrations of ZDV.     

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution, and Skin Decontamination

Glyphosate Skin Binding, Absorption, Residual Tissue Distribution,and Skin Decontamination. Wester, R. C., Melendres, J., Sarason,R., McMaster, J., and Maibach, H. I. (1991). Fundam. Appl. Toxicol.16, 725–732. Glyphosate is a broad-spectrum postemergencetranslocated herbicide. Its interactions with skin and potentialsystemic availability through percutaneous absorption was studiedby skin binding, skin absorption, residual tissue distribution,and skin decontamination. Glyphosate in a final formulation(Roundup) undiluted and diluted with water 1:20 and 1:32, wouldnot partition into powdered human stratum corneum (<1%).In vitro percutaneous absorption through human skin into humanplasma as receptor fluid was no more than 2% over a concentrationrange of 0.5–154 µg/cm² and a topical volume rangeof 0.014–0.14 ml/cm². Disposition of glyphosate followingiv administration of 93 and 9 µg doses to rhesus monkeyswas mainly through urine excretion, 95 ± 8 and 99 ±4% in 7 days, respectively. Percutaneous absorption in vivoin rhesus monkey was 0.8 ± 0.6% for the low dose (25µg/cm²) and 2.2 ± 0.8% for the high dose (270 µg/cm²).No residual ¹⁴C was found in organs of the monkeys euthanized7 days after the topical application. Washing the skin applicationsite with soap and water removed 90 ± 4% of applied dose,and washing with water only removed 84 ± 3% of applieddose. Both soap and water and water only were equal in abilityto remove glyphosate from skin over a 24 hr skin applicationperiod. About 50% of the initially applied dose could be recoveredafter 24 hr. Glyphosate is very soluble in water and insolublein most organics (octanol/water log P = –1.70) and thereforenot compatible with the lipid-laden stratum corneum. This isconsistent with the low skin binding and skin absorption andalso consistent with the efficient removal from skin with soapand water or water-only wash.     

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPase Activity in Rat Brain Synaptosomes

Toxaphene Inhibition of Calmodulin-Dependent Calcium ATPaseActivity in Rat Brain Synaptosomes. PRASADA RAO, K. S., TROTTMAN,C. H., MORROW, W., AND DESAIAH, D. (1986) Fundam. Appl. Toxicol.6, 648–653. Effect of toxaphene on Ca⁺²-ATPase activityin rat brain synaptosomes was studied in vitro and in vivo.Ca⁺²-ATPase in calmodulin-depleted synaptosomes was inhibitedin vitro to a maximum of about 50% at 150 µM toxaphenc.Substrate activation kinetics of Ca⁺²-ATPase in synaptosomesrevealed that toxaphene inhibited the enzyme activity noncompetetivelyby decreasing V_max values, without affecting the enzyme-substrateaffinity. Toxaphene inhibited the calmodulin activated Ca⁺²-ATPaseactivity in a concentration-dependent manner with an IC50 of10 µM, a concentration at which no significant effectwas observed on basal enzyme activity. Nuclear and P₂ fraction(synaptosomes) calmodulin levels were reduced significantlyin toxaphene-treated rats. The synaptosomal Ca⁺²-ATPase wasalso reduced to about 45% in toxaphene-treated rats and theactivity was restored to normal levels by the exogenously addedcalmodulin. These results suggest that toxaphene may cause synapticdysfunction by in terfering with calmodulin and its regulationof neuronal calcium.     

In Vivo Percutaneous Absorption of Boric Acid, Borax, and Disodium Octaborate Tetrahydrate in Humans Compared to in Vitro Absorption in Human Skin from Infinite and Finite Doses

Literature from the first half of this century report concernfor toxicity from topical use of boric acid, but assessmentof percutaneous absorption has been impaired by lack of analyticalsensitivity. Analytical methods in this study included inductivelycoupled plasma-mass spectrometry which now allows quantitationof percutaneous absorption of ¹⁰B in ¹⁰B-enriched boric acid,borax, and disodium octaborate tetrahydrate (DOT) in biologicalmatrices. This made it possible, in the presence of comparativelylarge natural dietary boron intakes for the in vivo segmentof this study, to quantify the boron passing through skin. Humanvolunteers were dosed with ¹⁰B-enriched boric acid, 5.0%, borax,5.0%, or disodium octaborate tetrahydrate, 10%, in aqueous solutions.Urinalysis, for boron and changes in boron isotope ratios, wasused to measure absorption. Boric acid in vivo percutaneousabsorption was 0.226 (SD = 0.125) mean percentage dose, withflux and permeability constant (K_p) calculated at 0.009 µg/cm²/hand 1.9 x 10^–7 cm/h, respectively. Borax absorption was0.210 (SD = 0.194) mean percentage of dose, with flux; and K_pcalculated at 0.009 µg/cm²/h and 1.8 x 10^–7 cm/h,respectively. DOT absorption was 0.122 (SD = 0.108) mean percentage,with flux and K_p calculated at 0.01 µg/cm²/h and 1.0 x10^–7 cm/h, respectively. Pretreatment with the potentialskin irritant 2% sodium lauryl sulfate had no effect on boronskin absorption. In vitro human skin percentage of doses ofboric acid absorbed were 1.2 for a 0.05% solution, 0.28 fora 0.5% solution, and 0.70 for a 5.0% solution. These absorptionamounts translated into flux values of, respectively, 0.25,0.58, and 14.58     

Metabolic Alterations Induced by Topical Dimethylacetylenedicarboxylate

Metabolic Alterations Induced by Topical Dimethylacetylenedicarboxylate.KLAIN, G. J., BONNER, S. J., AND BELL, W. G. (1988). Fundam.Appl Toxicol 10, 730–735. The disposition of topical dimethylacetylenedicarboxylate(DMAD) in tissue and its effect on glucose metabolism were studiedin vivo, using skin grafted athymic nude mice, and in vitro,using excised pig skin. [¹⁴C]DMAD that penetrated skin graftswas distributed throughout the body. At 24 hr, the liver contained15.62% of the applied dose. The kidneys, lungs, brain, and theheart contained 12.73, 5.61,0.36, and 3.24% of the dose, respectively.One hour postapplication, DMAD markedly decreased [U-¹⁴C]glucoseoxidation and the syntheses of fatty acids and glycogen in thelivers and skin grafts. Similar effects were observed in excisedpig skin. In addition, the activities of hepatic glucose-6-phosphatedehydrogenase, isocitric and NADP-mahc dehydrogenase, and acetyl-CoAcarboxylase were significantly reduced in DMAD-treated mice.In contrast, no effect was observed on the activity of glucokinase.The data indicate that DMAD rapidly penetrates the skin andcauses aberrations in the activities of the glycogenic. lipogenic,and tricarboxylic acid metabolic pathways.     

Dose-dependent Disposition of n-Hexane in F-344 Rats After Inhalation Exposure

Dose-dependent Disposition of n-Hexane in F-344 Rats After InhalationExposure. Bus, J.S., Deyo, D. and Cox M. (1982). Fundam. Appl.Toxicol. 2:226-229. The purpose of this study was to assessthe disposition of radioactivity in rats after single inhalationexposures to varying concentrations of [l,2-¹⁴C]-n-hexane. MaleFischer 344 rats were exposed to 500,1000,3000 or 10,000 ppm¹⁴C-n-hexane for 6 hr and the elimination of radioactivity followedfor 72 hr after exposure. The disposition of radioactivity wasdose-dependent, with 12, 24, 38 and 62% of the acquired bodyburden excreted as n-hexane by the lung with increasing exposureconcentration. In contrast, 38, 31, 27 and 18% of the body burdenof radioactivity was recovered as expired ¹⁴CO₂ and 35,40,31and 18% was recovered in the urine with increasing n-hexaneconcentration. Radioactivity remaining in the tissues and carcass72 hr after exposure represented 6.1,8.8, 7.4 and 5.4% of thebody burden for the respective exposures. The dose-dependent'eliminationof radioactivity was apparently due in part to an inhibitionof n-hexane metabolism, reflected by a decrease in total ¹⁴CO₂and urinary ¹⁴C excretion after 10,000 ppm exposure comparedto the 3000 ppm exposure.     

Evaluation of Changes in the Secretion of Immunoactive Inhibin by Adult Rat Seminiferous Tubules in Vitro as an Indicator of Early Toxicant Action on Spermatogenesis

Evaluation of Changes in the Secretion of Immunoactive Inhibinby Adult Rat Seminiferous Tubules in Vitro as an Indicator ofEarly Toxicant Action on Spermatogenesis. Allenby, G., Foster,P. M. D., and Sharpe, R. M. (1991). Fundam. Appl. Toxicol 16,710–724. A method for culturing isolated seminiferoustubules (ST) from adult rats for 1–3 days has been developedand optimized rigorously on the basis of the secretion of immunoactiveinhibin under basal conditions and after maximal stimulationwith rat FSH or dibutyryl cyclic AMP. The effect on these culturesof three known testicular toxicants was assessed. Of these,two are thought to act on the Sertoli cell, meta-dinitrobenzene(mDNB) and nitrobenzene (NB), while the third, methoxy aceticacid (MAA), is thought to act on pachytene spermatocytes. Inaddition, the effect of a possible testicular toxicant, 3-mononitrotoluene(3-MNT), was investigated. These data were compared with thoseobtained using cultures of immature rat Sertoli cells (SC) orSC + germ cells and with data on the effect of equivalent dosesof the compounds on the secretion of immunoactive inhibin invivo. In studies designed to optimize conditions for the secretionof immunoactive inhibin by ST in culture, significant effectswere found of the type of culture medium used, the durationof culture, the total and individual length of tubules used,etc. All subsequent studies with toxicants utilized optimalconditions. Addition of either mDNB or NB to ST cultures at10^–5 or 10^–3 m, or MAA at 10^–4 m, stimulatedbasal secretion of immunoactive inhibin by two- to fourfoldon Days 1, 2, or 3 of culture while FSH or dibutyryl cyclicAMP-stimulated secretion of immunoactive inhibin was eitherunaffected or was enhanced to a small extent. At the same doses,mDNB or NB also enhanced secretion of immunoactive inhibin bySC cultures, although these effects were more variable and ofsmaller magnitude than the effects on ST cultures. In contrast,addition of up to 10^–3 m MAA to cocultures of SC + germcells had no effect on the secretion of immunoactive inhibin.Exposure of rats in vivo to levels of mDNB, NB, or MAA similarto those which stimulated secretion of immunoactive inhibinin vitro resulted in a two-to fourfold increase in the levelsof immunoactive inhibin in testicular interstitial fluid (IF)at 1 and 3 days post-treatment, and this was associated withearly impairment of spermatogenesis (as judged by testis weight).In contrast to these effects, addition of 3-MNT to ST or SCcultures had no effect except at 10^–3 m, when the secretionof immunoactive inhibin was increased marginally. Treatmentof rats with an equivalent dose of 3-MNT in vivo resulted indeath, but exposure to the highest nonlethal dose (1 g/kg) hadno significant effect either on spermatogenesis or on the levelsof immunoactive inhibin in testicular IF. In view of these findings,it is concluded that modulation of the secretion of immunoactiveinhibin by isolated ST from adult rats has considerable potentialas an in vitro screening method for investigating potentialadverse effects of chemicals on spermatogenesis, and thus meritsmore detailed evaluation. Moreover, because of the agreementbetween in vitro and in vivo findings, measurement of the levelsof immunoactive inhibin levels in vivo in testicular IF (orblood) may also be useful in the detection of early adverseeffects of chemicals on spermatogenesis.