In vivo disposition of p-substituted phenols in the young rat after intraperitoneal and dermal administration

Pergamomt

Food and Chemical Toxicology 35 (1997) 697-704

In Vivo Disposition of p-Substituted Phenols

in the Young Rat after Intraperitoneal and Dermal Administration M. F. HUGHES*t and L. L. HALL:I: tManTech Environmental Technology, Inc., Research Triangle Park, NC 27709 and :l:NationalHealth and Envi::onmentalEffects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA (Accepted 4 February 1997)

A~tract--Tiae objective of this study was to examine the 120-hr disposition of phenol and four p-substituted congeners after ip and dermal administration in the 29-day-old female rat. The dermal absorption was very high (66-80% of the dose) for phenol, cyanophenol, heptyloxyphenol and nitrophenol, but minimal for hydroxybenzoic acid (2%). The major portion of the dose for all of the phenols not absorbed dermally in 24 hr was washed from the skin. Only minor amounts (1-2%) were detected in the treated skin at 120 hr. Urinary excretion was the predominant means of elimination for these phenols and occurred primarily within 24 hr after dermal and ip administration. However, the excretion of heptyloxyphenol after administration by both routes differed from that of the other compounds, with more of it detected in the faeces. The profile of metabolites in urine (collected at 12-24hr) from the animals dennally treated with phenol, cyanophenol, heptyloxyphenol and nitrophenol showed only peaks that eluted earlier than the parent compound, which suggests that conjugates or more polar metabolites were formed and excreted. The difference in dermal absorption between hydroxybenzoic acid and the other phenols may be due to potential ionization of the p-substituted carboxylic acid group of hydroxybenzoic acid. This suggests that, at least for the phenols examined in this study, physicochemical characteristics other than just lipophilicity can affect in vivo dermal absorption. © 1997 Elsevier Scie,~ce Lid

]INTRODUCTION The absorption of chemicals through the skin is an area of research tlaat has been extensively studied for a number of years. Dermal absorption can represent a major pathway for chemicals to enter the body, particularly in cases of occupational and environmental exposure, because the skin is constantly exposed to its surroundings. The skin is also a convenient site for the administration of drugs, as evidenced by the use of transdermai drug delivery systems. Many factors can affect the extent of dermal absorption of a chemical. Some of these factors include the physicochemical properties of the chemical, the attributes of the vehicle and the characteristics of the skin (Franklin et al., 1989; Wester and Maibach, 1986). One important element in dermal absorption is the lipid and water solubility properties of the chemical applied onto the skin. The stratum corneum, the outermost layer of skin, is essentially a lipid barrier, whereas the viable epidermis, which lies below the stratum corneum, is basi*Author for correspondence at: US EPA, NHEERL, MD74, Research Triangle Park, NC 27711, USA.

cally an aqueous environment (Guy and Hadgraft, 1991). Thus, the ability of a chemical to be absorbed through skin and enter the systemic circulation is in part determined by its ability to partition into both lipid and water environs. The age of the skin may also influence dermal absorption, but this effect is not absolutely distinct. The dermal absorption of some chemicals, such as carbofuran (Shah et aL, 1987a), dinoseb (Hall et al., 1992), 2,3,7,8-tetrachloro-p-dioxin and 2,3,4,7,8pentachlorodibenzofuran (Banks et al., 1990), has been shown to be affected by age, whereas dermal absorption of other chemicals, such as chlordecone, folpet and permethrin (Shah et al., 1987b), shows no age effect. This effect on dermal absorption may be due in part to changes in the anatomy, biochemistry and physiology of the skin as it ages (Kligman and Balin, 1989). Our laboratory has been interested in studying the effect of age on dermal absorption. These studies were originally initiated because of concerns over the exposure of young agricultural workers to pesticides (EPA, 1980). National recognition of the perils of pesticide exposure to children has been raised in the past few years (NRC, 1993). Because

0278-6915/97/$17.00 + 0.00 © 1997 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(97)00035-5

698

M.F. Hughes and L. L. Hall

Table 1. Molecular weight, octanol/water partition coefficient (log P) and acid dissociation constant (pKa) of five phenols Compound

Molecular weight

Phenol 94.1 4-Cyanophenol 119.1 4-Nitrophenol 139.1 4-Heptyloxyphenol 208.3 4-Hydroxybenzoic 138.1 acid

IogP

pKa

1.46 1.60 1.91 4.75 1.58

9.92 7.96* 7.15 10.84" 4.58 (COOH) 10.03 (OH)*

*Calculated by Pallus Software. children have a greater surface-to-volume ratio, are in various stages of developing physically and mentally, a n d - - i n the case of very young children-have an immature detoxification system, dermal exposure to chemicals may place children at a higher risk of their potential toxic effects than adults. Phenol is a common chemical used in industrial processes, is found in many commercial products, and is a contaminant in water, air and soil (Babich and Davis, 1981). Thus the potential exists for dermal exposure and subsequent absorption of phenol. There are several published reports on the in vitro and in vivo dermal absorption of phenol in man (Baranowska-Dutkiewicz, 1981; Bucks et al., 1988; Feldmann and Maibach, 1970; Piotrowski, 1971) and laboratory animals (Hinz et al., 1991; Hughes and Hall, 1995; Hughes et al., 1993 and 1994). The physicochemical characteristics of phenol and several of its substituted congeners, some of which are used as chemical intermediates, drugs and disinfectants, are well characterized (Table 1) (Hansch and Leo, 1979; Hinz et al., 1991; Leo et al., 1971). This has led to their use as model compounds in many dermal absorption studies (Hinz et al., 1991; Hughes et al., 1993 and 1994; Huq et al., 1986; Jetzer et al., 1986; Roberts et al., 1977 and 1978). There are limited data on the in vivo dermal absorption of chemicals in the young age group. This study investigated the effect of structure and chemical properties of various para-substituted phenols, which serve as model compounds, on the permeability of young rat skin. The dermal absorption and subsequent disposition results were compared with the disposition of the phenols after ip administration. The latter route of administration was considered to result in complete systemic absorption of compound. The data generated should increase the information base on the relationship of chemical structure and dermal absorption, as well as giving an indication of the permeability of chemicals in the young.

MATERIALS AND M E T H O D S

Uniformly Jac-ring labelled phenol, p-cyanophenol, p-nitrophenol, p-hydroxybenzoic acid and pheptyloxyphenol (sp. act. I00 pCi/~mol for phenol, 58.6 p C i / p m o l for the other compounds) were pur-

chased from Moravek Biochemicals (Brea, CA, USA). The radiochemical purity of the phenols was greater than 98% as determined by radiochromatography (see below). Flo-Scint II, Carbosorb, Instagel and Permafluor V were obtained from Packard Instruments Co. (Downers Grove, IL, USA). HPLC-grade methanol and water were purchased from Burdick and Jackson (Muskegon, MI, USA). Ether was obtained from Fisher Chemical Co. (Fair Lawn, N J, USA). Trifluoroacetic acid was purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were of the highest grade commercially available. A Hewlett Packard (Palo Alto, CA, USA) 1090 Liquid Chromatograph with a Hypersil ODS Cts column (Hewlett Packard, 100 x 2.1 mm, 5 #m particle size) and a Radiomatic Flo-One/fleta radiochromatography detector model A280 (Packard) were used to analyse the phenols. The chromatography solvents were 1.5% (v/v) aqueous trifluoroacetic acid (A) and methanol (B). The system was run under isocratic conditions (95% A/5% B) with the flow rate set at 0.5 ml/min for the analysis of hydroxybenzoic acid, cyanophenol, nitrophenol and phenol. Column effluent was mixed with Flo-Scint II (1.5 ml/min) in a 0.05-ml flow cell for detection of radioactivity. A water/methanol gradient was used for heptyloxyphenol analysis. Initial conditions were set at 40% methanol and held for 5 min postinjection. The solvent mixture was then changed linearly to 100% methanol over 5 min and held for an additional 5 min. Initial conditions were then reestablished. The retention times of the phenols were hydroxybenzoic acid 6min, nitrophenol 7.7min, phenol 7.2 min, cyanophenol 7.2 min and heptyloxyphenol 11 min. Female Fischer 344 rats were purchased from Charles River Laboratories (Raleigh, NC, USA). The animals were maintained according to the National Institutes of Health Guide on the Care and Use of Laboratory Animals and were initially housed in polycarbonate cages with woodchip bedding and kept on a 12-hr light/dark cycle. The room temperature was kept at 25°C (+2 °) with a relative humidity of 50% (__+10%). The animals were provided with Purina Rat Chow No. 5001 (St Louis, MO, USA) and water ad lib. 3 days before dosing the rats were placed individually in metabolism cages (Nalgene, Rochester, NY, USA) and provided with Dustless Precision Pellets (BioServe Inc., Frenchtown, N J, USA) and water ad lib. 24 hr before dosing, the animals designated for dermal exposure were lightly anaesthetized with CO2 (Clifford, 1984) and the hair on their dorsal skin surface was removed with an electric clipper (blade size 40, Oster Corp., Milwaukee, WI, USA). The clipped skin was then washed with a small volume of acetone to remove sebaceous gland secretions and other debris. A restraint consisting of latex tubing and wire was placed around the animal behind the forelegs (Bartek et al., 1972) and kept on throughout the experiment.

Intraperitoneal and dermal disposition of phenols Animals were dosed with [14C]phenol or one of the para-substituted congeners by ip (2.5 #g, approx. 1 pCi) or 6ermal (5 pg, 3.9 #g/cm 2, approx. 2 pCi) routes. The ip dosed animals received each compound in 0.05 ml water-propylene glycol-ethanol (5/4/1, by vol). The restraint was removed from the animals designated for dermal treatment. The animals were lightly anaesthetized with CO2 and supplemented with ether when required. Once the animal was anaesthetized, a circular area (1.27 cm 2) was marked with a permanent marker on the clipped area. The chemical in 10~1 ethanol was applied in small drops within the marked area. Once the ethanol had dried, a 5-ml plastic cup was glued over the tre~.ted skin with cyanoacrylate adhesive (Loctite Cerp., Newington, CT, USA) to protect the treated :~ite. This resulted in occlusion of the treated skin and was done because of our concerns about the volatility of phenol. The restraint was then put back on the animal. The animals were 29 _ 2 days old and their weight ranged from 52 to 74 g on the day of dosing. The rats were returned to the metabolism cages after dosing. Urine and faeces were collected at 4, 8, 12, 24, 48, 72, 96 and 120 hr, weighed after collection and stored at -70°C until analysed. Because a small volume of urine was excreted by the rats at the 4-12-hr time points, all of it was mixed with Instagel and analysed for radioactivity in a Packard 2260 liquid scintillation counter. For the remaining

699

urine samples, aliquots (100 ~1) were analysed for radioactivity after mixing with scintillant. Faeces were air-dried, weighed, pulverized, and combusted in a Packard D-306 oxidizer before analysis for radioactivity. 24 hr after dosing, the dermally treated animals were lightly anaesthetized with CO2 and supplemented with ether when required. The top of the plastic cup was cut to provide an opening over the treated area. A l ml wash solution consisting of liquid Ivory soap/water ( l / l ) was applied to the treated skin with an automatic pipette. Using forceps, a cotton ball was gently rubbed on the skin to absorb the soap solution and chemical. The soap wash was repeated twice and was followed by two (l ml) water washes (with cotton ball), and finally a dry wash with a cotton ball. The cotton ball from each wash (total of five) was transferred to a scintillation vial and analysed for radioactivity (Bucks, 1990). Another plastic cup was glued over the original cup and the animals were returned to the metabolism cages. The animals were killed by CO2 asphyxiation at 120 hr after treatment. A sample of untreated skin was removed from the dermally treated animals. The plastic cup was removed, placed in a scintillation vial and analysed for radioactivity. The treated skin area was then cut away. The carcasses were frozen in liquid nitrogen and homogenized in a Waring blender. The skin and samples of the

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Fig. 1. Excretion of chemical-derived radioactivity after administration of hydroxybcnzoic acid (ll), cyanophenol (A), heptyloxyphenol (r"l), nitrophenol (0), and phenol (<)). (A) urinary excretion after ip administratio:a of compound; (B) faecal excretion after ip administration; (C) urinary excretion after dermal application; (D) faecal excretion after dermal application.

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700

M.F. Hughes and L. L. Hall

whole-animal homogenate were weighed, cornbusted, and analysed for radioactivity. Aliquots of the 12-24 hr urine collected from the dermally treated animals were concentrated in v a c u o and reconstituted in water. Portions of this concentrated urine were incubated with 6 N HC1 at 37°C overnight. These samples were analysed by HPLC, as described above. The urine from the ip-treated animals and the hydroxybenzoic acid dermally-treated animals were not analysed because of the low number of counts in the urine at this timepoint. The results are expressed as percentages of the recovered dose, which is defined as the sum of radioactivity excreted in the urine and faeces and that detected in the carcass. For the dermally-treated animals, the recovered dose also includes the skin wash and treated and untreated skin. The recovered dose (mean + SD) was 97.1 + 5.4% for the ip-treated animals and 98.5 _ 19.8% for the dermally-treated animals. Comparisons between chemicals were made using the Tukey-Kramer multiple comparisons test only if the analysis of variance was significant (P < 0.05) (InStat, Graphlad Software, San Diego, CA, USA).

RESULTS

Urinary excretion of chemical-derived radioactivity after ip administration of phenol, cyanophenol, hydroxybenzoic acid and nitrophenol was rapid, with 80-90% of the dose excreted within 4 hr. The elimination in urine of these compounds was essentially complete by 8-12hr after administration (Fig. 1A). Heptyioxyphenol-derived radioactivity was excreted in lower amounts in the urine compared with the other phenols after ip administration, with 35% of the dose eliminated by 4 hr and 55% by 24 hr. The maximal rates of urinary excretion of the phenols after ip administration ranged from 20 to 23% of the dose/hr for hydroxybenzoic acid, phenol, cyanophenol and nitrophenol (Table 2). The rates for the latter compounds were significantly greater than that for heptyloxyphenol (approx. 8% of the dose/hr). The maximal excretion rates for all of the phenols were attained within 4 hr postexposure.

Table 2. Maximal rate of urinary excretion of phenols after ip and dermal administration Route

Chemical Hydroxybenzoic acid Phenol Cyanophenol Nitrophenol Heptyloxyphenol

ip

Dermal

19.6_+ 1.0 (4)

0.04 ± 0.01 b'c (24)

22.2 ± 0.4 (4) 23.2 _ 0.4 (4) 21.6 + 1.5 (4) 8.4 ± 2.5a (4)

10.2 _+2.5 (4) 7.4 ± 2.4 (4) 6.9 _ 2.0 (4) 3.4 ± 1.40 (8)

Values are mean percentage of close excreted in urine/ hr ± SD of three or four animals. Time (hr) of maximal excretion in parentheses. aSignificantly lower than all other compounds administered ip (P < 0.001); bSignificantlylower than phenol administered dermally (P < 0.001); CSignificantlylower than cyanophenol and nitrophenol administered dermally (P < 0.01); dSignificantly lower than phenol administered dermally (P < 0.01); Tukey-Kramer multiple comparisons test. Approximately 2-4% of the dose of phenol, hydroxybenzoic acid and nitrophenol was excreted in the faeces by 24hr after ip administration (Fig. 1B). Cyanophenol had the lowest and heptyloxyphenol the highest faecal excretion of the five phenols, with less than 1% and 30% of the dose eliminated by 24 hr, respectively. The 120-hr cumulative excretion of phenol, cyanophenol, hydroxybenzoic acid, and nitrophenol after ip administration ranged from 87 to 97% in urine and from 1 to 4% in faeces (Table 3). A significantly lower and higher percentage of heptyioxyphenol was excreted in the urine (60%) and faeces (33%), respectively, than was observed with the other phenols. The carcasses of the ip-treated animals were analysed for radioactivity (Table 3) and approximately 0.5% of the dose was detected in the phenol-, cyanophenol- and nitrophenol-treated animals. A significantly higher percentage of the dose (7-10%) was detected in the carcasses of heptyloxyphenoland hydroxybenzoic acid-treated animals. After dermal administration, the urinary excretion of the phenols was lower than in the iptreated animals. For phenol, cyanophenol and nitrophenol, approximately 30-40% of the dose was excreted in the urine by 4 h r (Fig. 1C) and increased to 60-70% by 24hr. The urinary ex-

Table 3. Disposition of chemical-derivedradioactivity 120 hr after ip administration Chemical Location Body Urine Faeces

Hydroxybenzoic acid

Phenol

Cyanophenol

Nitrophenol

Heptyloxyphenol

10.2_+ 1.3a 86.5 ± 1.3c'd 3.4 ± 0.4

0.5_+0.3 96.4 ± 1.8 3.1 -+ 2.0

0.4±0.2 98.3 ± 0.6 1.3 ± 0.7

0.4+0.1 94.5 ± 2.8 5.0 ± 2.8

7.1 ±0.3 b 59.7 -+ 3.4e 33.2 -+ 3.6~

Values are mean percentage of dose + SD of three or four animals. a Significantlygreater than all other compounds (P < 0.001); b Significantly greater than phenol, cyanophenol and nitrophenol (P < 0.001); CSignificantlylower than phenol and cyanophenol (P < 0.001); aSignificantlylower than nitrophenol (P < 0.01); eSignificantlylower than all other compounds (P < 0.001); Tukey-Kramer multiple comparisons test.

lntraperitoneal and dermal disposition of phenols

701

Table 4. Disposition of chemical-derivedradioactivity 120 hr after dermal application Chemical Location Untreated skin Treated skin Wash" Body Urine Faeces

Hydroxybenzoic acid

Phenol

Cyanophenol

Nitrophenol

Heptyloxyphenol

< 0.005

< 0.005

< 0.005

< 0.005

< 0.005

1.8 + 0.2

2.2 ___2.5

0.9 + 0.2

0.7 + 0.3

0.9 + 0.5

95.9 _+0.3~ 0,28 _+0.01 1.9 _+0.3d 0.04 + 0.19

17.9 _+5.8 0.8 + 0.9 76.8 + 8.2 2.3 + 1.1

33.2 ___6.7b 0.2 _+0.1 64.7 + 3.7 1.0 ___0.5

29.8 _+3.5~ 0.4 _+0.1 66.3 + 7.5 2.8 + 2.5

31.1 ___3.1¢ 0.38 _+0.04 53.4 "t- 2 . 5 e'f 14.2 + 0.9a

Values are mean percentage of the dose _+SD of three or four animals. *Skin wash 24 hr after dermal application. ~Significantlygreater than all other compounds (P < 0.001); bSignificantlygreater than phenol (P < 0.01); ¢Significantly greater than pher~ol IP < 0.05); dSignificantlylower than all other compounds (P < 0.001); ~Significantlylower than phenol (P < 0.001); 'Significantlylower than nitrophenol (P < 0.05); Tukey-Kramer multiple comparisons test. cretion of heptyloxyphenol had a noticeable lag, with 7% of the dose excreted by 4 hr and 50% by 24hr. Excretion of hydroxybenzoic acid in the urine was lowest of that of all the compounds after dermal application, with less than 1% of the dose eliminated in 24 hr. The maximal rates of urinary excretion of the phenols after demlal administration (Table 2) ranged from 0.4% (for hydroxybenzoic acid) to 10% of the dose/hr (for phenol). The rate for phenol, cyanophenol and nitrophenol was significantly greater than the rate for hydroxybenzoic acid. The rate for phenol was also significantly greater than that for heptyloxyphenol. The time for the maximal excretion rate also varied, with 4 hr for phenol, cyanophenol and nitrophenol, 8 hr for heptyloxyphenol and 24 hr for hydroxybenzoic acid. Faecal excretion of radioactivity by 24 hr after dermal administration of hydroxybenzoic acid, cyanophenol, phenol and nitrophenol ranged from less than 1% to 2% of the dose (Fig. 1D). By 24hr, heptyloxyphenol had the highest faecal excretion of the five phenols, with approximately 12% of the dose eliminated by this time. After dermal exposure, the 120-hr cumulative urinary and faecal excretion of phenol, cyanophenol and nitrophenol r~nged from 65 to 77% and 1 to 3% of the dose, respectively (Table 4). Cumulative excretion of heptyloxyphenol was significantly lower in urine and higher in faeces, with 53 and 14% of the dose respectively, compared with phenol, cyanophenol and nitrophenoi. Hydroxybenzoic acid had the lowest cumulative percentage of the dose excreted in the urine and faeces, with 2 and less than 0.1%, respectively. The amount excreted in urine for hydroxybenzoic acid was significantly lower than that for the other phenols. 24 hr after dermal application of the phenols, the treated skin was washed to remove unpenetrated compound (Table 4). This wash removed significantly greater amounts of hydroxybenzoic acid than that of the other compounds. The amounts of cyanophenol, nitrophenol and heptyloxyphenol

removed by the wash were also significantly greater than the amount of phenol removed. In the dermally treated animals, the carcass, treated-skin site and a piece of untreated skin were analysed for radioactivity (Table 4). Less than 1% of the dose of the phenols was detected in the carcass and untreated skin. In the treated skin, 0.7-2.2% of the dose was detected, with nitrophenol having the lowest and phenol the highest percentage of the dose at this site. Summation of the percentage of the dose of the phenols excreted in urine and faeces can serve as a rudimentary measure of their absorption through skin. Figure 2 displays the absorption of these compounds over time after dermal administration. Phenol had the highest absorption by 24 hr (75% of the dose), followed by nitrophenol, cyanophenol and heptyloxyphenol (62-66% of the dose). Less than 1% of the dose of hydroxybenzoic acid was absorbed by 24 hr. The total absorbed dose of the phenols applied dermally can be approximated by including the radioactivity in the carcass at 120hr with the cumulative urinary and faecal excretion data. Phenol had the highest absorbed dose, with 80.0+8.1% and was followed by nitrophenoi 10090-

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Fig. 3. Chromatographic profiles of urine from rats excreted 12-24 hr after dermal administration of [14C] labelled (A) cyanophenol, (B) heptyloxyphenol, (C) nitrophenol and (D) phenol. The arrow indicates the retention time of the parent compound. (69.5 + 6.8%), heptyloxyphenol (68.0 + 2.8%) and cyanophenol (65.9 + 6.7%). Absorption of hydroxybenzoic acid (2.3 + 0 . 3 % ) was significantly lower than that of the other compounds. The radiochromatograms of concentrated urine from animals treated dermally with cyanophenol, heptyloxyphenol, nitrophenoi and phenol are shown in Fig. 3. In all cases there was one large predominant early eluting peak. No parent was detected in any of the analysed urine. The early peak in the cyanophenot, nitrophenol and phenol samples, but not the heptyloxyphenol sample, was sensitive to acid treatment. Peaks coeluting with parent were found in the acid-sensitive samples, suggesting that the early peaks were conjugates linked to the phenolic group.

DISCUSSION Considerable amounts of phenol, cyanophenol, nitrophenol and heptyloxyphenol (66-80% of the dose) were absorbed through the skin of the 29day-old rats. In contrast, a minimal amount of hydroxybenzoic acid (2% of the dose) was absorbed. However, once all five phenols penetrated the skin, the major portion of the absorbed dose was excreted in the urine. This excretion occurred primarily within 24hr, indicating that the compounds were not retained by the animals after their dermal absorption. The compounds excreted in urine (except for hydroxybenzoic acid), at least

between 12-24hr after dermal administration, appeared to be in the form of a conjugate or more polar metabolite than parent compound. There were some differences in the disposition of the phenols after ip compared with dermal administration. Although urinary excretion was the major means of elimination, regardless of the route of exposure, it was faster after ip administration, as evidenced by the higher maximal rates of urinary excretion; furthermore, the urinary elimination of the ip-administered compounds was basically complete within 8-12hr postexposure. The maximal urinary excretion rates of the dermally treated animals were lower and occurred at later time points for hydroxybenzoic acid and heptyloxyphenol; furthermore the urinary excretion for the five compounds was not complete until 24 hr after dermal administration. This effect on urinary excretion of the dermally administered compounds was probably due to a hindrance in absorption by the skin. The predominant barrier to absorption in skin is the lipid-rich stratum corneum, the outermost epithelial layer of skin, which is not found in other tissues or organs. In contrast, after ip administration, the chemical has only to traverse the blood vessels within the peritoneal cavity in order to be absorbed. Only a small percentage of the dose of the phenols was detected in the carcass of the dermallytreated animals. Whereas similar results were observed with phenol, cyanophenol and nitrophenol

Intraperitoneal and dermal disposition of phenols after ip administration, 7-10% of the dose of hydroxybenzoic acid and heptyloxyphenol was retained in the carcass. The reason for this difference is not known, but perhaps the two chemicals were sequestered in the peritoneum. Heptyloxyphenoi, which was the most lipophilic phenol used, may have diffused into the fat of the peritoneum. In the case of hydroxybenzoic acid, the carboxylic acid functional group, which has a pKa of 4.6, may have ionized in the peritoneum, retarding its absorption. The excretion of beptyloxyphenol differed from that of the other phenols after dermal and ip administration. Although urinary excretion of this chemical was still greater than faecal excretion, the percentage in the urine was lower and that in the faeces higher than observed with the other compounds. This difference is also reflected in the significantly lower maximal urinary excretion rate of heptyloxyphenol than the other compounds after ip administration and compared with phenol after dermal administration. A glucuronide conjugate, which could be formed by the coupling of glucuronic acid with the phenolic group on heptyloxyphenol, would have a molecular weight of more than 300, making it a candidate for biliary excretion. Thus the mechanism of heptylox~phenol excretion in the rat differs from that of the other phenols, because it may be more readily eliminated in the bile than the other phenols. The in vivo disposition of phenol after dermal administration is similar between the 29- and 90-dayold female rat (Hughes and Hall, 1995). Approximately 80°,/0 of the dose of phenol was absorbed in 120 hr in the former and 72 hr in the latter animal. In both age groups, urinary excretion was the major means of elimination of the compound and it occurred primarily within 24 hr postexposure. The profile of urinary metabolites excreted by the animals of these two age groups was also comparable. Finally, a very minor amount of compound was retained in the animals of both ages. The in vivo dermal absorption of the phenols used in this study, except for hydroxybenzoic acid, were very similar. On the basis of the percentage of the dose absorbed alone, it appears that the lipophilicity of these similarly absorbed phenols did not overtly influence their dermal absorption. Heptyloxyphenol is approximately 1000 times more lipophilic than phenol, yet these two compounds differed in absorption by only 15%. Although the maximal urinary excretion rate for heptyloxyphenol was significantly lower than that for phenol after dermal absorption, this difference is probably due to the higher biliary excretion of heptyloxyphenol. In addition, cyanophenol and nitrophenol differ significantly from heptyloxyphenol with respect to lipophilicity, yet their cumulative absorption was very similar. What :may have influenced the dermal absorption of hydroxybenzoic acid--even though its lipophilicity is similar to that of cyanophenol,

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nitrophenol and phenol--is the ionization potential of its carboxylic acid group (pKa of 4.58). Huq et al. (1986) have shown that, for several substituted phenols, their in vitro dermal absorption is reduced when they are ionized. Perhaps the carboxylic acid group on hydroxybenzoic acid is ionizing on the skin after application, leading to reduced dermal absorption of the compound. In summary, four of the five phenols examined were able to penetrate the skin of 29-day-old rats to an extent greater than 65% of the dose. The dermally absorbed dose for all the compounds was primarily excreted in urine and not retained in the body. The results suggest, at least for the phenols examined in this study, that the ionization potential of the p-substituted functional group may have a pronounced effect on the dermal absorption of the compound. Acknowledgements--The authors thank Brenda Edwards and Carol Mitchell for their technical assistance. Although the research described in this article has been funded wholly by the US Environmental Protection Agency under contract number 68-D2-0056 to ManTech Environmental Technology, Inc., it has not been subjected to Agency review. Therefore, it does not necessarily reflect the views of the Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. REFERENCES

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