Pulmonary and percutaneous absorption of 2-propoxyethyl acetate and 2-ethoxyethyl acetate in beagle dogs.

A comparison was made of the absorption and elimination rates of 2-propoxyethyl acetate (PEA) and 2-ethoxyethyl acetate (EEA) following inhalation, dermal application or IV administration. Male beagle dogs were exposed to 50 ppm PEA or EEA for 5 hr, and breath samples were collected during the exposure and a 3-hr recovery period. Both compounds were rapidly absorbed through the lungs. After 10 min of exposure, the concentrations of the parent compounds in the expired breath were 5 to 10 ppm (80-90% absorption) and reached plateau values at about 3 hr of 13 ppm for PEA (74% absorption) and 16 ppm for EEA (68% absorption). Post-exposure breath samples declined exponentially to 0.5 ppm and 2 ppm after 3 hr for PEA and EEA, respectively. Expired concentrations of PEA were slightly, but significantly (p less than 0.025), lower than those of EEA at corresponding times during the exposure. After IV dosing with 1 mg/kg [ethyl-1,2-14C]PEA, the urine contained 61% and 88% of the dose in 4 and 24 hr, respectively. [14C]EEA was eliminated more slowly, with 20% and 61% of the dose appearing in the urine in 4 and 24 hr, respectively. Blood elimination half-lives were 1.6 hr for [14C]PEA and 7.9 hr for [14C]EEA. Only trace amounts of 14CO2 (less than 1%) or volatile materials (less than 0.1%) were detected in the expired air with either compound. For studies of percutaneous absorption, [14C]PEA or [14C]EEA was added to undiluted compound and applied in a glass cell to a shaved area on a dog's thorax for 30 or 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
The ethylene glycol ethers have been used extensively in industry and consumer products for a number of years. They are highly miscible with water and organic solvents and have found application in coatings, dyes, insecticides, soaps and cosmetics. Exposure may therefore occur both by skin contact and inhalation.
In general, this class of compounds exhibits low to moderate toxicity and only slight skin irritation, even though most are readily absorbed percutaneously (1). Recent reports, however, have indicated that some ethylene glycol ethers and their acetic acid esters produce reproductive effects in laboratory animals (2)(3)(4)(5). Both 2-ethoxyethanol and 2-ethoxyethyl acetate have been reported to produce teratogenic effects and testicular changes in rodents (2,5,6). After repeated, high oral *Biochemistry Section, Health, Safety, and Human Factors Laboratory, Eastman Kodak Company, Rochester, NY 14650. tAuthor to whom all correspondence should be addressed. doses (2200-4400 mg/kg), 2-propoxyethyl acetate produced testicular atrophy in rats (7). 2-Propoxyethanol administered orally for 6 weeks at approximately 1500 mg/kg did not produce reproductive effects in male rats (2). In view of these findings, a comparison was made of the absorption and elimination rates of two glycol ether derivatives, 2-propoxyethyl acetate and 2-ethoxyethyl acetate, in dogs after inhalation, dermal application or intravenous administration.

Animals
Male beagle dogs weighing 10 to 13 kg were obtained from Marshall Research Animals, Inc. (North Rose, NY). Dogs used for skin application and intravenous (IV) administration experiments were fasted for 16 hr before use. A suitable area of skin on the dog's thorax was shaved one day prior to a skin absorption experiment. Atropine sulfate and Ace-Promazine were administered as pre-anesthetics. Dogs were anesthetized with sodium pentobarbital for the duration of the experiment. Dogs used for inhalation experiments were not fasted prior to use.

Methods
Inhalation Exposure. Inhalation exposures were conducted at a concentration of 50 ppm with either PEA (299 mg/m3) or EEA (270 mg/m3). Two animals, housed in separate cages, were exposed in each experiment. Four animals were exposed to each compound.
The exposure facility consisted of a 32,000 L interior room, equipped with an independent, adjustable air supply system. The exposure room was maintained at a slight negative pressure. Filtered air was supplied to the exposure facility at a rate of 10,000 L/min which resulted in 19 air changes per hour.
The test substance was metered into a 5-L roundbottomed flask by using a dual syringe pump (Modern Metalcraft, Midland, MI). The flask was heated to 110°C, and the vaporized compound was flushed from the flask with a stream of dried nitrogen (50 L/min). The vapor was carried into the room air supply via 1/2 in. stainless steel tubing. The room concentration of test compound was monitored continuously with a Beckman Model 400 Hydrocarbon Analyzer (Beckman Instruments, Inc., Fullerton, CA). The hydrocarbon analyzer was calibrated with a 50 ppm standard of each test compound. The time-weighted average exposure concentrations, calculated from the output of the hydrocarbon analyzer, were 50.3 + 1.6 ppm for PEA and 50.8 ± 1.1 ppm for EEA (x ± SD). A sample of room air was collected during each exposure and assayed by gas chromatography to verify the room concentrations.
All sample handling systems used in these experiments were fabricated of either Teflon, glass or stain-less steel. Expired air samples were collected utilizing a close-fitting mask fabricated from Teflon. The mask was constructed with a sampling port connected to a length of Teflon tubing, a breathing port, and a pressure indicator. At sampling times, the mask was strapped to the animal's head, and a 25-mL repeating dispenser (Brinkmann Instruments, Westbury, NY) was connected to the sampling port of the mask. A 750-mL Teflon gas sampling bag was connected to the outlet of the repeating dispenser. A breath sample was aspirated from the mask on the upstroke of the dispenser and dispensed into the gas sampling bag on the downstroke. Pooled end tidal breath samples (approximately 30 breaths) were collected during the late phase of exhalation.
A breath sample was collected from each dog before it entered the inhalation chamber and after 10, 20, 40, 60, 90, 120, 180, 240 and 300 min of exposure. The dogs were removed from the chamber and breath samples were collected at 10, 20, 40, 60, 90, 120 and 180 min after exposure.
Breath and room atmosphere samples were analyzed on a Varian 2100 gas chromatograph (Varian Instrument Division, Palo Alto, CA) equipped with a flame ionization detector. Gaseous samples were injected through a 25-mL sampling valve (Carle Instruments, Inc.). Gas chromatographic separation was achieved on a 6-ft glass column packed with 20% Carbowax 20M on 80/100 Gas-Chrom Q at 75°C. For quantitation, sample peak areas were compared to those of external standards. The standards were prepared by vaporizing a measured volume of test compound with air metered through a Singer No. 802 Flowmeter (Singer, American Meter Division) into a Teflon gas sampling bag.
Statistical evaluation of the data was accomplished with a program developed by the Statistical Analysis System Institute (8).
Intravenous Administration. For intravenous administration experiments, the radioactive compound was added to a solution of the nonradioactive compound in saline to give a final concentration of 5 to 10 mg/mL. Approximately 3 mL of this solution (containing about 10 ,uCi) was administered via an in-dwelling forelimb intravenous catheter at a dose level of 1 mg/kg body weight. Expired air was collected continuously into a Teflon bag by means of an endotracheal tube connected to a two-way valve and passed through silica gel and sodium hydroxide traps to absorb expired volatile organic materials and carbon dioxide, respectively.
A Packard Tri-Carb scintillation spectrometer (Model 2660, Packard Instruments, Downers Grove, IL) was used for the determination of radioactivity. Radioactive samples were dissolved in Eastman Ready-to-Use II scintillation fluid (Eastman Kodak Company, Rochester, NY). A Packard sample oxidizer (Model 306) was used for the measurement of radioactivity in blood samples.
Skin Exposure. For dermal application an appropriate quantity of radioactively labeled compound was added to 25 mL of undiluted nonlabeled compound and a portion (40 ,uCi, 15 mL) was transferred to a sealed, glass absorption cell secured to the dog's thorax with surgical tape and adhesive (Skin Bond, Howmedica Inc., Largo, FL). Cotton gauze inside the cell facilitated a uniform distribution of the solution. The absorption cell measured 15 cm long, 3 to 4 cm wide and 1.5 cm deep with a skin contact surface area of 55.6 cm2. Exposures were carried out for 30 or 60 minutes.
Expired air, blood and urine samples were collected and analyzed as described above. Absorption rates were calculated after correction for incomplete excretion of material using the data obtained from IV experiments.

In Vitro Percutaneous Absorption
Whole skin was removed from the thorax of beagle dogs at necropsy, and kept moist with saline under refrigeration until used. Subcutaneous fat was removed and sections (approximately 1.5 cm diameter) cut to fit across a glass diffusion cell (Franz diffusion cell, Crown Glass Co., Somerville, NJ). The skin contact area was 0.9 cm2. The lower chamber of the cell was filled with isotonic saline maintained at 37°C. The absorption rates of lt14C]PEA and [14C]EEA were determined by placing an excess of compound (0.3 mL, 1.5 puCi) in the upper chamber of the cell and measuring its appearance in the lower chamber hourly from 2 to 7 hr.
The values obtained at each time point were corrected for loss of material due to sampling. Six to eight cells were run for each compound using skin from each of two dogs. The absorption rates were calculated by linear regression analysis of the data from the time that steady state absorption was obtained.
An estimate of the integrity of the skin sample in each cell before and after exposure to [14C]PEA or [14C]EEA was obtained by determination of the permeability of the preparation to tritiated water. These measurements also provided an indication of the potential of each compound to cause irreversible, skin damage that would lead to an increase in permeability. The damage ratio was calculated as the ratio of the tritiated water permeability after skin contact with the glycol ether to the initial tritiated water permeability.

Inhalation Exposures
Analysis of the end tidal air samples during exposure to 50 ppm of PEA or EEA indicated that both compounds were rapidly absorbed through the lungs. The breath concentrations of PEA and EEA showed a rapid increase with the duration of exposure and reached a plateau after about 3 hr. After 10 min of exposure to PEA, 5 ppm PEA was detected in the end tidal air, indicating that 90% of the inhaled vapor was absorbed. PEA concentrations in breath reached a plateau at about 13 ppm which indicated that 74% of the inhaled PEA was absorbed. After 10 min exposure to EEA, 9 ppm EEA was detected in the breath (ca. 80% absorption). The EEA concentration reached a plateau at about 16 ppm, indicating that 68% of the inhaled compound was absorbed.
Post-exposure breath samples showed a rapid, exponential decline for both compounds. At 10 min, the PEA breath concentration was 5 ppm and declined to 0.5 ppm at 3 hr post-exposure. The breath concentration of EEA was 7 ppm at 10 min and decreased to 2 ppm at 3 hr post-exposure.
The serial expired air PEA concentrations were consistently lower than the EEA concentrations at corresponding times during and after the exposure (Fig. 1). These findings were statistically significant (p < 0.025) during the exposure. Linear regression analysis of the curves during the exposures showed no significant differences in the slopes, indicating that there was a similar rate of equilibration in the lungs for these two compounds.   There was no GC evidence for the presence of volatile metabolites of either PEA or EEA in the expired air.

Intravenous Administration After intravenous (IV) administration of [14C]PEA
(1 mg/kg), dogs eliminated about 88% of the radioactivity in the urine within 24 hr. The majority of this (61% of the dose) was eliminated in the first 4 hr (Table 1, Fig.  2).
After IV administration of [14C]EEA (1 mg/kg) about 61% of the radioactivity was excreted in the urine within 24 hr. Only approximately 20% of the dose was eliminated in the first 4-hr period (Thble 1, Fig. 2).
The blood concentrations of radioactivity (expressed as nmole equiv./mL) after IV administration of

Skin Exposure
The rates of percutaneous absorption and the elimination of radioactivity following 30-min or 60-min skin exposures to [14C]EEA (110.5 mmoles) or [14C]PEA (97.9 mmoles) are summarized in Table 2. Radioactivity was detected in the urine at 4, 8 and 24 hr after 30-min and 60-min exposures to both compounds ( Table 2). After exposure to [14C]PEA, urinary excretion of radioactivity was greatest at 4 hr and declined rapidly during the remaining collection period. After exposure to [14C]EEA, urinary excretion of radioactivity was similar in the first two collection periods and was still substantial overnight (Table 2). These patterns of urinary 14C excretion were similar to the respective excretion patterns seen after IV administration. No measurable concentrations of radioactivity were detected in the blood of dogs exposed for 60 min to For each compound, the amounts absorbed were similar after 30-min and 60-min exposures ( Table 2).
Small amounts of 14CO2 were detected in the expired air after exposure to both compounds.

In Vitro Percutaneous Absorption
The absorption of [14C]PEA and [14C]EEA through dog skin in vitro is shown in Figure 4. The lag times (time taken to reach steady state absorption) were calculated by extrapolation of the linear portion of each plot to the ordinate (Fig. 4, broken lines). The values obtained were 1.2 hr (PEA) and 1.6 hr (EEA). The slopes of the absorption plots indicate rates of absorption of 292 nmole/cm2/min (2.3 mg/cm2/hr) and 167 nmole/cm2/min (1.5 mg/cm2/hr) for EEA and PEA, respectively ( Table 3)   bSee methods for explanation (n = 8 or 9). cWater applied to skin instead of glycol ether. EEA nor PEA altered the diffusion properties of the skin preparations (Table 3).

Discussion
The inhalation studies demonstrate that PEA and EEA are rapidly absorbed through the lungs. At equilibrium (about 3 hr) about 70% of the inhaled vapor was absorbed by the dogs. Both compounds appeared to be removed quickly from the blood, as indicated by the rapid decline in the post-exposure breath concentrations (Fig. 1). That the expired PEA concentrations were significantly lower than the EEA concentrations during the exposure may be a reflection of the more rapid blood clearance and urinary excretion rates for PEA (Tables 1 and 2 The different elimination characteristics of PEA and EEA may result from differences in the metabolism of the compounds. At present, no data are available on the metabolism of PEA. Ethoxyacetic acid and N-ethoxyacetylglycine have been identified as metabolites of ethoxyethanol in rats after inhalation and oral administration. The combined excretion of the two metabolites was estimated to be 30% of the oral doses (9). Other ethylene glycol ethers, methoxyethanol, butoxyethanol and 2-(2-ethoxyethoxy)ethanol, have also been shown to be metabolized by oxidation to the corresponding alkoxyacetic acid (10)(11)(12). Dealkylation of [1-14C-ethoxy]2-ethoxyethanol has also been reported (13). Thus it is reasonable to expect that PEA would undergo the same metabolic reactions as these related compounds. Different excretion rates for PEA and EEA may therefore reflect quantitative differences in metabolic products.
Although the excretion rates for PEA and EEA were markedly different, the total amount of each compound absorbed through the skin was similar ( Table 2). The fact that, for each compound, approximately equal amounts of radioactivity were absorbed in 30 and 60 min may result from the relatively short exposure periods used in these experiments. Percutaneous absorption studies in vitro indicated absorption lag times of 1.2 hr (PEA) and 1.6 hr (EEA) with dog skin before steadystate absorption occurred, after which time EEA was absorbed at a faster rate than PEA (2.3 mg/cm2/hr vs. 1.5 mg/cm2/hr) ( Fig. 4 and Table 3).
The data from the in vivo dog experiments may be used to estimate PEA or EEA uptake after human exposure, assuming that the rate of absorption in man is similar to that seen in dogs. Thus, after inhalation exposure for 1 hr at a concentration of 50 ppm, about 270 mg of PEA or 220 mg of EEA would be absorbed through the lungs. This is equivalent to about 2 g of material in an 8-hr day. After immersion of both hands for 30 to 60 min, about 580 to 750 mg of PEA or 640 mg of EEA would be absorbed through the skin. Extrapolating from the clearance rates in dogs, the absorbed PEA would be cleared more rapidly than EEA.
These absorption rates are similar to those obtained in our laboratory for other lipid-soluble chemicals, such as methyl n-butyl ketone and n-butanol (14,15).